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These methods are used primarily for field experiments, including shovelomics and soil coring.

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Plant sciences articles within Nature Methods

Technology Feature | 19 June 2024

How’s your health, forests?

Assessing forest health and resilience is an urgent, many-methods task that takes global collaboration, data collected at multiple scales and a willingness to be surprised.

• Vivien Marx

This Month | 06 October 2023

Chlamydomonas reinhardtii : a model for photosynthesis and so much more

The green alga Chlamydomonas reinhardtii is a useful reference organism for studying photosynthesis, cilia and the cell cycle. Like many other algae, it exhibits daily rhythms in gene expression and behavior that are in sync with the rising and setting of the sun.

• Sunnyjoy Dupuis
•  &  Sabeeha S. Merchant

Technology Feature | 16 July 2021

Exploring the diverse, intimate lives of plants

New ways to assess plant–microbe interactions can yield unexpected paths to biodiversity.

Technology Feature | 24 February 2021

Model organisms on roads less traveled

Beyond the well-known pantheon of model organisms are others. A shift is underway to level the playing field.

Article | 29 June 2020

Optogenetic control of gene expression in plants in the presence of ambient white light

PULSE is an optogenetic tool that consists of two modules with different wavelength sensitivities. Their interplay enables optogenetic access to gene expression in plants independently of ambient light.

• Rocio Ochoa-Fernandez
• , Nikolaj B. Abel
•  &  Matias D. Zurbriggen

In Brief | 05 May 2020

A pan-plant protein complex map

• Arunima Singh

Brief Communication | 18 September 2017

Autofocusing MALDI mass spectrometry imaging of tissue sections and 3D chemical topography of nonflat surfaces

A mass spectrometry imaging system combined with a laser triangulation system provides simultaneous topographic and molecular information for 3D biological samples and eliminates height-related artifacts in imperfect tissue sections.

• Mario Kompauer
• , Sven Heiles
•  &  Bernhard Spengler

Article | 26 June 2017

CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping

CrY2H-seq, a Cre recombinase reporter-mediated yeast two-hybrid method coupled with next-generation sequencing, enables ultra-high-throughput screening of transcription factor interactions in Arabidopsis thaliana .

• Shelly A Wanamaker
• , Renee M Garza
•  &  Joseph R Ecker

Tools in Brief | 31 October 2016

A plant metabolite spectral library

Technology Feature | 29 June 2016

Plants: a tool box of cell-based assays

Cell-based assays are less routine for plant biologists than for researchers who work with animal or human cells, but that is changing.

Article | 16 November 2015

Quantitative characterization of genetic parts and circuits for plant synthetic biology

Quantitative analysis of promoter-repressor pairs in plants will allow the design of predictable gene circuits in multicellular organisms.

• Katherine A Schaumberg
• , Mauricio S Antunes
•  &  Ashok Prasad

Brief Communication | 02 February 2015

Reporters for sensitive and quantitative measurement of auxin response

Two fluorescent reporters allow high-resolution visualization of auxin response; DR5v2 is more sensitive than existing auxin response reporters, and a ratiometric version of DII-Venus named R2D2 allows for accurate quantification of auxin input.

• Che-Yang Liao
• , Wouter Smet
•  &  Dolf Weijers

Methods in Brief | 30 December 2014

A powerful haploid tool for plant genetics

Tools in Brief | 28 August 2014

Watching auxin make its move

Brief Communication | 22 September 2013

Accounting for technical noise in single-cell RNA-seq experiments

A statistical method that uses spike-ins to model the dependence of technical noise on transcript abundance in single-cell RNA-seq experiments allows identification of genes wherein observed variability in read counts can be reliably interpreted as a signal of biological variability as opposed to the effect of technical noise.

• Philip Brennecke
• , Simon Anders
•  &  Marcus G Heisler

Article | 30 September 2012

A microfluidic device and computational platform for high-throughput live imaging of gene expression

RootArray and RootArray Controller comprise a robust pipeline for acquisition and analysis of gene expression data in plant roots over time.

• Wolfgang Busch
• , Brad T Moore
•  &  Philip N Benfey

Brief Communication | 01 April 2012

Integrated genetic and computation methods for in planta cytometry

Automated cell segmentation in time-lapse confocal images of growing Arabidopsis combined with ratiometric imaging of fluorescent gene expression reporters permits the correlation of cellular properties with gene expression in the growing plant.

• Fernán Federici
• , Lionel Dupuy
•  &  Jim Haseloff

Methods in Brief | 27 March 2012

Faster genetic mapping in plants

Research Highlights | 28 February 2012

Reporting plant hormone levels: a disappearing act

A fluorescent protein fusion reports levels of the plant hormone auxin by succumbing to its degradation signal.

Brief Communication | 30 October 2011

Enhanced Y1H assays for Arabidopsis

An almost-complete, sequence-verified collection of Arabidopsis thaliana root stele transcription factors is reported. The authors use it in the enhanced yeast-one hybrid (eY1H) assay to map gene regulatory interactions in the plant. Also in this issue, Reece-Hoyes et al . describe the eY1H pipeline.

• Allison Gaudinier
• , Lifang Zhang
•  &  Siobhan M Brady

News & Views | 01 July 2010

The inside view on plant growth

With a combination of microscopic and computational methods, the lineage of cells produced by divisions in the meristems of growing plants can now be tracked over time.

• Edgar P Spalding

Article | 13 June 2010

Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution

A set of computational and imaging approaches, called MARS-ALT, permits three-dimensional tracking of plant tissue development, including cell lineaging, at cellular resolution. It is applied to the study of floral development in Arabidopsis .

• Romain Fernandez
• , Pradeep Das
•  &  Christophe Godin

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• v.15(1); 2022

Tool and techniques study to plant microbiome current understanding and future needs: an overview

a Department of Plant Pathology, School of Agriculture, SMPDC, University of Lucknow, Lucknow, India

Prem Chandra

b Department of Environmental Microbiology, Babasaheb Bhimrao Ambedkar (A Central) University, Lucknow, India

Microorganisms are present in the universe and they play role in beneficial and harmful to human life, society, and environments. Plant microbiome is a broad term in which microbes are present in the rhizo, phyllo, or endophytic region and play several beneficial and harmful roles with the plant. To know of these microorganisms, it is essential to be able to isolate purification and identify them quickly under laboratory conditions. So, to improve the microbial study, several tools and techniques such as microscopy, rRNA, or rDNA sequencing, fingerprinting, probing, clone libraries, chips, and metagenomics have been developed. The major benefits of these techniques are the identification of microbial community through direct analysis as well as it can apply in situ . Without tools and techniques, we cannot understand the roles of microbiomes. This review explains the tools and their roles in the understanding of microbiomes and their ecological diversity in environments.

Introduction

Microorganisms have a variety of roles in both human and environmental life. Despite their small size, these organisms have a significant impact on society, either positively or negatively [ 1 ]. Small organisms, on the other hand, can be found in almost any environment, including water, soil, and air, making microorganisms pervasive [ 2 ]. Microbe research is difficult due to our lack of understanding of them and their environments. Because microorganisms occur in large quantities in the natural environment, conventionally based methods do not provide enough data to fully comprehend microbes and their impact on the ecosystem, including plants [ 3 ]. However, due to a lack of relevant and up-to-date instruments and methodologies for understanding these microbes, numerous microbial species have remained unknown till today [ 4 , 5 ].

Different techniques/procedures are available for characterization and identification of microbes, such as isolation, purification of visible colonies, and microbe rearing, however, all methods are applied at the laboratory desk, and these methods are old [ 6 , 7 ]. Some techniques and instruments, such as culture technique and microscopy, have been widely employed in the past, but they provide a limited picture of the microbial world [ 8 , 9 ]. Many bacteria seem the same under a microscope, and many won’t thrive outside of their natural habitats. Because of a lack of tools and methodologies, only around 1% of microbes have been discovered. This is because certain microbes are viable but non-culturable (VBNC) while others are found in extreme environments such as very high or low temperature, pH, pressure, salinity, and so on [ 10 , 11 ]. As a result, early scientists only investigated a small number of bacteria, and a certain region of microbial habitat refers to just those microbes that have been produced in a microbial laboratory. However, increased culture is required for a complete investigation of microorganisms, which can only be done in the lab [ 12 , 13 ]. For the study of microbial diversity, there are commonly three methodologies. 1) methods that are culture-specific 2) Techniques that are not culture-specific 3) methods based on molecular biology. Culture-dependent approaches lose the majority of microbial species, making it impossible to investigate microbial ecology [ 14 , 15 ], but culture-independent and molecular techniques allow us to research microbial ecology more easily [ 16 ]. For bacterial identification, PCR amplification of the universal 16S rRNA gene is often utilized. 16 sRNA sequencing offers data at the taxonomic level of bacterial species, and it is a commonly used technology [ 17 , 18 ]. The comparable rRNA gene known as 18 sRNA is used to research higher microorganisms such as fungus [ 19 ]. If 18 sRNA does not provide enough information on the fungi, we go on to the internally transcribed spacer (ITS). ITS gives complete data for fungus taxonomy identification [ 20 , 21 ]. Although neither technique can identify a new species, this is a significant drawback of both techniques because they both rely on primers. Although both procedures cannot identify new species, this is a significant limitation of both techniques because both techniques rely on primers that detect just the target species/organisms [ 22–24 ].

All realms of life, on the other hand, are populated in a complex environment. Prokaryotic grazers, such as protozoa, fungi, nematodes, and oomycetes, can be important plant symbionts or destructors, while others are prokaryotic grazers found in most soils around the world [ 25 , 26 ]. In agricultural soils, the archaea domain participates in metabolic activities such as ammonia production, oxidation, and methanogenesis. In the environment, bacteria in the last domain serve a variety of activities, including plant growth and development, pathogen defense, bioremediation, biodegradation, and other industrial sectors [ 27 ]. They also operate in the manufacturing and service industries, producing ethanol, enzymes, acids, fragrances, and medications, among other things. Bacteria interact with the host plant and other bacteria in the ecosystem, therefore capturing as much variation of a microbiome as possible is critical [ 28 ]. To accomplish so, international methodologies such as metagenomics, meta-transcriptomic, meta-proteomic, and metabolomics must be used, which allow for the simultaneous appraisal and judgment of indigenous microflora across all domains of life [ 29 ]. Metagenomics is the direct extraction and cloning of genetic material from samples to determine the genomic diversity of microbes (culturable and unculturable), whereas metaproteomics, metatranscriptomics, and metabolomics, respectively, provide a sketch of community-wide protein abundance, gene expression, and metabolic activities [ 30 , 31 ].

In this review, we will look at the know-how and methods that have been used in the isolation and characterization of bacteria and microbiomes on a morphological and molecular level. We’ll also learn how to use metagenomics to identify VBNC organisms. These methods can also be used to investigate microbiomes, or microbe populations, in various contexts [ 32 ].

Plant microbial community

To address the importance of the soil-associated plant microbiome to plant trait expression and ecosystem functions, the aspects of the plant microbiome covered in this overview are limited to the rhizosphere, rhizoplane (epiphytes), and internal endosymbiosis (endophytes) of the belowground organs of the plant [ 33–35 ]. However, this ignores the rhizosphere microbiome’s effects on aboveground interactions, including herbivory, pollination, and seed predation, as well as pathogen attack from aboveground structures [ 36 , 37 ].

Plant-microbe interactions are important for plant growth and yield, and they have gotten a lot of attention recently [ 38 ]. This type of interaction is seen in all regions of plants, according to microbiologists and ecologists, but it is called a plant microbiome when it occurs in a specific portion of the plant [ 39 ]. “A plant microbiome is a specific place/region/habitats, such as roots, leaves, stems, and floral sections, where varied bacteria exist and display various interactions with the plants,” according to the definition [ 40 ]. These interactions may be mutual/beneficial/harmful given in Figure 1 .

Role of the plant microbiome.

Types of plant microbiome

Plant microbiomes are diverse, containing both beneficial and harmful rhizospheric microorganisms, as well as endophytic and pathogenic pathogens [ 41 ]. Plant microbiome is categorized into three kinds based on their occurrence and interactions with the plant: 1) Rhizospheric plant microbiome; 2) Phyllospheric plant microbiome; and 3) Endophytic plant microbiome.

Rhizospheric plant microbiome

Soil has a variety of microorganisms, which divide into two zones based on their availability to the plant [ 42 ]. The rhizosphere is a zone characterized by increased microbial mass and soils that directly surround plant roots. The rhizosphere is further separated into two parts: a) edaphosphere (one side bordered by soil region) and b) histosphere (the other side surrounded by plant tissues), both of which play a vital role in rhizosphere development [ 43 , 44 ]. Rhizospheric soils have a high water-holding capacity, indicating that there are many mutual interactions between microorganisms and plant roots in the soil, as well as improved nutrient availability. Rhizoplane refers to soil particles that are closely adhered to the root surface [ 45 , 46 ].

Fungi, protozoa, archaea, nematodes, oomycetes, bacteria, algae, and viruses are all frequent creatures found in the rhizosphere, and these organisms are referred to as rhizo-microbiomes [ 37 , 47 ]. These creatures dwell in the rhizosphere and feed on the plant’s nutrients (organic acids, sugars, amino acids, fatty acids, vitamins, and growth hormones) [ 48 ]. Plant growth-promoting rhizobacteria (phosphate solubilizing bacteria, nitrogen-fixing bacteria, biological control bacteria), cyanobacteria, fungi, mycorrhiza, protozoa, and pathogenic bacteria, fungi, virus, and nematodes are among the organisms that have been well studied for their beneficial effects on plant growth and development [ 27 ]. Another type of bacteria to consider is Pseudomonas aeruginosa , which improves plant growth and production but can cause sickness in people [ 38 , 49 ]. Plant growth and fitness are influenced by rhizospheric microbiomes, which can be beneficial or harmful. It is positively influenced by phytohormone secretion (indole-3 acetic acid, gibberellin), production of siderophore and ammonia, solubilization of phosphate, zinc, and potassium, and indirectly by decomposition of detritus, nutrient cycling, pathogen inhibition, secretion of stress hormone (ACC deaminase), and stimulation of the plant immune response [ 50 ]. However, certain bacteria function as pathogens, reducing crop productivity [ 51 ].

Phyllospheric plant microbiome/aerial plant surface microbiome

The phyllosphere is the world’s second-largest microbial habitat. By colonizing severe, stressful, and changing settings, the microbial community in this region performs a dynamic function [ 52 , 53 ]. It is a significant point of entry for phytopathogens into plant tissues, where they cause disease. Furthermore, they offer a unique location for easily comprehending the interaction between microbiota and plants, as well as the methods by which distinct microbial populations sustain their populations in nature [ 54 , 55 ]. In comparison to the rhizosphere or endophytic zone, the phyllosphere has a low nutritional content [ 56 ].

On the phyllosphere, microbial population colonization is different, but it is influenced by the leaf’s stomata, hairs, and veins [ 57 ]. On the leaf surfaces, 107 microorganisms per cm 2 colonize [ 58 ]. The phyllosphere is a much more interesting place, where microbes live in the presence of large fluctuations in temperature, radiation, moisture, and light throughout the day and night. Plant metabolism changes as a result of these environmental factors and the phyllosphere microbiome are affected [ 59 ].

Endophytic/ root interior plant microbiomes

Endophytes are microorganisms that dwell inside plant tissue, whereas the endosphere is the surrounding environment [ 60 ]. Endophytic bacteria are typically thought to be non-disease causing agents because they cause no obvious symptoms on plants [ 61 ], but researchers have recently uncovered several pathogens that are dependent on the host genotype and environmental factors.

Endophytes are thought to have started in the rhizosphere, although they exhibit unique characteristics from rhizospheric microbes, implying that not all rhizospheric microbes can penetrate the plant [ 62 ]. When bacteria penetrate a plant, they change their physiological/metabolic processes and adapt to the host’s inner environment [ 3 ].

Tools and techniques to the understanding of plant microbiome

There are new methodologies and procedures available to assist in the research of microorganisms that reside in a biome, allowing for accurate microbial ecology values [ 63 ]. The abundance of species, population size, species consistency, and species distribution are all factors in microbial ecology [ 64 ]. Measurement of microbial diversity is routinely done using morphological, biochemical, and molecular approaches. Molecular-based approaches, for example, can provide taxonomy-level information [ 65 ].

Some molecular approaches, including G + C percentage, restriction fragment length polymorphism (RFLP), DNA hybridization, and community-level physiological profiles (CLPP), are useful for identifying aquatic populations without providing any data [ 66 ]. Many DNA fingerprinting techniques, such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), repetitive PCR, arbitrarily PCR, and terminal-restriction length polymorphism (T-RFLP), are now available and widely used in the identification of microbial species by retaining polymerase chain reaction from environmental samples [ 67 , 68 ]. Researchers, on the other hand, can’t utilize diversity metrics since microbiomes don’t have a specific diversity and fluctuate as the environment changes [ 69 ]. First and foremost, the origin of the community must be known to comprehend the secret of microbes and plants in a specific habitat [ 70 ]. Different tools and strategies are required to do this. Table 1 lists the most important instruments and strategies for learning about plant microbiomes.

Major tools and techniques for studying of plant microbiome and their merits and demerits [ 96 ].

Researchers employ a variety of microscopes to study the plant microbiome, including light, compound, dark field, bright field, confocal, and fluorescence microscopes [ 71 ]. However, because these microscopes have a smaller focusing point, they are not suitable for in-depth research. Electron microscopes, such as the scanning electron microscopy (SEM) and transmission electron microscope (TEM), can overcome these restrictions since they have a high-resolution power attached to the usage of an electron beam, as opposed to light or compound microscopes [ 72 ]. Microorganisms and plants on the surface and inside the cell can be spotted using an electron microscope [ 73 ]. Furthermore, the microbiome’s habitat, niche, host, and behavior can all be explored [ 74 ]. The microscope aids in the colonization of microorganisms on or inside the plant surface, as well as the understanding of their role in the plants [ 75 ]. Because it is a basic requirement of microorganisms for sustenance, and because of the nature of the habitat, effective colonization is a vital point in plant-microbe interactions [ 3 ]. Thus, plant colonization has long been an important issue in studies on the routes and roles of these organisms with plants [ 76 ], and microscopy aids in the viewing of microorganisms in their natural habitat and relationship with plants [ 77 ]. Furthermore, this method enables the tracking of microorganisms [ 78 ].

Nucleic acid extraction

Understanding the plant microbiome requires the use of nucleic acid extraction, which is one of the most significant technologies in biology. A nucleic acid extraction is an old approach that has been well refined in the twenty-first century [ 79 ]. Nucleic acids are extracted in three processes, depending on the sample and downstream application: a) breaking the samples (tissue or cells); b) removing lipids, proteins, and other contaminants from the nucleic acid; and c) transferring the nucleic acid to a buffer solution for storage [ 80 ]. However, numerous commercial molecular kits for nucleic acid extraction are currently available on the market. However, the identification of DNA and RNA can be done using both traditional and kit approaches, and both methods are used to extract quantitative and qualitative nucleic acid analyses [ 81 ]. Furthermore, it is expected that the same standardized procedure is employed, as each method will have its preferences in terms of nucleic acid quality and amount [ 82 , 83 ].

Nucleic acid hybridization

In the study of the plant microbiome, nucleic acid hybridization (DNA-DNA and RNA-DNA) is a useful technique [ 84 ]. Hybridization reaction occurs when two harmonizing single-stranded nucleic acids form a partial or entire double-stranded nucleic acid by a specific-sequence interface [ 85 ]. Nucleic acid is analyzed quantitatively and quantitatively utilizing specific probes in this approach [ 86 ]. The probe, which is recognized sequences spanning in specificity from domain to species and is identified with markers at the fifty end position, is often taken [ 87 ]. There are a variety of nucleic acid hybridization procedures available, with FISH (fluorescent in situ hybridization) being one of the most popular [ 88 ].

This method can be used to investigate the spatial dispersion of a microbial population in a different place [ 89 ]. However, due to a lack of sensitivity and the presence of high copy numbers in comparison to dominant species in a sample, we cannot directly extract nucleic acid from environmental materials using this method [ 90 ]. As a result, analytical tools can be used to overcome these constraints, and PCR is a good option. Membrane hybridization is another prominent technique utilized by researchers in addition to FISH [ 91 ]. Denatured RNA or DNA is immobilized on inner support in such a way that self-annealing is prevented while the residual sequence (bound) is present for hybridization with tagged probes in the membrane method (single or double-stranded). Furthermore, the membrane is extensively washed to remove the unattached probe, and a low case of matched hybrids follows the nucleic acid hybridization reaction [ 92 ].

Polymerase chain reaction

One of the most essential techniques for increasing or amplifying a certain target sequence of DNA is the polymerase chain reaction (PCR) [ 93 ]. In PCR, the targeted sequences are chosen from the nucleic acid sequence, such as repeated sequences or specific genes [ 94 ]. The PCR process takes at least 35–40 cycles to complete and is divided into three parts based on temperature: One cycle consists of 1) denaturation, 2) annealing, and 3) elongation. 16S rRNA gene primers are known as universal or species-strain primers [ 95 ].

It’s a promising PCR amplification for bacterial species identification and phylogenetic reasons [ 96 ]. The PCR employs nonspecific dyes (SYBER Green I and SYBER Gold) that bind to double-stranded DNA [ 97 ]. PCR develops numerous variants based on the type of sample to justify the isolation and quantification of live bacterial numbers at the same time. Multiplex PCR is a fantastic example for separating mixed bacterial pathogens from a sample as well as differentiating multiple species belonging to the same genus [ 65 , 98 ].

The earliest PCR, on the other hand, is unable to detect live or dead bacterial cells. However, this problem can now be rectified using a cutting-edge technology known as reverse transcriptase PCR (RT-PCR) [ 99 ]. The reverse transcriptase enzyme powers RT-PCR. This technology, on the other hand, is sensitive yet does not require pre-enrichment operations, and it also takes less time than traditional PCR. Furthermore, VNC cells cannot be cultivated using standard PCR or simple laboratory methods, but we can detect them using RT-PCR [ 100 , 101 ].

Exonuclease activity generates a luminous and detectable signal in RT-PCR. A signal is created with each PCR cycle. The generated signal enables real-time detection through real-time PCR. When a signal permits a specific threshold level, the signal is transformed into forecast target gene numbers based on a pre-established calibration line with ordinary target DNA [ 102 ]. RT-PCR also assesses the magnitude of gene effects in local settings as well as the degree of gene expression in microhabitats. As a result, it may be possible to more precisely map microflora and their utility to the soil, plants, and other places using this technique [ 103 ].

DNA fingerprinting

Because every organism has unique DNA, DNA fingerprinting is a unique approach for identifying an organism based on DNA properties. It is primarily utilized in forensic sciences, although it is currently used in a variety of fields, including the study of plant-microbe interactions [ 104 ]. In the fingerprinting techniques, restriction fragment length polymorphism (RFLP), polymerase chain reaction-restriction fragment length (PCR-RFLP) [ 105 ], denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), temperature gradient gel electrophoresis (TGGE), single-strand conformational polymorphisms (SSCP), ribosomal internal spacer analysis (RISA), length heterogeneity-PCR (LH-PCR) are involved [ 106 , 107 ].

These strategies aid in the study and comparison of data from microbial communities in a sample. However, some PCR-based approaches for microbial community characterization were outmoded, but many new post-PCR analytical techniques have emerged in recent years [ 108 ]. The main advantages of this method are that it allows for a comparison of the microbiome’s morphology, composition, and diversity in samples such as soils. Another advantage of this technique is that it can distinguish between viable and nonviable cells in the microbial population, which is something that no other fingerprint technology can do [ 109 ].

Denaturing gradient gel electrophoresis

DGGE is a very useful technology for detecting the microbial population directly from environmental materials, which means it does not require microbe rearing. PCR-DGGE, phylogenetic DGGE, and functional gene DGGE are the three most common forms. Only a few samples are required for microbial community characterization in PCR-DGGE [ 110 ]. This method detects specific clusters of microorganisms from various plant zones, such as roots and leaves.

On polyacrylamide gels with denaturing gradients, the principle of PCR-DGGE is carried out. However, while this technique was originally developed for mutation analysis, it is currently employed in a wide range of applications, such as detecting microbial communities in environmental samples [ 111 ]. This technique has the advantage of simultaneously observing many samples and assessing microbial communities based on ecological and historical differences [ 112 , 113 ].

Phylogenetic study of bacteria using 16 sRNA genes is commonly employed in PCR-DGGE currently. This is not a new notion, as similar techniques have been in use since 1990 [ 114 ]. The single species of the community can be identified using phylogenetic DGGE [ 115 , 116 ] by removing DGGE bands from the gel and sequencing them, or by creating clone libraries of 16S rRNA that are separated using DGGE [ 115 ]. Muyzer et al . [ 117 ] reported that the 16S rRNA gene is employed as a molecular biomarker for microbial species in a population. The most important thing to remember when using partial 16S rRNA gene sequencing is to be cautious when interpreting the results. V4-V5 are appropriate sections for phylogenetic analysis when compared to full-length sequence data [ 118 ]. However, the DGGE technique has some limitations, such as microbial DNA extraction and community analysis from environmental samples. Another limitation is that while one strain produces only one band, in some species two or more bands have been observed, and thus we cannot estimate the true microbial diversity data [ 110 ].

The data of soil health, quality, and function is provided by the functional gene-based DGGE. We may simply link reduced soil microbial diversity to poor soil functioning this way [ 119 ]. The study of coding proteins genes entangled in critical biome practices has gotten a lot of interest in the past few years [ 120 ]. Furthermore, functions in which the genes are accommodated by one or more species of bacteria were postulated. In comparison to extremely unnecessary bacteria species/groups, interruption inducing bacteria species/groups affect the functioning of soil [ 121 ].

Furthermore, functional genes for nitrogen fixation, denitrification, and other processes are abundant in bacterial species. Since the previous few decades, there has been a surge in interest in gene databases, including primer design, gene function, and gene identification [ 122 ]. The gene producing nitrate reductase narG, nitrite reductases nirS and nirK, encoding dinitrogenase reductases nifH, and amoA encoding the ammonia monooxygenase have all been used as substitutions to track changes in soil functional gene diversity [ 123 , 124 ]. The nifH has recently been utilized to investigate the influence of GM white spruce on soil N 2 fixation communities. However, the authors failed to mention the GM plant’s significant impact on N 2 fixation ecosystems [ 125 ]. Another study used the PCR-DGGE approach to positively trace the phlD gene, which codes for diacetyl phloroglucinol (DAPG), an antagonistic chemical generated by pseudomonads [ 126 ].

Clone libraries

A clone library is a collection of DNA fragments that have been cloned into vectors and have been used by researchers to identify and extract those DNA fragments that they are interested in studying further [ 127 ]. cDNA libraries and genomic DNA libraries are the two types of libraries that exist. The cDNA library is made up of clones and contains reverse-transcribed mRNA, but it lacks DNA sequences corresponding to genomic areas that aren’t expressed, such as 5’ and 3’ noncoding regions, and introns. Genomic libraries, on the other hand, contain huge amounts of DNA in the form of bacterial, bacteriophage, or other synthetic chromosomes [ 128 ].

Clone libraries provide immediate access to information on the microbiome’s targeted gene sequences. This method involves joining PCR-generated replicons to a suitable vector plasmid [ 129 ]. In addition, using the transformation procedure, the synthesized DNA is cloned into an appropriate host such as Escherichia coli . Following transformation, plasmid extraction can be used to remove cloned replicons from the inserted vector, which can then be sequenced and studied using databases [ 130 ]. Chimeras (a single bacterial cell with two different genotypes) are generally eliminated during this procedure.

Because the strain’s sequences are evaluated separately, this technique is far superior to phylogenetic analysis or fingerprinting techniques [ 131 ]. The capacity to directly obtain and evaluate novel strains is a major benefit of this technology, which improves our understanding of the soil microbial population [ 132 ]. However, other researchers claim that clone libraries are a time-consuming method because many techniques have progressed in recent years, allowing for a more comprehensive understanding of bacteria and functional genes in a microbiome [ 109 ].

DNA microarrays

For studying the soil microbiome, DNA microarray is an excellent technique. The term “DNA chip” or “biochip” is also used to describe it. This technique is used to examine the genetic constitutions of several sections of an organism’s genome or to evaluate gene expression levels at a large number of genes at the same time [ 133 , 134 ]. The sample’s DNA is extracted and amplified using PCR in this procedure. A microarray is used to analyze additional DNA samples that have been tagged. A thick range of oligonucleotide probes is inserted on the microarray, ranging from 10 to 1000 [ 135 ]. Probes could be 16S rRNA gene fragments or functional gene fragments. The DNA samples must be homogeneous to the probes on the chip for them to bind or hybridize. The chip’s signals are digitally analyzed after binding [ 136 ]. Chip contributes to our understanding of phylogenetic diversity, functional genes, and community composition in this way. When the material is extremely complex, such as soil, however, analysis can be difficult [ 137 ].

Cross-hybridization, on the other hand, is a serious concern with microarrays. At least 11 or more short oligonucleotides have been designed to overcome the challenge, allowing dissimilar matches to be distinguished from similar perfect matches [ 138 , 139 ]. According to the number of probes and design [ 140 ], there are two types of microarrays: a) geochips and b) phyloarrays. Over 24, 000 probes cover over 10,000 genes scattered among more than 150 functional categories enmeshed in carbon, sulfur, phosphorous, and nitrogen cycling on the geochips [ 141 , 142 ]. They’re utilized to collect soil samples. Geochips are utilized on Antarctic soils to analyze various cycles such as carbon, nitrogen, and other elements. Microarray binding holds a lot of promise, including the possibility of creating a “universal microarray.” It explains the state and condition of the soil [ 143 ].

Next generation OR omics tools and techniques

Metagenomics is the most recent technology to be developed. It’s a cutting-edge technique. Metagenomics is the study of bacteria’ whole genomes retrieved from environmental materials. Metagenome gives a demonstrated understanding of species composition, genetic diversity, inter-species interactions, and species evolution in the context of typical civilizations’ environments [ 30 ]. Several sophisticated omics sequencing techniques, like pyrosequencing and Illumina sequencing, were established before the last several decades. These methods are useful in metagenomics and metatranscriptomics research.

For example, soil samples were identified using 454-based pyrosequencing and DNA or RNA extraction [ 144 ]. Multi parallel sequencing by synthesis is used in this technique, with luciferase enzyme detection and pyrophosphate release detection of the produced light [ 145 ]. Both Illumina and pyrosequencing, on the other hand, go through three steps of traditional sequencing: template preparation, library preparation, and actual capillary sequencing [ 146 ]. Due to multi parallelity, the sequencer can generate anything from 100 to millions of 450 base pair (bp) readings in a single run [ 147 ].

The sensitivity of next-generation sequencing, when collected straight from a soil sample, is investigated for its competence and impartiality [ 148 ]. As a result, the depiction and conclusion bias of the results are determined by the extraction of DNA from the soil sample [ 149 ]. Although the 454 sequencers are used directly for the development of base reads because it produces longer reads, the Illumina sequencer may be utilized to fill in gaps in the 454 generated sequence data because of its high throughput [ 150 , 151 ].

Metabolomics

Metabolomics research aims to learn more about a biological system’s small molecule metabolites under specific conditions. Primary and secondary metabolites make up the metabolome in general. Plant defence mechanisms have a large diversity of secondary metabolites compared to other complex biological systems [ 152–154 ]. Herbivores and microorganisms find the majority of them poisonous or repulsive. Metabolomic compound analysis yields metabolic profiles and fingerprints, as well as the identification of novel biomarkers, which can be combined into microbiome research for a more holistic understanding of the plant microbiome [ 155 ].

Analytical technologies used in metabolomics

Nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS) are the most common technologies utilised in metabolomics (LC-MS). MS-based approaches are substantially more sensitive than NMR at detecting metabolites [ 156 ]. MS samples, on the other hand, necessitate extensive preparation, and detection is limited to metabolites that can ionise into the detectable mass range. For chemicals that are difficult to ionise or dissolve, or that require derivatization for MS, NMR has certain advantages [ 157 ].

Targeted and untargeted techniques to metabolomics have hitherto been separated, however this may change in the future [ 158 ]. Pre-processing, annotation, post-processing, and statistical analysis are all steps in the analysis of data generated using these technologies (NMR and MS). These procedures are generally customised to the analytical technology [ 159 ]. To adjust discrepancies in peak shape width and location caused by noise, sample differences, or instrument parameters, pre-processing procedures are used. There is currently no gold standard pipeline for data pre-processing [ 160 ].

A metabolite must be compared to at least two orthogonal properties of an authentic chemical standard evaluated in the same laboratory using the same analytical methodologies as experimental data, according to the Metabolites Standard Initiative (MSI) [ 161 ]. Because most metabolites do not have chemical criteria, they cannot be completely identified. As a result, MS annotation tools are classified into several levels of annotation. Metabolites can be detected using NMR by simply comparing data from internet databases [ 162 ]. This restricts the results to the content of the relevant databases [ 163 ].

Metatranscriptomics

Metatranscriptomics is a method for identifying active genes or pathways in a microbial community by sequencing the total message RNA (mRNA). This procedure entails extracting total RNA from microbial communities, eliminating ribosomal RNA (rRNA) to obtain high amounts of mRNA transcripts, reverse transcribing mRNA into cDNAs, ligating adapters, and sequencing with NGS [ 164 ]. Metagenomics and metatranscriptomics are frequently used jointly to give assembled genomes as templates for transcript mapping. Top Hat and HISAT, as well as Cufflinks and HTSeq, have been developed for this purpose. Metatranscriptomics has been successfully applied to a wide range of settings, including soil, sediment, gut microbiomes, and activated sludge. It’s a useful method for deducing community function and activity, as well as identifying novel pathways in uncultured microorganisms [ 165 ].

The study of proteins in a microbial community extracted from an environmental sample is known as metaproteomics. Metaproteomics, unlike other -omics techniques, gives direct evidence for proteins, post-translational modifications, protein-protein interactions, and protein turnover, all of which represent the structure, dynamics, and metabolic activities of microbial communities [ 166 ]. Metaproteomics mostly use mass spectrometry-based proteomics technologies [ 167 , 168 ]. Metaproteomics has been used in plant microbiome studies to assess bacterial communities in the phyllospheres of tree species in a pristine Atlantic Forest [ 169 ], to investigate the response of the plant PGPB Bacillus amyloliquefaciens FZB42 to the presence of plant root exudates [ 170 ], and to determine the differences in soil protein abundance in plant sugarcane and rat Despite its success, metaproteomics in the plant microbiome is limited due to reduced protein expression in plant microbial samples and limited database information [ 171 ].

Metaproteomics is a technique for assessing microbial activity in an ecosystem at a certain moment using protein expression [ 172 ]. Metaproteomics, unlike metagenomics and metatranscriptomics, uses liquid chromatography tandem mass spectrometry (LC-MS/MS). The procedure begins with protein extraction, followed by LC-MS/MS 1generation of MS spectra, and finally, comparison of spectra with peptides from thousands of proteins from various taxonomic groups [ 173 ]. These comparisons can be made in two ways: by exploring current protein/peptide databases or by comparing to theoretical peptide spectra created in silico from metagenomes from the same sample or from similar environments [ 174 , 175 ]. Metaproteomics is a potent technique for deciphering active metabolic processes in various contexts in a more direct manner than metagenomics or metatranscriptomics. This method has been used to study soils [ 176 ], sediments, marine habitats, freshwater systems [ 177 ].

Environmental samples such as air and seawater can be monitored for hazardous compounds using biosensors [ 178 ]. Nucleic acid amplification techniques, mass spectrometry methods, and receptor-ligand binding assays are the three most used approaches for multiplex detection from complicated matrices. By improving signal transduction, nanotechnology has been used in biosensors to improve sensitivity and performance of assays [ 179 ]. Nanotechnology is the process of creating structures, gadgets, and systems that have unique properties that can be controlled by changing the size and form of materials at the nanoscale scale [ 180 ].

• To utilize these instruments to arrest and study the accumulative multifaceted data and information, new ways and approaches for evidence progress are required to build new methods and approaches [ 181 ].
• Tools and procedures improve the research of microbial communities and evidence of these communities [ 113 ].
• It aids in time management and data organization.
• Some approaches, such as metagenomics, allow us to glimpse into the hidden microbial world, which aids in the study of viable but nonculturable (VBNC) bacteria [ 30 ].
• Tools and techniques can be used to understand the connection between microbiomes and their hosts, as well as the link between symbiotic, mutualistic, and commensalism variety and functions [ 182 ].
• Modern omics approaches to aid in the development of novel tactics and the conduct of research to obtain a detailed report on the microbiome and its expression, as well as the level of plant genome expression monitoring [ 183 ].
• The finding of numerous new sequences, the ultra-high throughput of sequences, and the lack of preferences are all advantages of metagenomics [ 184 ].
• Although the huge sequence of data obtained will necessitate the use of sophisticated bioinformatics software for processing, metagenomics does not [ 185 ].
• Tools and procedures are also useful in the study of microbial metabolites and their interactions with plants [ 186 ].
• The use of approaches can be seen in changes in diversity as a result of specific treatments, as well as in management strategies for soil diversity and production [ 187 ].

Disadvantages

• When working with a complex sample, such as soil, tools can cause issues.
• One of the biggest drawbacks of this method is the high cost of the instrument used in microbiome research [ 32 ].
• Until now, important tools that will be employed by the majority of researchers are unknown, implying that a researcher’s lack of skills is a big difficulty in comprehending the plant microbiome [ 188 ].
• For a microbiome researcher, data interpretation, such as metagenomics, is a big difficulty [ 189 ].
• An omics technology like pyrosequencing or Illumina is a lengthy procedure [ 190 ].

Concluding remarks

We provide an overview of almost traditional molecular methods for accessing the soil microbiome in this review paper, including microscopy, nucleic acid extraction and hybridization methods, fingerprinting, PCR, and PCR-based techniques, as well as information on the development of a novel method and its application to environmental samples. Microarrays and metagenomics, for example, are new approaches that aid in the research of microbiomes that are visible and hidden in the world. The information on microbiome communities is derived from omics methods’ data. Furthermore, the roles of microorganisms in a given community may be understood. Metagenomics and metatranscriptomics aid in the in-depth investigation, and this is an interactive method that occurs in this microbial habitat. As a result of the application of molecular tools and methodologies, we have made significant progress in our knowledge of microbial communities in microbiomes. The nature of the sample, the collection, and the habitat are all important aspects of molecular identification. However, given the overall study of these techniques used to study variegated microbial communities of soil and the analytical power suggested by devouring culture microorganisms, it is strongly recommended to use a polyphasic analytical method to analyze soil and other environmental samples in these types of studies. The authors also mentioned the plant microbiome and how many different types of microbiomes exist in nature.

In addition, the positive and negative roles of microorganisms are reviewed in this review work. When studying the microbial ecology of natural habitats, ancient culture-based techniques are overpowering, but they are exceedingly unfair when studying microbial samples. Microbial ecology studies employing culture nondependent molecular procedures have ushered in a new era of microbial variety, thanks to recent advancements in the omics era and sequencing technologies.

Acknowledgments

P.C. thanks to the Department of Environmental Microbiology, BBAU, Lucknow and Enespa thank to Department of Plant Pathology, School for Agriculture, SMPDC, University of Lucknow, Lucknow for the writing of this manuscript.

Funding Statement

There are no funding agency available here.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Chapter 6: Breeding Methods

Asheesh Singh; Arti Singh; and Anthony A. Mahama

The results of breeding and selection may be new varieties or clones that are superior to currently used standard commercially grown genotypes (checks) according to some criterion or criteria, or populations that are superior to previous ones. Several breeding strategies exist and though some methods are generally commonly accepted, different methods are applied in different crops as they are more efficient and effective based on the type of mating of different crops, resources and objectives. In other words different breeding strategies are deployed and used to maximize superiority per unit cost and time. Also depending upon the goals of the breeding program, different strategies may be used simultaneously or at different stages of the program.

• Identify and describe different plant breeding methods relevant to crops grown in Africa
• Mention and describe innovation used to enhance backcross breeding method
• Explain innovation used to enhance recurrent selection method

Methods Used in Self-Pollinated Crops

In self-pollinated crops, the following breeding methods are commonly used to develop pure-line cultivars:

• Bulk method
• Pedigree methods
• Single Seed Descent
• Doubled Haploid

Example of self-pollinated crops in which these methods are used include: common bean, soybean, cowpea, groundnut, rice, wheat, barley, millet, and sorghum.

In specific situations, for example, when a breeding program is converting pure-lines to contain a specific gene or 2-3 genes (of qualitative inheritance), the backcross breeding method is used.

The doubled haploid method is not used in legume crops as these species have so far been recalcitrant to tissue culture and haploid induction and rescue.

Methods Used in Cross-Pollinated Crops

In cross-pollinated crops, the following breeding methods are used to develop cultivars:

• Recurrent selection (for example, maize)
• Development of hybrids: a 2-step process where first inbred lines are developed and assessed for their specific combining ability, followed by crossing of the inbred lines (generally, 2 inbred lines, but can be 3 or 4) to produce hybrid, as for example, maize, rice, sorghum, cotton.

Few self-pollinated species (such as rice, sorghum, and cotton) have some level of outcrossing and expression of heterosis, which is exploited to develop hybrid cultivars.

Recurrent selection methods are used to develop open-pollinated varieties or synthetics.

Methods Used in Clonal Crops

The crop species that can be clonally propagated present unique advantages:

• Heterosis can be fixed in F 1 and in subsequent crop production cycles, and its clones can be propagated to preserve the high yield advantage.
• Farmers can harvest the crop and use the vegetative plant part to grow the next crop. For example, potatoes, sugarcane, cassava.

In breeding clonal cultivars, hybridization is made between two clones and a large F 1 population (remember that parental clones are heterogeneous and heterozygous) is screened as each F 1 is unique and different from other F 1 s. This process is repeated over different crop cycles to identify the superior clone for release as a new cultivar.

Breeding Methods Used in Major Crops

Pedigree method.

The pedigree method of breeding is used in development of both self-pollinated (to develop pure-lines) and cross-pollinated crops (to develop inbreds). It is one of the most commonly used breeding methods. Selection of highly heritable traits is practiced in early generations on individual plants. Yield testing is generally done once homozygous lines are developed (Fig. 1). However, in an early generation testing procedure or a modified pedigree method, yield testing is done in early generations while within-family selection is still ongoing.

Explanation of steps in Fig. 1

• Select in F 2 and later generations.
• Selected F 3 plants (or seed from inflorescence of selected plants) grown in next season (in winter nursery if available).
• Selected F 3 rows (or selected plants within rows) grown as F 4 in rows (or yield plot).
• Selected F 4 plants (or seed from inflorescence of selected plants) grown in next season (in winter nursery if available) as F 5 .
• Repeat this process until selection is effective (remember, additive genetic variance among lines increases but decreases within lines as selfing is used).
• Bulk harvest the last generation when a row is grown (and appears homogenous), F 6 or F n and plant in the next season as a yield plot.
• Grow through successive seasons of yield testing to select the genotypes that are superior to checks.
• Pedigree information is kept to maintain family information, which allows selecting more plants from families that are superior performing or to advance families for yield testing if those families are superior.

Additional notes

• Number of plants/row and population sizes vary between programs and some estimates can be obtained from text books or plant registration documents. These numbers will depend on the objective of the cross, number of crosses made per year, available resources (technical, infrastructure).
• Selection for other specific traits is simultaneously happening (on harvested seed, or specific nurseries).
• Single plants or inflorescence per plant are selected at each generation, but in some visibly inferior rows, breeder may not make any within rows selection (i.e., practice among row selection).
• Selection can be practiced in winter nursery if genetic correlation is high among home location and off-season location (i.e. winter/dry season nursery locations).
• A breeder may combine two or more methods of breeding and these methods will then be called modified pedigree (or modified bulk, or modified single seed descent etc.).

Bulk Method

Bulk method allows natural selection to act and remove undesirable genotypes from the population (i.e., per cross) (Fig. 2). The choice of growing environment will dictate what kinds of traits will be selected for or against, therefore care needs to be exercised to use environments that are suitable for realizing the objectives of the program.

Explanation of steps in Fig. 2

• Generations are advanced to homozygosity through bulks.
• It is a low cost, less technical method of breeding.
• Natural selection is used to remove undesirable plants.
• Artificial selection environment can be used to select for a trait of interest. Bulks can be grown in a disease or another stress nursery to select for that trait. Markers can also be utilized to select for desirable traits to constitute the bulks. These variations will make the scheme as a modified bulk method.
• Early generation testing of bulk may be done for yield testing and to make a decision on retention of populations based on ranking among populations.
• In modified bulk method, single plants or inflorescence per plant are selected at each generation; while in bulk method, plants from the entire population are harvested and seeded (all or sub-sample of seed) in next generation.
• Lighter shade yield plot = grown, tested, not selected; darker shade yield plot = grown, tested, selected and advanced to next generation testing.

Single-Seed Descent Method

Single Seed Descent (SSD) was developed as a breeding method to rapidly advance lines to homozygosity so that selection can be practiced on homozygous lines (Fig. 3). The original intent of this method was to maintain a large population size to mimic the genetic variation in F 2 generation for effective selection. However, this method is now used to reduce the time to develop cultivars. (Sleper and Poehlman, 2006).

Explanation of steps in Fig. 3

• Generations are advanced to homozygosity rapidly. In case of small grain crops (such as wheat, barley, oats), three seasons can be completed in artificial growing conditions (greenhouse etc.), and limited space is needed to keep a population size of 250-300 seed per cross.
• If true single seed descent is practiced (where one seed per plant is grown in successive generations, population size is reduced in each cycle due to losses due to no germination and emergence. As an alternative modified, single seed descent can be used where 2-3 seed per plant are planted in hill plots in each cycle, and 2-3 seed from each hill are collected from an inflorescence.
• SSD plots can be grown in a disease or another stress nursery to select for that trait.
• It is a cheaper, less technical method of breeding. Rapid inbreeding and homozygosity is achieved.
• No need for record keeping of individual plants while advancing through SSD.
• Open circle = single plants (or hills in modified SSD) per population.

Doubled Haploid Method

Doubled haploids (DH) are created by generating haploid plants from microspores (androgenesis) or unfertilized eggs or ovules (gynogenesis). Haploid plants are then subjected to a chemical treatment (with colchicine) to double their chromosome number to produce homozygous diploid plants.

Doubled haploids are generated from heterozygous plants, typically F 1 plants derived from crossing of two pure-lines or inbred lines. DH can also be developed from selected F 2 individuals from a cross. This method is used in development of both self-pollinated (to develop pure-lines) and cross pollinated crops (to develop inbreds). Process is shown in Fig. 4.

• Generations are advanced to homozygosity in single generation. DH genotypes are true homozygous.
• Specialized lab is needed to create doubled haploids. Can be generated through a service provider.
• Population size is an important consideration because only one generation of meiosis occurs (at F 1 ).
• This method is suitable for marker assisted breeding to select for traits that are fixed.
• Can develop cultivars most quickly. If sufficient seed is available, can go to advanced yield trial in season 3.
• It is becoming a preferred method of inbred line development in maize.

Backcross Breeding Method

The backcross breeding method is used if the objective is to introgress a gene into an elite cultivar or breeding line. Examples are disease resistance gene(s) and herbicide tolerance gene(s) (Fig. 5). By crossing to the recurrent (adapted) parent, the newly developed cultivar will contain the majority of the recurrent parent genome and only the gene of interest from the donor parent.

If the gene to transfer is recessive ( rr ), progeny of crossing with RR recurrent parent will segregate as RR and Rr , and therefore progenies are selfed for one generation to determine the Rr type versus RR types ( RR are discarded) before making the next backcross. With the application of molecular markers, this extra step has become redundant and F 1 plants can be grown, DNA extracted from young plant tissue to determine Rr and RR types. RR types can be removed and crosses can be made with Rr types.

For a backcross breeding program, if the gene to be moved comes from an unadapted or related species, the breeder has to be aware of inadvertently bringing in undesirable genes linked to the desired target gene (termed linkage drag). Larger population sizes will need to be grown to identify recombinants. Innovations, e.g. marker assisted backcrossing, marker assisted recurrent selection, and genomic selection, exist that reduce the need for large population sizes.

Innovations in Backcross Breeding

Marker-assisted recurrent selection, reliability for selection.

Steps of Marker-Assisted Recurrent Selection

• One generation of phenotypic selection in the target environment is conducted,
• Markers with significant effects are used to predict the performance of individual plants, and
• Several generations of marker-only selection are performed in a year-round nursery or greenhouse

Comparison between Conventional and Marker-Assisted Backcrossing

Early Generation Testing

Early Generation Testing (EGT) describes the procedure for selecting superior lines or families before they are homozygous. It also refers to a specific use where a genetic worth of a population is determined by analyzing yield data from a segregating (early generation) plot and removing entire populations. EGT is used in self- and cross-pollinated species.

In the pedigree breeding method we looked at individual plant selection for highly heritable traits in early generations. With high heritability, individual plant selection is still effective, for example traits such as plant height, disease resistance, and morphological traits. Several breeding programs, however, follow a modified method (such as modified pedigree method), in which yield testing is started in an early generation (for example, F 3 or F 4 ) to make selections. The early generation lines are grown on yield plots (2 or 4 row plots), therefore, more resources are required to handle EGT. Nonetheless, EGT allows elimination of materials (lines) that are inferior due to use of replication and multi-environment testing. Also, selection for lower heritability can be practiced to discard inferior lines.

Other breeders may choose to perform a yield test on populations derived from early generation bulks to identify superior bulks (inferior bulk populations are removed completely from further generation advancement). Thus, EGT testing in this scenario can be done for one or 2 generations followed by selection of superior plants, and then starting yield testing of these lines.

Cytoplasmic Male Sterility Systems

Plant breeders working with cytoplasmic male sterility (CMS) systems will aim to develop new ‘B-lines’ and ‘R-lines’. In crops where CMS system is used to produce hybrids, different ‘R’ restorer genes are identified and breeders will improve ‘R-lines’ that will be used as males in creation of hybrids. ‘B-lines’ and ‘R-lines’ are developed using the self-pollinated breeding methods we learned about earlier in this module (pedigree, bulk, SSD, DH etc., or a modified method that combines more than one method in the development of breeding line of cultivar).

An outline of a CMS system is shown in Fig. 8. Note that the ‘R’ and ‘r’ genes are in the nucleus and the ‘S’ and ‘F’ genes are in the cytoplasm.

A breeder who develops ‘B-lines’ will use the backcross method to develop ‘A-lines’ using available CM sterility genes. A hybrid cultivar is produced by crossing of ‘A-lines’ with ‘R-lines’. The A/B and R gene pools are considered separate gene pools (reproductive gene pools) similar to heterotic gene pools we learned about in maize systems.

Hybrid Cultivars

In the chapter on Steps in Cultivar Development, we looked at the development of maize hybrids using two-way crosses. Crosses are made within a heterotic group to develop superior inbred lines in the heterotic group. These inbred lines are crossed to testers from other heterotic groups to decide on the best specific combing ability. This process is repeated for all heterotic groups that the breeding institution or company works with internally.

For evaluation, superior inbred lines from dissimilar heterotic groups are crossed to produce hybrids. Several 100 or 1000’s of hybrids are evaluated each year to finally pick the most superior hybrid(s) for commercial release based on their performance and target area of adaptation (maturity, stress, environment etc.).

Hybrid seed is produced by growing inbred female rows (say 6 to 8) from one heterotic group and inbred male rows (1 or 2) from a dissimilar heterotic group interspersed among the sets of female rows, and de-tasseling the female rows (that is, removing male inflorescences from female plant rows) before pollen shed. Manual or mechanical tools are used to de-tassel (prior to pollen being ready or shed to avoid any selfing of plants of the inbred female line). Cobs from female rows are harvested and these constitute the hybrid seed. In some programs, but routinely done in private seed industries, the male rows are usually destroyed when pollination is completed to avoid contamination from cobs from inbred male plants if allowed to grow and produce cobs.

Recurrent Selection

In recurrent breeding and selection, parents of a crop species are crossed to develop populations using various mating designs described in the chapter on “Refresher on Population and Quantitative Genetics.” Based on one or more selection criteria, and using within family and among family selection strategies, individuals are selected and inter-mated to produce the next generation. This procedure of selection can continue for an indefinite amount of time, hence the term “recurrent”. Recurrent selection method is employed in order to achieve the following:

• The goal of recurrent selection is to improve the mean performance of a population of plants and to maintain the genetic variability present in the population.
• The underlying principle of recurrent selection is to increase the frequency of desirable genes that the breeder is attempting to improve.
• Recurrent selection is used to improve populations in cross pollinated species. Open pollinated varieties are one type of cultivar developed using recurrent selection.

Comparison: Mass Selection versus Phenotypic Recurrent Selection

• Mass selection : Female plants are selected after pollination with unselected and selected pollen source.
• Phenotypic recurrent selection : Male and female are both controlled. ONLY selected plants are intercrossed to obtain seed for the next cycle of selection. Expected genetic gain from selection of only the female parent is one-half compared to expected genetic gain when both parents are selected.

Note that the terms mass selection and phenotypic recurrent selection are sometimes used interchangeably and one would have to look at the breeding scheme for details in order to determine which method is being referred to.

Comparison: Genotypic versus Phenotypic Recurrent Selection

The difference between genotypic and phenotypic recurrent selection is that Genotypic Recurrent Selection is selection based on progeny performance (combining ability), while Phenotypic Recurrent Selection is selection based on the phenotype of the individual.

Phenotypic Recurrent Selection Issues

There are several problems with selecting individual plants in the field:

• Micro-environment variability does not permit assessing breeding value.
• Competition effect due to uneven planting.

Solutions to these problems include:

• Gridding designs (selecting plants within a grid)
• Not selecting plants that have missing neighbors

The generalized recurrent selection method consists of the following steps:

• development of a base population (for selection).
• evaluation of individuals from the population
• selection of superior individuals from the population
• intercrossing the selected individuals to form a new population.

Development of Base Population

A base population can be an existing population (for example a maize synthetic) which may not have been previously selected for your trait of interest.

More commonly, a base population will come from outstanding families from a recurrent selection program. It may also be created with elite inbred lines. Smaller number of inbred lines will ensure use of elite material that are similar morphologically, but inbreeding depression will be greater.

Superior inbred lines are identified based on their performance in multi-location tests and superior general combining ability (specific combining ability is not as important in the performance of OPV; it is most important if one is developing a hybrid cultivar).

These superior inbred lines are crossed using an appropriate mating design from among available designs (for example, diallel design).

Evaluation of Individuals

Individuals are evaluated for selection when advancing generations following crossing, and the type of cross made play a key role in the phenotypic and genotypic schemes employed in selecting individuals (Table 1).

Progeny are produced by self-fertilizing the individuals that are evaluated for selection.

Full-sib families are created by crossing the individuals to be evaluated in pairwise combinations. Since in each pairwise cross both parents are common for that family, individuals of that family are full-sibs.

Half-sibs are formed by crossing the individuals to be evaluated to a common parent (which can be a population or an inbred line as a tester. Since all progeny have the tester as a common parent, they are half-sibs.

Population Improvement

As the name implies, the breeding populations creating from crossing parents, need to be improved in performance of the desired traits, in order to continue to make progress in breeding programs. Different methods are used for population improvement, and depending on the breeding program’s specific project goals, can described as intra population or interpopulation improvement.  Table 2 shows some methods that are used.

Recurrent Phenotypic Selection

Steps include:

• Plant a population (space planting individuals to facilitate note taking on individual plants).
• Evaluate for trait of interest and identify the best individuals (higher heritability such as flowering time or morphological traits are suitable for this method).
• Harvest seed of the best individuals and reconstitute seed to form the next cycle of recurrent selection.
• In this example, pollen control can be exerted if the trait can be evaluated prior to flowering. Undesirables can be removed before they contribute pollen to the rest of population; and this ability to control parental pollen helps improve the response to selection.

Recurrent Half-Sib Selection

• An intra-population improvement method: cross the individuals in a population to a common tester (population per se, or inbred tester), evaluate the half-sib progeny of each plant, select the best individuals, and intercross the selected individuals.
• The main step is evaluation of individuals through their half-sib progeny. There are numerous variations within and among crops based on what is used as a tester (population vs inbred), parental control, intercrossing.
• Where possible, it is desirable to control both parents. This can be achieved by evaluating in one season and recombining in another generation (in winter nursery or second season). This necessitates an extra season but genetic gain per year will be higher. While the half-sib are being evaluated, the remnant seed of the individual needs to be kept as reserve so that this seed can be used if the individual is selected based on the half-sib performance to intermate and create material for the next cycle of selection.
• In maize, obtaining selfed and half-sib seed from the same plant can be accomplished by self pollinating the single ear on the individual to be tested and using pollen from that individual to pollinate several individuals of the tester (bulk of population per se, or inbred line). The ears on the tester, bulked together from that individual as pollen source, represent the half-sib family to be tested for that individual.
• Recombining selfed progeny will require three seasons: (1) selfing and crossing to the tester, (2) evaluation, and (3) intercrossing selfed progeny.

Recurrent Half-Sib Examples

Female parent selected; population used as tester..

• Start with a random mating population
• Harvest ears of each plant (say, 200). Grow 200 half-sib progeny plots (with checks) at multiple locations (can be unreplicated or replicated). Traits of interest is yield (for example). At one location, grow in isolation as seed source for the next cycle. At this location, select plants within a half-sib row. At other locations, use for testing.
• At the location with isolation, grow the male rows (bulk seed of all half-sib families) adjacent to female half-sib rows. De-tassel the female rows.
• At the location where grown in isolation harvest ears from each selected plant by hand. Make selections to pick the best half-sib families. These ears will form the next cycle seed.
• Season 2, conduct random mating of selected plants.
• Repeat steps
• One can use an inbred line as tester instead of bulk seed of population used as male.

Female and male parent selected; population used as tester.

Cycle 0 : (intermate population)

• Harvest ears from each plant (selection may be performed)
• Divide the seed of half sib plants into two: part 1 for next season field testing, part 2 for remnant to reconstitute selected half-sibs.

Season 1 : Each half sib (using part 1 seed) is a separate entry in replicated or unreplicated trials with 2 or more locations, with checks.

• Select superior half-sib families based on performance. These selections will be used in crossing.

Season 2 : remnant seed (part 2 of seed bag) of selected individuals is used for intercrossing to form next cycle.

Cycle 1 : Seasons 3 and 4 – repeat as above.

Recurrent Half-Sib (Testcross Progeny)

• Start with an intermated population
• Season 1: plants in an intermated population are selfed and pollen used for selfing and pollinating a tester.
• Season 2: testcross progeny are evaluated in replicated tests. Selections made to identify superior performing progenies.
• Season 3: selfed seed of selected families are used to form the next intermating cycle. Cycle is repeated as above.

Recurrent Full-Sib Process

The main steps are listed below.

End of first year:

• Season 1: Make paired crosses between individuals in the population.
• Season 2: Evaluate the full-sib families in the field and identify the best families.
• Season 3: Recombine (intercross) the best families using remnant seed from the first season.

Start of second year:

• Season 4: Begin the second cycle with paired crosses between individuals in the population.

An advantage is the completion of one cycle per year. A disadvantage is less recombination between cycles of selection.

Recurrent Full-Sib Example

Start with an intermated population. Make selections.

• Season 1: paired crosses are made between pairs of selected plants in a population. Seed is divided into two parts: Part 1 is for field testing, and Part 2 is to reconstitute next cycle.
• Season 2: Part 1 seed used to plant field tests. Evaluate full-sib in field tests (single or multiple locations, unreplicated or replicated, with checks). Select superior families based on performance.
• Season 3: Part 2 seed used to intercross selected families. Intermated seed is used to form the next cycle.
• Cycle 2: Seasons 4, 5, 6.

Recurrent Selection Among Selfed Families

• Season 1: S 0 plants from the population are selfed to produce S 0:1 lines.
• Season 2: Evaluate the selfed progenies in field (for trait of interest).
• Season 3: Use the remnant S 1 seed from season 1 to intercross selected lines.

This completes cycle 1 and S 0 plants are obtained. The cycle is repeated as described above in season 4-6 for cycle 2, and so on.

Variation can include more than one generation of selfing if more seed is required for evaluation.

Reciprocal Recurrent Selection

Reciprocal recurrent selection (RRS), as a breeding method for open-pollinated crops was first proposed by Comstock et al. 1949 to take advantage of both additive and dominance genetic effects. In brief, plants from one population are mated to plants of another population, and selection of individuals for the next cycle of selection is based on the performance of progeny in hybrid combination. For this breeding method, each cycle requires one generation for selection of individuals and a second generation for intermating of selected individuals to produce materials for the next generation. RRS is a procedure to improve both the general and specific combining ability of two populations simultaneously, and steps involved are as below:

• Plants are selected in each of two populations
• Plants of population#1 are selfed and outcrossed as the tester to the selected plants in population#2 to generate test cross progeny.
• Plants of population#2 are selfed and outcrossed as the tester to the selected plants in population#1.
• The resulting test cross progenies are evaluate in each season. Superior plants are identified based on their test cross performance. Selfed seed from these selected plants are used to intercross within each population to generate materials for the next generation.
• Cycle is repeated.

Maize Open Pollinated Varieties (OPV)

Development of a maize opv.

Maize OPV Cultivar Evaluation

OPV Advantages and Disadvantages

Clonal Cultivar Methods

Since each clone breeds true (i.e., no gene segregation because no sexual recombination), breeding programs can evaluate a clone in several different tests simultaneously (field testing, disease nursery etc.). In clonal crop breeding, each cross produces unique and distinct F 1 seed (true seed). True seed plants are transplanted into field testing and selection commences to identify which F 1 of F 1 ’s are suitable for cultivar release. Step-wise reduction process is used to discard undesirable F 1 clones each testing season (remember, clones can be propagated for more extensive testing once smaller number of desirable clones are identified. Shown in Table 4 below is an example of sugarcane cultivar CP 03-1912 developed in Florida.

Approximately 10% culling rate was practiced in each season after 2003. As seasons advance, clones are grown in replicated yield trials at several locations and comparisons with standard checks is made to identify which clones to advance to the next stage of testing.

Synthetic Cultivar

Synthetic cultivars are formed by using clones of inbred lines in pre-determined proportions for released to farmers. Farmers can use a synthetic for several generations (as open-pollinated population) but once inbreeding depression causes yield reduction, farmers need to use seed from the breeding institution or company. Therefore synthetics are reconstituted regularly by the breeder. Maize is an example where synthetics have been developed. In crops with self-incompatibility, synthetics are the preferred types of cultivars as the method exploits heterosis for a few generations.

Clones or inbred lines used in the formation of synthetic are chosen on the basis of their general combining ability. Crossing is made to ensure random pollination allowing gametes of each component (clone of inbred line) to be equally represented.

Canola Council of Canada . 2014. Canola varieties.

The Maize Program. 1999. Development, Maintenance and Seed Multiplication of Open-Pollinated Maize Varieties – 2nd edition. Mexico, D>F.: CIMMYT.

Comstock, R. E, H.E. Robinson, and H.P. Harvey. 1949. A breeding procedure designed to make maximum use of both general and specific combining ability. J. Am. Soc. Agron. 41: 360-367.

Gilbert et al. 2011. Registration of ‘CP 03-1912’ sugarcane. JPR 5(3): 319-324.

International Rice Research Institute. 2006. Molecular breeding, Lesson 1: Marker assisted breeding for rice improvement.

Chapter 6: Breeding Methods Copyright © 2023 by Asheesh Singh; Arti Singh; and Anthony A. Mahama is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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• asexual plant propagation
• plant propagation techniques
• plant tissue culture
• sexual plant propagation
• tissue culture advantages
• tissueculture

Blog > Seven Methods of Plant Propagation

Seven Methods of Plant Propagation

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Each type of plant propagation technique has its own advantages and disadvantages and you choose them based on the plant you are trying to grow and the purpose you want to achieve.

Introduction

We all desire to create a small garden and decorate our home with beautiful and fascinating indoor plants. However, many of us have fail to understand how we can really do this. Especially, growing a specific plant seems the most challenging task to do. Some of us buy plants from cultivators or garden centers or nurseries but fail to maintain them! This costs us a lot of money, time, and effort put into the process. 

You must know that different plants require different approaches to grow and develop in natural environment. For example, some can only be grown using seeds, while the other (such as the rose plant) can be grown faster by using the cutting method of plant propagation.

Each type of plant propagation technique has its own advantages and disadvantages and you choose them based on the plant you are trying to grow and the purpose you want to achieve. 

In this article, we will cover seven methods of plant propagation that you can use to grow your desired plants. It will help you to create your own small kitchen/home garden and save your money. These seven methods include: seed propagation, cutting, layering, division, grafting, budding, and tissue culture technique.

Let’s get started!

What do we mean by Propagation?

Propagation is simply multiplication or production of plants, which you can do by using your own plants! Because of the commercialization of crops, several techniques have been developed to grow plants. All techniques are designed to achieve specific goals, like uniformity in crops, increased productivity, disease-resistant plants, and plants with desired characters.

Mainly these techniques are divided into two categories depending on the means of propagation: Sexual means of propagation and asexual means of propagation. Let’s have a look at each of them in detail.

Sexual Propagation

Sexual propagation of plants involves the union (fertilization) of pollen and egg leading to seed formation. So, it can also be called ‘seed propagation’. It’s an old, easy, simple, and effective technique for ornamentals or flowering plants, vegetables, fruits, and medicinal plants. It allows for genetic diversity in plant species and creates new varieties and cultivars of plants. Also, seeds can be stored for a long period of time.

The disadvantages of this technique are delayed flowering and fruiting, plants that do not produce seeds can not be propagated by this method, identical plants can not be produced, and mass production is harder to achieve.

Considering these disadvantages, asexual propagation methods are being developed and followed by several culturists and hobbyists worldwide.

Asexual Propagation

Asexual propagation of plants can also be called ‘vegetative propagation’ because it involves the use of vegetative parts of plants like leaves, stems, roots, or modified organs. It’s the best method to use to clone your plants, which means to produce plants identical to their parents. It involves methods like cutting, division, layering, grafting, budding, and tissue culture techniques. These techniques are commercially exploited mainly to produce horticulture plants.

The demerits of the asexual means of propagation are: difficulty in producing new varieties, the practice and skillsets required to follow these methods, and plants being more prone to any kind of stresses.

This is cutting the vegetative part of the plant (leaf, stem, and root) and then planting it again to regenerate the whole plant. The three types of cutting are named after the plant part being detached/cut:

• Stem cutting
• Leaf cutting
• Root cutting

The technique is also categorized based on the type of stem the parent plant has, such as herbaceous cuttings (for herbaceous plants), softwood cuttings (for evergreen shrubs and conifers), and hardwood cuttings (for deciduous and evergreen shrubs). 

Source: https://www.majordifferences.com/2013/03/differen...

2. Division

This is a suitable technique for perennials (plants that live for more than two years). It involves dividing the plant by digging and moving it to an already prepared site. This helps the plant to rejuvenate and reduce water and nutrient competition. The technique is commonly used to grow herbaceous perennial plants and sometimes woody shrubs (in this case division should only be performed when the plants are in dormant phase). While dividing the plant part, ensure it doesn't get damaged. 

Dividing perennial plants by using a garden fork.

Source: https://www.gardenersworld.com/plants/five-method...

3. Layering

In this technique, the attached and bent branch of the plant is covered with soil and allowed to root. After the emergence and development of roots that specific part of the plant is cut and allowed to grow as a new plant. This is called ‘layering’.

Different types of layering technique include:

• Simple Layering: A shoot or branch is bent, a part of it is buried in the soil, and the tip of he plant stick out.
• Compound Layering:  Similar to simple layering, but with a longer, flexible branch. Cover parts with soil and leave parts exposed alternately.
• Tip Layering:  For plants with long branches, put the tip of a new branch into the ground to grow.
• Mound Layering:  When the plant is dormant, cut it at ground level and cover it with soil.
• Air Layering:  Here, the new roots grow above the ground. Choose a branch and remove a strip of bark, then cover the area with a special soil and a plastic cover.

A schematic diagram showing a simple layering process.

Source: https://www.groworganic.com/blogs/articles/how-to...

4. Grafting

This involves cutting a twig of one plant and joining it with the stem of another plant in such a manner that they form a unit and function as one plant. It is a bit of a complex process but allows you to bring the desired character to your plant. However, be sure to sterilize your hands and tools to make sure you don’t transfer any infections during the process.

To obtain successful union of attached plants,  ensure the following factors :

• The rootstock and scion chosen for the process are compatible
• Both plants parts are at a suitable physiological stage
• Cambium of both the parts are in contact
• Union point is not dry and there's no infection

A schematic diagram of the grafting technique.

Scion: the upper portion of the graft.

Rootstock: the lower portion which is providing root.

Source: https://www.toppr.com/ask/question/explain-grafti..

In this method, a cut is made in the rootstock and a single bud with little or no wood is inserted into it in such a way that they unite and grow as a new plant.

Source: https://www.toppr.com/ask/question/bud-grafting-i...

6. Tissue Culture

This is the most recent and advanced technique in which plant tissues are grown in media under controlled and sterile conditions/environments. It is extensively used for commercial purposes to produce clones of plants or mass produce plants. It also provides several advantages over all the traditional methods explained above.

Advantages of tissue culture technique:

• It allows for the production of clones or exact copies of the mother plant.
• Plants with desired traits or characters can be grown using this technique.
• It is beneficial in propagating plants without seeds.
• It allows the production of plants in a shorter period of time compared to traditional techniques.
• Plants that are difficult to grow by traditional methods can be grown by this method.
• Disease-free plants can be produced.
• Mass production of plants is possible with this technique.
• Enhance productivity.
• Easy transportation of plants.

Which technique you should choose depends on what type of plant you want to propagate, the purpose of your propagation, and how much time and effort you can put into the process. So, make your choice and get started working on your greeneries!

Want To Be An Expert In Tissue Culture Application? Plant Cell Technology Can Help!

Plant Cell Technology is helping tissue culturists around the world by providing unique and world-class products and services that streamlines their tissue culture workflows. It has MS media , agar, gellan gum, Plant Preservative Mixture (PPM) , culture vessels, Biocoupler (TM) , and masks in its store to facilitate your processes.

And, that’s not it! Plant Cell Technology also offers consultation services to culturists of all sizes that help to get instant solutions to your tissue culture problems.

However, if you're willing to learn directly from an expert, we've got you covered! 

Our comprehensive tissue culture master classes are designed to learn everything about tissue culture, ranging from theoretical concepts to hands on experience on explant and media preparation, multiplication, rooting, and acclimation of plants. Additionally, it also covers advanced tissue culture techniques, such as shoot apical meristem and synthetic seed production to make you the next pro in the industry.

All our masterclasses are taught by tissue culture experts having experience of 10-30 years in the field. They not only help you learn the tissue culture concepts but also share their personal success and failure stories to learn from them and start your tissue culture journey with a bang.

Seems something long awaited? So, why wait? Sigh up for your choice of tissue culture master class TODAY and excel in your tissue culture game!

• https://ncert.nic.in/vocational/pdf/kegr103.pdf
• https://resourcecentral.org/plant-propagation-meth...
• https://en.wikipedia.org/wiki/Plant_propagation
• https://extension.umaine.edu/gardening/manual/prop...
• https://en.wikipedia.org/wiki/Plant_tissue_culture
• https://www.toppr.com/ask/question/explain-graftin...
• https://www.groworganic.com/blogs/articles/how-to-...
• https://www.gardenersworld.com/plants/five-methods...
• https://www.majordifferences.com/2013/03/d

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Microbe Notes

Plant Tissue Culture: Definition, Media, Steps, Types, Uses

Plant tissue culture is the in-vitro aseptic culture of cells, tissues, or whole plants under controlled nutritional and environmental conditions, often to produce clones of plants.

The technique primarily relies on plant cells’ totipotency which is the capacity of a single cell to express the whole genome during cell division. The ability of cells to change their metabolism, growth, and development is just as significant and essential for the regeneration of the entire plant. 

Plant tissue culture technology is being widely used for large-scale plant multiplication. In addition to being used in research, they are now essential for plant propagation, disease eradication, and the generation of secondary metabolites.

Plant Tissue Culture History

Gottlieb Haberlandt, in 1902 tried to cultivate individual palisade cells from leaves in knop’s salt solution supplemented with sucrose. The cells sustained for a month stored starch but ultimately did not divide. Despite his failure, he is considered the father of plant tissue culture since his experiment set the stage for developing tissue culture technology. Similarly, Roger J. Gautheret, a French scientist, had encouraging results with culturing cambial tissues of carrots in 1934.

Table of Contents

Interesting Science Videos

Plant Tissue Culture Conditions

The choice of medium is based on the types of plant species; explants are used for culture for optimal response. All the nutrients required for a plant’s proper growth and development should be present in the plant tissue culture media. Macronutrients, micronutrients, vitamins, other organic ingredients, plant growth regulators, a carbon source, and in the case of a solid medium, a few gelling agents make up the majority of its composition. Similarly, hormone levels and culture variables like temperature, pH, light intensity, and humidity also play an important role in the success of tissue culture.

• The minerals consist of macronutrients such as nitrogen, potassium, phosphorus, calcium, magnesium, and sulfur, and micronutrients such as iron, manganese, zinc, boron, copper, molybdenum, and cobalt. 
• Vitamins are necessary for the healthy growth of plant cultures. The vitamins like thiamine (vitamin B1), pyridoxine (B6), and nicotinic acid (niacin). Other vitamins such as biotin, folic acid, ascorbic acid (vitamin C), and vitamin E (tocopherol) are sometimes added to media formulations. 
• Plants also require an external carbon source; sugar. The most commonly used carbon source is sucrose. Other sources used are glucose, maltose, and sorbitol. 
• The pH of the culture medium remains vital as it influences the uptake of various components of the medium and regulates a wide range of biochemical reactions. Most media are adjusted to a pH of 5.2–5.8. A higher pH may be required for certain cultures.

Plant Tissue Culture Media

• The most popular medium for in vitro vegetative propagation of various plants is Murashige and Skoog medium (MS medium) . For culturing, either a solid or liquid medium can be employed. 
• McCown’s woody plant medium (WPM) has been widely used for tree tissue culture.
• Knudson’s medium is used for orchid tissue culture and fern tissue culture.

Plant Tissue Culture Growth regulators

• Plant growth regulators (PGRs) are crucial for determining the development of plant cells and tissues in a culture medium. 
• The most commonly used plant growth regulators are auxins, cytokinins, and gibberellins.
• The high auxin concentration often favors the development of roots. The most commonly used auxins are IAA (indoleacetic acid), IBA (indolebutyric acid), NAA (naphthaleneacetic acid), and 2,4-D (2,4 dichlorophenoxyacetic acid). 
• Cytokinins promote cell division and shoot growth. The most commonly used cytokinins are BAP (benzylaminopurine), zeatin, Isopentenyl adenine (2-ip), and kinetin. Cytokinins are generally dissolved in dilute HCl or NaOH.
• A clump of undifferentiated cells called a callus develops when auxin and cytokinin levels are balanced.

Plant Tissue Culture Vessels 

• Another critical aspect in plant tissue cultures is the management of the gaseous hormone ethylene. In closed culture vessels used for in vitro plant growth, ethylene builds up and is often detrimental to the cultures. The addition of ethylene biosynthetic inhibitors such as silver nitrate, AVG (aminoethoxyvinylglycine), and silver thiosulphate have been shown to increase the formation of shoots.
• Cultures are grown in walk-in growth rooms or growth chambers. Humidity, light, and temperature must be controlled for the proper growth of cultures. 
• A 16-hour light photoperiod is optimal for tissue cultures, and a temperature of 22 – 25⁰C is used in most laboratories. 
• Cool white fluorescent lamps also supply a light intensity of 25–50 µmol m-2 s-1. 
• Relative humidity of 50–60% is maintained in the growth chambers. Some cultures are also incubated in the dark. 
• Cultures can be cultivated in various containers, including test tubes, flasks, Petri dishes, and bottles.
• The preservation of a sterile environment is necessary for effective tissue culture. The laminar flow hood is used for all tissue culture work. A dust filter and a high-efficiency particulate air (HEPA) filter are used in the laminar flow hood to filter the air. The hood must be kept spotless, which can be accomplished by wiping it with alcohol that contains 70% of the alcohol.
• The surfaces of plant tissues naturally contain a variety of bacteria and fungi. Before tissue culture, it is crucial to thoroughly clean the explant since contaminants can proliferate in the culture media. They also compete with the plant tissue for nutrients, depriving it of those nutrients. Bacteria and fungi can rapidly surpass plant tissues and destroy them. 
• Explants are commonly surface-sterilized using sodium hypochlorite, ethanol, and fungicides when using field-grown tissues. 
• The type of tissue usually decides the time of sterilization. Leaf tissue requires a shorter sterilization time than seeds with a hard seed coat.

Plant Tissue Culture Types

Callus culture .

A callus is an unorganized mass of cells that develops when cells are wounded. When the explant is cultivated on media that promote the development of undifferentiated cells, a callus is formed. The majority of callus cells are formed with the aid of auxins and cytokinins. Using plant growth hormones, callus can multiply continuously or be directed to develop organs or somatic embryos. 

Cell Suspension Culture

Small fragments of loose friable callus can be cultured as cell suspension cultures in a liquid medium. Cell suspensions can be maintained as batch cultures grown in flasks for long periods. A portion of callus tissue can be transferred into a liquid medium, and when subjected to continuous shaking, single-cell cultures and suspension cultures can be cultivated from callus cultures. The growth rate of the suspension-cultured cells is generally higher than that of the solid culture. 

Anther/Microspore Culture

The culture of anthers or isolated microspores to produce haploid plants is known as anther or microspore culture. Embryos can be produced via a callus phase or be a direct recapitulation of the developmental stages characteristic of zygotic embryos. Compared to traditional breeding methods, microspore culture enables the creation of homozygous plants in a very short time. These homozygous plants are useful tools in plant breeding and genetic studies. 

Protoplast Culture

Protoplasts contain all the components of a plant cell except for the cell wall. Protoplasts can be used to create somatic hybrids and regenerate whole plants from a single cell. Cell walls of explant can be removed either mechanically or enzymatically. Protoplasts can be cultured either in liquid or solid medium. Protoplasts embedded in an alginate matrix and then cultured on a solid medium have better success rates of regeneration. Although protoplasts appear to be a very appealing method for regenerating plants and transferring genes, they are extremely delicate.

Embryo Culture

It is a technique in which isolated embryos from immature ovules or seeds are cultured in vitro. For species whose seeds are dormant, resistant, or prematurely sterile, embryo culture has been used as a helpful tool for direct regeneration. In plant breeding programs, embryo culture goes hand in hand with in vitro control of pollination and fertilization to ensure hybrid production. In addition, direct somatic embryos and embryogenic calluses can be produced from immature embryos.

Meristem Culture

Using apical meristem tips, it is possible to produce disease-free plants. This technique can be referred to as meristem culture, meristem tip culture, or shoot tip culture, depending on the actual explant used. Plant apical meristems make good explants for the cultivation of virus-free plants. Hence, this method is usually used to eliminate viruses in many species.

Regeneration Methods of Plants in Culture

It includes two methods:

• Organogenesis

Somatic Embryogenesis

Organogenesis .

In plant tissue culture, it refers to the formation of either shoot or root. The equilibrium of auxin and cytokinin and the tissue’s capacity to react to phytohormones during culture are key factors in in-vitro organogenesis. In-vitro organogenesis can be of two types: 

• Indirect organogenesis
• Direct organogenesis
• Indirect organogenesis involves the formation of organs indirectly via a callus phase. For the production of transgenic plants, induction of plants through a callus phase has been used. Either the callus is transformed, plants are regenerated, or the primary explant is transformed, and the callus is formed, and then shoots are cultivated from the explant. It is more important for transgenic plant production.
• Direct organogenesis involves direct bud or shoots formation from the tissue without a callus stage. Plants are usually propagated by direct organogenesis for improved multiplication rates and production of transgenic plants but mainly for clonal propagation. 

Somatic embryogenesis is a nonsexual developmental process that produces a bipolar embryo with a closed vascular system from the somatic tissues of a plant. It has become one of the most powerful techniques in plant tissue culture for mass clonal propagation. Somatic embryogenesis may occur directly or via a callus phase. For clonal propagation, direct somatic embryogenesis is preferred since there is less chance of somaclonal mutation.

Indirect somatic embryogenesis is usually used in the selection of desired somaclonal variants and for the production of transgenic plants.

Encapsulated somatic embryos are known as synthetic seeds. Synthetic seeds have multiple advantages. They are easy to handle, they can potentially be stored for a long time, and there is potential for scaleup and low cost of production.

Rooting of shoots

The success of acclimatization of a plantlet greatly depends on root system production. Rooting of shoots can be achieved in vitro or ex-vitro.

Ex vitro rooting involves pretreating the shoots with phenols or auxins and then planting them directly in soil under high humidity, which significantly lowers the cost of manufacturing. This technique also allows simultaneous acclimation of the rooted shoots.

In vitro rooting consists of rooting the plants in axenic conditions. Despite the cost factor, in vitro rooting is still a common practice in many plant species.

Several factors are known to affect rooting. The most important factor is the action of endogenous and exogenous auxins. Phenolic compounds are also known to have a stimulatory effect on rooting. Phloroglucinol, a root promoter, is reported effective in root development. Catechol, a strong reducing agent, has been reported to regulate IAA oxidation.

Acclimation / Acclimatization

Once plants are generated by tissue culture, they have to be transferred to the greenhouse or field. This requires that the plants be hardened-off before transfer to the field. To reduce water loss during acclimatization, plants are initially transferred to a greenhouse or growth chamber. The relative humidity outside the vessels is often significantly lower than the humidity inside the vessels. Once the plants are acclimatized under greenhouse conditions, they are ready for transfer to the field.

Advantages of Plant Tissue Culture

• Totipotency, nutrition, metabolism, division, differentiation, and preservation of plant cells.
• Morphogenesis and plant regeneration from individual cells or tissues through organogenesis or somatic embryogenesis.
• Variations were generated through in vitro culture.
• Evolution of haploids through anther and pollen culture, including ovule culture.
• Wide hybridization programs through ovule, ovary, and embryo cultures to overcome both pre-zygotic and post-zygotic sterility mechanisms.
• Micropropagation of plant materials.
• In vitro selection of mutants tolerant to biotic and abiotic stresses.
• In vitro culture and secondary metabolite biosynthesis.
• Plant genetic engineering using DNA transfer and in vitro culture techniques.

Disadvantages of Plant Tissue Culture

• Labor-intensive and expensive process.
• Vulnerable to many environmental factors.
• Cardoza, V. (2008). Tissue Culture: The Manipulation of Plant Development. In N. C. J. Stewart (Ed.), Plant Biotechnology and Genetics (pp. 113–128). John Wiley & Sons, Inc.
• Hussain, A., Qarshi, I. A., Nazir, H., & Ullah, I. (2012). Plant Tissue Culture: Current Status and Opportunities . Intech Open. https://www.intechopen.com/chapters/40180
• ICAR. (2022). Principles of Plant Biotechnology . https://pravara.in/wp-content/themes/twentyseventeen/essentials/pdf/elearn/Principles-of-Plant-Biotechnology.pdf

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Dibyak Kapali

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Article Contents

Introduction, materials and methods, acknowledgments, author contributions, supplementary data, dive curated terms, the m 6 a reader ect8 is an abiotic stress sensor that accelerates mrna decay in arabidopsis.

Zhihe Cai, Qian Tang and Peizhe Song contributed equally.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors ( https://academic.oup.com/plcell/pages/General-Instructions ) is Guifang Jia ( [email protected] ).

Conflict of interest statement. The authors declare no conflict of interests.

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Zhihe Cai, Qian Tang, Peizhe Song, Enlin Tian, Junbo Yang, Guifang Jia, The m 6 A reader ECT8 is an abiotic stress sensor that accelerates mRNA decay in Arabidopsis, The Plant Cell , 2024;, koae149, https://doi.org/10.1093/plcell/koae149

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N 6 -methyladenosine (m 6 A) is the most abundant mRNA modification and plays diverse roles in eukaryotes, including plants. It regulates various processes, including plant growth, development, and responses to external or internal stress responses. However, the mechanisms underlying how m 6 A is related to environmental stresses in both mammals and plants remain elusive. Here, we identified EVOLUTIONARILY CONSERVED C-TERMINAL REGION 8 (ECT8) as an m 6 A reader protein and showed that its m 6 A-binding capability is required for salt stress responses in Arabidopsis ( Arabidopsis thaliana ). ECT8 accelerates the degradation of its target transcripts through direct interaction with the decapping protein DECAPPING 5 within processing bodies. We observed a significant increase in the ECT8 expression level under various environmental stresses. Using salt stress as a representative stressor, we found that the transcript and protein levels of ECT8 rise in response to salt stress. The increased abundance of ECT8 protein results in the enhanced binding capability to m 6 A-modified mRNAs, thereby accelerating their degradation, especially those of negative regulators of salt stress responses. Our results demonstrated that ECT8 acts as an abiotic stress sensor, facilitating mRNA decay, which is vital for maintaining transcriptome homeostasis and enhancing stress tolerance in plants. Our findings not only advance the understanding of epitranscriptomic gene regulation but also offer potential applications for breeding more resilient crops in the face of rapidly changing environmental conditions.

As one of the most essential internal chemical modifications in eukaryotic mRNA, N 6 -methyladenosine (m 6 A) plays important roles in numerous processes, including chromatin maintenance, RNA processing and metabolism, translation efficiency (TE), as well as other biological events ( Jia et al. 2011 ; Zheng et al. 2013 ; Liu et al. 2014 ; Ping et al. 2014 ; Wang et al. 2014 , 2015 ; Xiao et al. 2016 ; Pendleton et al. 2017 ; Xu et al. 2021 , 2022 ). The dynamic regulation of m 6 A modification involves the well-coordinated efforts of methyltransferases and demethylases and m 6 A-binding proteins that participate in entire RNA lifecycle ( Jia et al. 2011 ; Zheng et al. 2013 ; Liu et al. 2014 ; Ping et al. 2014 ; Xiao et al. 2016 ; Pendleton et al. 2017 ; Knuckles et al. 2018 ; Yue et al. 2018 ; van Tran et al. 2019 ; Xu et al. 2021 ). In mammals, m 6 A modifications have been associated with a wide range of biological processes, including embryonic development, stem cell differentiation, and cancer progression ( Zheng et al. 2013 ; Geula et al. 2015 ; Xu et al. 2017 , 2021 ; Yoon et al. 2017 ; Yankova et al. 2021 ). However, the understanding of the precise mechanisms underlying how m 6 A influences transcription processes and subsequent fate determination in plants remains incomplete.

In Arabidopsis ( Arabidopsis thaliana ), disruption of the m 6 A writer core subunit leads to developmental defects and even embryonic lethality ( Zhong et al. 2008 ; Bodi et al. 2012 ; Shen et al. 2016 ; Růžička et al. 2017 ; Sun et al. 2022 ; Wang et al. 2022 ; Zhang et al. 2022a ). Conditional complementation of these mutants has unveiled the regulatory role of m 6 A in various aspects of plant development ( Bodi et al. 2012 ; Wong et al. 2023 ). Two m 6 A demethylases in Arabidopsis, ALKB HOMOLOG 9B (ALKBH9B) and ALKBH10B, have been identified, and they are associated with stress response, floral transition, and viral infection ( Duan et al. 2017 ; Martínez-Pérez et al. 2017 , 2021 ; Tang et al. 2021 , 2022 ). Arabidopsis has 13 YT521-B homology (YTH) domain-containing proteins predicted to function as m 6 A-binding proteins. Among them, EVOLUTIONARILY CONSERVED C-TERMINAL REGION 2, 3, and 4 (ECT2, ECT3, and ECT4, respectively) have been characterized as m 6 A readers, collectively contributing to the regulation of leaf morphogenesis and abscisic acid (ABA) response with genetic redundancy ( Arribas-Hernández et al. 2018 , 2020 ; Scutenaire et al. 2018 ; Wei et al. 2018 ; Song et al. 2023 ). Mechanistic studies have revealed that ECT2, ECT3, and ECT4 form a complex in the cytoplasm and interact with poly(A) binding proteins, POLY(A) BINDING PROTEIN 2 and 4 (PAB2 and PAB4), to enhance the stability of their bound m 6 A-modified mRNAs ( Song et al. 2023 ). The longer isoform of CLEVAGE AND POLYADENYLATION SPECIFICITY FACTOR 30 (CPSF30-L) is another identified m 6 A-binding protein in Arabidopsis, which regulates floral transition, ABA response, and nitrogen signaling ( Hou et al. 2021 ; Song et al. 2021 ). It recognizes m 6 A-modified far upstream elements (FUE) of polyadenylation signal, influencing poly(A) site selection within liquid-like nuclear bodies ( Song et al. 2021 ).

Disrupting m 6 A writers or erasers in Arabidopsis has revealed that m 6 A also plays a role in mRNA degradation ( Luo et al. 2014 ; Anderson et al. 2018 ; Govindan et al. 2022 ). However, whether other m 6 A readers participate in this regulatory process remains to be well-investigated. The research is necessary to elucidate the precise mechanism involving how m 6 A is related to mRNA degradation in plants.

The m 6 A modification is emerging as an epitranscriptomic mark that regulates gene expression, and it exhibits the potential to swiftly adapt to environmental stresses. When mammalian cells are subjected to heat shock or hypoxia, ∼5% to 10% of m 6 A sites in mRNAs undergo dynamic changes ( Liu et al. 2023 ). Similarly, Arabidopsis also displays dynamic changes in m 6 A levels after 6 h treatment with 150 m m NaCl ( Hu et al. 2021 ). Nevertheless, the mechanisms by which m 6 A responds to environmental stresses in both mammals and plants remain elusive. The regulation of m 6 A modification hinges on 3 key categories of proteins: the m 6 A writers, erasers, and readers, and it has been observed that the expression level of m 6 A writer subunits increases after high-concentration NaCl treatment ( Hu et al. 2021 ). Nonetheless, the specific m 6 A reader proteins responsible for facilitating a rapid response to environmental stresses remain a mystery.

Considering that the detailed mechanism of most m 6 A reader proteins remains unclear and it could be an enormous work to investigate them individually, especially because of the potential functional redundancy observed before ( Song et al. 2023 ), we initially focus on ECT8, which exhibits the strongest response to external abiotic stress. It suggests that ECT8 could be a promising point of entry for studying the function of m 6 A modification during rapid signaling transmission in Arabidopsis and it is worthy to explore whether ECT8 has other intriguing regulatory functions.

In this study, we discovered that ECT8, an m 6 A reader protein, serves as a sensor of abiotic stresses in Arabidopsis. ECT8 exhibits a strong binding affinity to m 6 A-modified mRNA through recognition with conserved tryptophan residues, and it primarily localizes within processing bodies (P-bodies) in the cytoplasm, where it exerts its regulatory functions. Combining analysis of formaldehyde crosslinking and immunoprecipitation (FA-CLIP)-sequencing, RNA-seq, and mRNA lifetime sequencing confirmed that ECT8 accelerates the degradation of targeted mRNAs. More specifically, ECT8 directly interacts with the decapping protein DECAPPING 5 (DCP5), contributing to the accelerated degradation of m 6 A-modified mRNA. Under salt stress, the increased abundance of ECT8 enhances its binding capability, thereby amplifying the degradation of ECT8-bound mRNAs. On the other hand, disruption of ECT8 leads to increased expression levels of negative regulators of salt stress, resulting in the elevated sensitivity to salinity. Collectively, our findings demonstrate that ECT8 serves as an abiotic stress sensor by accelerating mRNA decay in Arabidopsis, which is important for transcriptome homeostasis maintenance and stress tolerance. This work provides valuable insights into the molecular mechanisms governing m 6 A modification and its role in plant stress responses, with broad implications for agriculture and environmental adaptation.

ECT8 is an m 6 A-binding protein

To unravel the biological roles of ECT8 in Arabidopsis, we began with an extensive protein sequence alignment of the YTH domain, revealing high sequence similarities between ECT8 and YTH domain-containing family proteins (YTHDF1 to 3) in mammals, as well as a resemblance to the known Arabidopsis m 6 A reader protein, ECT2 ( Supplementary Fig. S1A ) ( Scutenaire et al. 2018 ). Additionally, the sequence similarities between the YTH domain of ECT8 and YTHDF1 to 3 were 69.7%, 70.4%, and 69.7%, respectively, while the similarity to ECT2 was 81.7% ( https://www.bioinformatics.org/ ). This alignment and structural analysis highlighted the importance of 3 conserved tryptophan residues (located at positions 343, 404, and 417 in ECT8) critical for m 6 A recognition within YTH domain, forming a hydrophobic aromatic cage similar to other YTH domain proteins, such as YTHDF2 ( Fig. 1A ).

ECT8 is an m 6 A-binding protein in Arabidopsis. A) Structure of ECT8 simulated by AlphaFold (AF-Q9FPE7-F1) comparing with the published structure of YTH-YTHDF2 in complex with m 6 A (PDB: 4RDN). The ligands for binding m 6 A nucleotide are highlighted. B) EMSA assay showing the binding ability of GST-ECT8 with RNA probe containing m 6 A-modified UGUAA motif but not with unmethylated probe. Each lane was loaded with varying concentrations (shown below the triangle panel) of protein and a consistent amount of RNA oligo with a final concentration of 4 n m . C) EMSA assay showing abolished binding affinity of GST-ECT8m toward both methylated and unmethylated RNA probe with UGUAA motif. Each lane was loaded with varying concentrations (shown below the triangle panel) of protein and a consistent amount of RNA oligo with a final concentration of 4 n m . D) In vitro RIP-UPLC-MS/MS showing that m 6 A is enriched in ECT8-bound mRNA compared with input and the flow-through fractions. Data are presented as means ± Se , n = 3 independent experiments, each with 3 technical replicates. *** P < 0.001 and **** P < 0.0001 by 1-way ANOVA. E) In vivo FA-RIP-UPLC-MS/MS showing that m 6 A is enriched in ECT8-Flag-bound RNA but not in ECT8m-Flag-bound RNA compared with IgG-bound RNA, separately. Data are presented as means ± Se , n = 3 independent experiments, each with 3 technical replicates. ns, not significant and **** P < 0.0001 by 1-way ANOVA.

In order to investigate ECT8’s ability to bind m 6 A modifications, we expressed and purified glutathione- S -transferase (GST)-tagged full-length wild-type (WT) ECT8 protein and a nonbinding variant (GST-ECT8m) with 2 mutations (W404A/W417A) ( Supplementary Fig. S1B ). Electrophoretic mobility shift assays (EMSA) with 5′-FAM-labeled RNA probes, both m 6 A-modified and unmodified, clearly demonstrated ECT8’s m 6 A-binding capability, a feature not observed with ECT8m ( Fig. 1, B and C ; Supplementary Fig. S1C ). For further research, we conducted an in vitro RNA immunoprecipitation (RIP) assay coupled with ultrahigh-performance liquid chromatography and triple–quadrupole tandem MS (UPLC-MS/MS) using GST-ECT8 protein and poly(A) + RNA extracted from Arabidopsis. Our findings showed a significant enrichment of m 6 A-modified poly(A) + RNA within the fraction bound by GST-ECT8, in contrast to the flow-through fraction ( Fig. 1D ). These results confirm ECT8's recognition of m 6 A modifications in vitro and emphasize the critical role of conserved tryptophan residues in forming a hydrophobic aromatic cage that is essential for ECT8’s m 6 A-binding ability.

To further confirm that ECT8 indeed serves as an m 6 A-binding protein in planta, we conducted in vivo RIP assay coupled with UPLC-MS/MS using 2 native genetic complementation lines generated in the T-DNA insertion ect8-1 mutant background: ProECT8:ECT8-FLAG/ect8-1 (termed as ECT8/ect8-1 ) and ProECT8:ECT8m-FLAG/ect8-1 (termed as ECT8m/ect8-1 ) transgenic plants ( Supplementary Fig. S2, A to D ). These lines, respectively, expressed WT ECT8 and the functionally impaired ECT8m (W404A/W417A) ( Supplementary Fig. S2B ). The results indicated that ECT8-FLAG-IP successfully isolated transcripts containing m 6 A modifications when compared with the IgG-IP control. On the other hand, both ECT8m-FLAG-IP and its corresponding IgG-IP control showed no such enrichment ( Fig. 1E ). These collective findings provide robust support for the conclusion that ECT8 functions as an m 6 A reader protein in Arabidopsis.

ECT8 is highly expressed in flowers, roots, and leaves, and ECT8 localizes in the cytoplasm

In our pursuit of uncovering the further molecular functions of ECT8, we explored its expression patterns in Arabidopsis. To achieve this, we generated proECT8:GUS transgenic plants and revealed that ECT8 is prominently expressed in vital anatomical regions, including flowers, roots, and leaves ( Supplementary Fig. S3A ). In roots, ECT8 displays specific expression patterns, with minimal presence in the meristematic zone but high expression levels in the root cap, mature zone, and elongation zone. Across other tissues, ECT8 primarily localizes to vascular tissues, while its expression diminishes notably in actively dividing areas like pollen and seeds, suggesting its role in responding to external stress in relatively mature tissues.

For a more comprehensive analysis of its tissue-specific expression pattern, we analyzed RNA-seq data from Arabidopsis RNA-seq database ( Zhang et al. 2020 ) ( http://ipf.sustech.edu.cn/pub/athrna/ ), which substantiated our findings from GUS staining assays ( Supplementary Fig. S3B ). We further fractionated nuclear and cytoplasmic portions using ECT8/ect8-1 seedlings. Using histone H3 (H3) as a nuclear marker and UDP-glucose pyrophosphorylase (UGPase) as a cytoplasmic marker, we found the evidence that ECT8 is primarily localized in the cytoplasm ( Supplementary Fig. S3C ).

Salt stress induces a significant increase in the transcript and protein abundance of ECT8

Analysis of an Arabidopsis RNA-seq database ( Zhang et al. 2020 ) revealed that the expression level of ECT8 is largely upregulated in response to various external abiotic stresses, including salt, oxidative, and drought stress, when compared with other YTH family proteins ( Supplementary Fig. S4A ). In contrast, ECT8 expression remained relatively stable under conditions such as darkness-induced stress. To delve deeper into our investigation of ECT8's response to external abiotic stress, we subjected WT seedlings to a 150 m m NaCl treatment. The results showed a continuous increase in mRNA and pre-mRNA expression of ECT8 over time during salt stress ( Fig. 2A ).

ECT8 is required for salt stress response in an m 6 A-dependent manner. A) RT-qPCR for the increase expression level of ECT8 mRNA and unspliced immature transcripts (pre-mRNA) during salt stress over time. TUB8 was used as negative control. Data are presented as means ± Se , n = 3 independent experiments, each with 3 technical replicates. *** P < 0.001, **** P < 0.0001 by 2-way ANOVA. B) Nuclear run-on assay indicating that the transcription rate of ECT8 is highly increased after 4 h NaCl treatment. ACTIN2 was used as negative control. Data are presented as means ± Se , n = 3 independent experiments, each with 3 technical replicates. ns, not significant, and * P < 0.05 by 2-way ANOVA. C) Protein immunoblot showing the relative expression level of ECT8 protein under mock and 150 m m salt treatment over time. ACTIN was used for loading control. kDa, kilodalton. D) Phenotypic analysis of salt response among WT, ect8-1 , ECT8/ect8-1 , and ECT8m/ect8-1 plants under mock control and 100 m m NaCl treatments. Representative images showing the morphology of 7-d-old seedlings. WT, wild-type. The scale bar is shown as white lines. E) Statistical analysis of germination rates in WT, ect8-1 , ECT8/ect8-1 , and ECT8m/ect8-1 plants under mock control and 100 m m NaCl treatment. WT, wild-type. Data are presented as means ± Se , n = 4 independent experiments, each with at least 35 seedlings. F) Phenotypic analysis of root length in WT, ect8-1 , ECT8/ect8-1 , and ECT8m/ect8-1 plants under mock control and 100 m m NaCl treatments. The 3-d-old seedlings grown on 1/2 MS plates are transferred to regular 1/2 MS medium and medium supplemented with 100 m m NaCl and cultivated vertically, respectively. Representative images showing the morphology of 7-d-old seedlings. WT, wild-type. The scale bar is shown as white lines. G) Statistical analysis of primary root length in WT, ect8-1 , ECT8/ect8-1 , and ECT8m/ect8-1 plants under mock control and 100 m m NaCl treatment. WT, wild-type. Data are presented as means ± Se , n = 4 independent experiments, each with at least 10 seedlings. ns, not significant, and *** P < 0.001 by 1-way ANOVA.

To further confirm that salt treatment increases ECT8 transcription, we conducted a nuclear run-on assay coupled with RT-qPCR to measure the relative in situ transcription rate of ECT8 in intact nuclei. The nuclear run-on assay was performed by supplying BrUTP to nuclei, and labeled transcripts were enriched by anti-BrdU beads. Indeed, we found the transcription accumulation of ECT8 was significantly increased by four times upon a 4 h salt stress treatment compared with normal conditions ( Fig. 2B ). Subsequently, we also observed an increase in the protein level of ECT8 under salt stress ( Fig. 2C ). These findings show an enhanced transcription rate and higher ECT8 protein abundance under salt stress conditions. As for m 6 A writer subunits and erasers, in contrast to ECT8 , no significant transcriptional changes were observed after the 4 h 150 m m NaCl treatment ( Supplementary Fig. S4, B and C ), suggesting that the m 6 A reader ECT8 is largely the main responder to salt stress instead of m 6 A writers, subunits, or erasers.

ECT8's m 6 A-binding capability is required for salt stress response

Subsequently, we further investigated the role of ECT8 in responding to salt stress. Under normal conditions, both the ect8-1 mutant and WT displayed indistinguishable germination and growth rates ( Fig. 2, D and E ). However, when exposed to varying high concentrations of NaCl (100 and 150 m m ), ect8-1 mutant plants exhibited delayed germination, reduced rates of green cotyledons and survival rate, and shorter primary root length compared with WT ( Fig. 2, D to G; Supplementary Fig. S4, D to F ). The hypersensitivity to salt stress in the ect8-1 mutant can be rescued by expression of WT ECT8, but not by expression of ECT8m ( Fig. 2, D to G ; Supplementary Fig. S4, D to F ). The results suggest that the m 6 A-binding ability of ECT8 regulates the response to salt stress.

ECT8 binds to the 3′ untranslated region of mRNAs with m 6 A modification under both normal and salt stress conditions

Considering that the function of m 6 A reader proteins relies heavily on their specific target genes, we conducted 2 biological replicated strand-specific FA-CLIP and 3 biological replicated strand-specific m 6 A-seq experiments under normal (mock) and 150 m m NaCl (salt) conditions to explore the molecular function of ECT8 ( Supplementary Fig. S5, A and B ). In the overlapping biological replicated sequencing results, we identified 18,111 m 6 A peaks ( Supplementary Data Set 1 ) and 7,065 ECT8-binding sites under mock condition ( Supplementary Data Set 3 ). Over 92% of ECT8’s binding sites overlapped with m 6 A peaks (6,535 ECT8- and m 6 A-binding sites), resulting in 5,479 identified ECT8- and m 6 A-targeted genes ( Fig. 3A ). Similarly, under salt condition, we identified 19,084 m 6 A peaks ( Supplementary Data Set 2 ) and 8,609 ECT8-binding sites ( Supplementary Data Set 4 ), revealing that over 95% of ECT8's binding sites harbor m 6 A peaks (8,240 ECT8- and m 6 A-binding sites) corresponding to 7,270 ECT8- and m 6 A-targeted genes ( Fig. 3B ). Taking the ECT8- and m 6 A-targeted genes into further analysis, we found that ECT8 predominantly interacts with mRNAs, with a concentrated binding distribution within the 3′ untranslated region (UTR) region under both conditions ( Fig. 3C ; Supplementary Fig. S5, C and D ). The known m 6 A motifs found in plants, URUAY (R = A or G, Y = C or U), and RRACH (R = A or G, and H is not G) were also identified in ECT8's binding sites ( Fig. 3D ).

ECT8 binds to mRNA 3′UTR regions under normal and salt stress conditions. A, B) Overlap between FA-CLIP-identified ECT8-binding sites and m 6 A peaks under normal A) and salt stress conditions B) . C) Metagene profile illustrating the region distribution of ECT8- and m 6 A-binding sites across the indicated mRNA segments under normal and salt stress conditions. 5′ UTR, 5′ untranslated region; CDS, coding sequence; 3′ UTR, 3′ untranslated region. D) Motifs identified by HOMER software based on the ECT8- and m 6 A-binding sites under normal and salt stress conditions. E) Distribution of the distance of ECT8- and m 6 A-binding sites under salt stress compared with those under normal condition. F) Venn plot depicting the overlap of ECT8- and m 6 A-targeted genes identified in normal and salt conditions. G) Cumulative plot combined boxplot showing the ECT8's binding ability toward 4,098 common ECT8- and m 6 A-targeted genes under normal and salt conditions. In boxplot, lower and upper hinges represent first and third quartiles, the center line represents the median, and whiskers represent ±1.5× interquartile range. P -values were calculated using Wilcoxon test. H) Boxplot indicating the m 6 A level of 4,098 common ECT8- and m 6 A-targeted genes under both conditions. Results have been calibrated with m 6 A spike-ins to diminish the difference of efficiency during immunoprecipitation in m 6 A-seq. Lower and upper hinges represent first and third quartiles, the center line represents the median, and whiskers represent ±1.5× interquartile range. P -values were calculated using Wilcoxon test. I) GO analysis of 4,098 common ECT8- and m 6 A-targeted genes identified in both normal and salt conditions. P -values were calculated from DAVID website ( https://david.ncifcrf.gov/ ).

Elevated ECT8 protein enhances its binding capacity to m 6 A-modified mRNAs under salt stress

To explore the regulatory function of ECT8 under salt stress, we subsequently investigated whether salt stress would alter ECT8's m 6 A-binding pattern and capability. We found that salt stress treatment did not disturb the m 6 A distribution pattern or ECT8's binding positions ( Fig. 3E ; Supplementary Fig. S5E ). Remarkably, over 60% of ECT8 and m 6 A-binding sites were overlapped between normal and salt conditions, corresponding to 4,098 ECT8- and m 6 A-targeted genes (termed as common ECT8- and m 6 A-targeted genes) identified in both conditions ( Fig. 3F ; Supplementary Fig. S5, F and G ). These indicate that ECT8 maintains a consistent m 6 A-binding pattern across both conditions.

We then used the common ECT8- and m 6 A-targeted genes identified in both normal and salt stress conditions for the evaluation of ECT8's binding capability under normal and salt condition. The results showed that ECT8 exhibits a significantly increased binding ability of common ECT8- and m 6 A-targeted genes under salt stress condition compared with normal condition, although overall m 6 A abundance does not differ between the conditions ( Fig. 3, G and H ; Supplementary Fig. S5H ). It suggests that the elevated ECT8 protein level induced by salt stress enhances its interaction with targeted transcripts.

To gain a deeper understanding of ECT8's role in response to external stress, we conducted gene ontology (GO) pathway analysis using common ECT8- and m 6 A-targeted genes. It revealed a distinct enrichment of pathways related to RNA metabolism, gene expression regulation, and responses to salt and cold stresses ( Fig. 3I ). Additionally, we also performed the GO analysis for the unique ECT8- and m 6 A-targeted genes specific to normal and salt condition ( Fig. 3F ; Supplementary Fig. S5, I and J ). Only the unique ECT8- and m 6 A-targeted genes specific to salt condition were involved in pathways related to osmotic changes and salt stress response. These results underscore the comprehensive role of ECT8 in regulating the expression levels of targeted transcripts under stresses.

ECT8 accelerates the degradation of m 6 A-modified mRNAs

To investigate gene expression regulation mediated by ECT8, we performed strand-specific poly(A) + RNA-seq on 12-d-old WT and ect8-1 seedlings under normal condition, ensuring high replicability between the replicates ( Supplementary Fig. S6A and Data Set 5 ). The identified transcripts were categorized into 3 groups: ECT8-targeted genes, ECT8- and m 6 A-targeted genes, and non-ECT8-targeted genes. Significantly, both ECT8-targeted genes and ECT8-and m 6 A-targeted genes exhibited higher transcript abundance than non-ECT8-targeted genes in ect8-1 compared with WT ( Fig. 4A ). Given that changes in gene expression are predominantly influenced by transcription rates and RNA stability, coupled with ECT8's cytoplasmic localization ( Supplementary Fig. S3C ), it is reasonable to posit that ECT8 plays a pivotal role in promoting the degradation of its target transcripts.

ECT8 facilitates the degradation of m 6 A-modified mRNAs . A) Cumulative distribution and boxplot of relative mRNA expression of 5,659 ECT8-targeted genes, 5,450 ECT8- and m 6 A-targeted genes, and 15,511 non-ECT8-targeted genes in ect8-1 compared with WT under mock condition. In boxplot, lower and upper hinges represent first and third quartiles, the center line represents the median, and whiskers represent ±1.5× interquartile range. WT, wild-type. P -values were calculated using Wilcoxon test. B) Cumulative distribution and boxplot of relative mRNA half-lives of 5,215 ECT8-targeted genes, 5,019 ECT8- and m 6 A-targeted genes, and 13,835 non-ECT8-targeted genes in ect8-1 compared with WT under mock conditions. In boxplot, lower and upper hinges represent first and third quartiles, the center line represents the median, and whiskers represent ±1.5× interquartile range. WT, wild-type. P -values were calculated using Wilcoxon test. C) Cumulative distribution and boxplot of relative mRNA half-lives of ECT8- and m 6 A-targeted genes with 3 or more binding sites (117), 2 binding sites (931), 1 binding site (4,164), and non-ECT8-targeted genes (13,835) in ect8-1 compared with WT under mock conditions. In boxplot, lower and upper hinges represent first and third quartiles, the center line represents the median, and whiskers represent ±1.5× interquartile range. WT, wild-type. P -values were calculated using Wilcoxon test.

Based on our investigation of ECT8, we performed transcriptome-wide mRNA lifetime sequencing on 7-d-old WT and ect8-1 seedlings at different time points with high replicability (0, 15, 30, 60, and 120 min) ( Supplementary Fig. S6B and Data Set 7 ). Our results showed that the transcripts of ECT8-targeted genes, as well as ECT8- and m 6 A-targeted genes, exhibited significantly longer half-lives in ect8-1 when compared with non-ECT8-targeted genes ( Fig. 4B ). Further results revealed that mRNAs with more ECT8-binding sites exhibited notably longer half-lives than those with fewer binding sites in ect8-1 compared with WT ( Fig. 4C ). It reinforces the hypothesis that ECT8 accelerates the degradation of its bound mRNAs, and this degradation process becomes more pronounced as the number of binding sites increases.

Considering the elevated ECT8 level under stress, we wonder whether there could be similar results under salt stress condition. Therefore, we performed strand-specific poly(A) + RNA-seq on 12-d-old WT and ect8-1 seedlings treated with 150 m m NaCl ( Supplementary Fig. S6A and Data Set 6 ). It revealed that disruption of ECT8 significantly increased mRNA abundance of ECT8-targeted genes and ECT8- and m 6 A-targeted genes compared with non-ECT8-targeted genes under salt condition ( Supplementary Fig. S6C ), consistent with the results under normal condition. These results further confirm that ECT8 promotes the degradation of its bound mRNAs under both normal and salt stress conditions.

Due to the cytoplasmic localization of ECT8, we explored its potential role in translation regulation. We first determined that ECT8 is exclusively localized in the nonribosome region ( Supplementary Fig. S7A ). Furthermore, ribo-seq results did not show significant differences in TE for non-ECT8-targeted genes, ECT8-targeted genes, and ECT8- and m 6 A-targeted genes between ect8-1 mutant and WT under normal growth condition ( Supplementary Fig. S7, B and C , and Data Set 8 ). These results confirm that ECT8 does not play a role in the overall translational regulation under normal condition.

ECT8 promotes m 6 A-modified mRNA decay through direct interaction with DCP5 in P-bodies

In human, YTHDF2 localizes in P-bodies and directly interacts with CCR4–NOT Transcription Complex Subunit 1 (CNOT1), a component of the deadenylase complex (CCR4–NOT complex), to promote the deadenylation of m 6 A-containg RNAs ( Du et al. 2016 ). Considering ECT8’s structural and functional similarities to YTHDF2, along with the presence of a disordered Prion-like domain (PrLD) at its N-terminus ( Fig. 1A ; Supplementary Fig. S8A ), it implies that ECT8 could engage in liquid–liquid phase separation (LLPS) to exert its mRNA decay function. Our results showed that ECT8 and P-body component DECAPPING 1 (DCP1) and DCP5 colocalized in liquid puncta ( Motomura et al. 2015 ) ( Fig. 5A ; Supplementary Fig. S8B ), confirming that ECT8 is localized in P-bodies.

ECT8 accelerates the degradation decay of m 6 A-modified mRNA through direct interaction with DCP5 within P-bodies. A) Confocal microscopy showing the colocalization of ECT8-GFP and DCP5-mCherry in P-bodies from protoplast coexpression experiment. Intensity traces (white lines) are analyzed by ImageJ and plotted at the right. Scale bar = 10  μ m. B) Y2H assay showing the physical associations between ECT8 and DCP5 in yeast cells on selective medium without tryptophan, leucine, histidine, and adenine. The full-length CDS of ECT8 and DCP5 were fused with wither the GAL4-AD or BD domain as indicated. AD, the activation domain expressed from pGADT7 ; BD, the binding domain expressed from pGBKT7 . 0-AD, the empty vector of pGADT7 ; 0-BD, the empty vector of pGBKT7.   C) BiFC assay showing the physical associations between ECT8 and DCP5 in N. benthamiana leaf cells. The puncta are highlighted using white arrows. Scale bars = 20  μ m. NYFP, N-terminal domain of YFP expressed from pBI121; CYFP, C-terminal domain of YFP expressed from pBI121; 0-NYFP, the empty vector of pBI121-NYFP ; 0-CYFP, the empty vector of pBI121-CYFP.   D) Cumulative distribution and boxplot of relative expression level of 5,692 ECT8-targeted genes, 5,479 ECT8- and m 6 A-targeted genes, and 16,667 non-ECT8-targeted genes in dcp5-1 compared with WT under mock conditions. In boxplot, lower and upper hinges represent first and third quartiles, the center line represents the median, and whiskers represent ±1.5× interquartile range. WT, wild-type. P -values were calculated using Wilcoxon test. E) Cumulative distribution and boxplot of relative mRNA half-lives of 2,756 DCP5-, ECT8-, and m 6 A-targeted genes, 5,019 ECT8- and m 6 A-targeted genes, and 13,835 non-ECT8-targeted genes in ect8-1 compared with WT under normal condition. In boxplot, lower and upper hinges represent first and third quartiles, the center line represents the median, and whiskers represent ±1.5× interquartile range. WT, wild-type. P -values were calculated using Wilcoxon test. F) Integrative genomics viewer (IGV) showing the m 6 A-seq and FA-CLIP sequencing results on AT5G13570 and AT1G79440 transcripts. FA-CLIP, formaldehyde crosslinking and immunoprecipitation. G) The RNA half-lives of AT5G13570 and AT1G79440 transcripts in 7-d-old WT and ect8-1 seedlings. External spike-ins were used as internal control. WT, wild-type. Data are presented as means ± Se , n = 2 independent experiments, each with 3 technical replicates.

Given that ECT8 facilitates mRNA degradation and localizes in P-bodies, we suspected that proteins interacting with ECT8 might play a role in mRNA degradation. We conducted yeast 2-hybrid (Y2H) to screen several candidates responsible for 5′ cap structure removal or deadenylation. Eventually, DCP5 and VARICOSE (VCS) were identified as potential interacting components in P-bodies of ECT8, but not others such as DCP1, DECAPPING 2 (DCP2), a scaffold protein in the CCR4–NOT complex (NOT1), EXORIBONUCLEASE4 (XRN4), or even ARGONAUTE 1 (AGO1), which is involved in miRNA-mediated posttranscriptional gene silencing ( Fig. 5B ; Supplementary Fig. S8, C and D ) ( Vaucheret et al. 2004 ; Goeres et al. 2007 ; Xu et al. 2007 ; Maldonado-Bonilla 2014 ; Bhat et al. 2020 ; Pereira et al. 2020 ). DCP5, in conjunction with other proteins such as VCS, DCP1, and DCP2, primarily functions in removing the protective 5′ cap structure from mRNA molecules within P-bodies, ultimately leading to mRNA degradation by XRN4 ( Xu and Chua 2009 ; Wawer et al. 2018 ). To validate this finding, we further conducted bimolecular fluorescence complementation (BiFC) by coexpressing ECT8 and DCP5 with split yellow fluorescent protein (YFP) in Nicotiana benthamiana leaves. The results demonstrated that the coexpression of ECT8 and DCP5 resulted in a strong reconstituted YFP signal in the cytoplasm ( Fig. 5C ), confirming the direct protein–protein interaction between ECT8 and DCP5.

Subsequently, we investigated whether DCP5 facilitates the function of ECT8 in mediating the degradation of m 6 A-modified mRNA. To achieve this, we analyzed published poly(A) + RNA sequencing data from the dcp5-1 mutant and its WT counterparts (GSE61812), categorizing the detected transcripts into 3 groups: ECT8-targeted genes, ECT8- and m 6 A-targeted genes, and non-ECT8-targeted genes. The analysis revealed that disruption of DCP5 significantly increases the mRNA abundance of ECT8-targeted genes and ECT8- and m 6 A-targeted genes compared with non-ECT8-targeted genes ( Fig. 5D ). This suggests that DCP5 promotes the degradation of mRNAs bound by ECT8. Furthermore, we identified 10,776 genes that were upregulated in the dcp5-1 mutant, which we refer to as DCP5-regulated genes. More than 60% of ECT8 and m 6 A target genes were overlapped with DCP5-regulated genes, collectively referred to as DCP5-, ECT8-, and m 6 A-targeted genes ( Supplementary Fig. S8E ). Our mRNA lifetime sequencing shows that DCP5-, ECT8-, and m 6 A-targeted genes had significantly prolonged mRNA half-lives in ect8-1 ( Fig. 5E ), indicating a coregulatory function of ECT8 and DCP5 in mRNA degradation.

Moreover, we selected 4 DCP5-targeted genes, AT5G13570, AT1G79440, AT3G45970, and AT1G03440, for further confirmation. Among them, AT5G13570 and AT3G45970 have been validated ( Xu and Chua 2009 ). The results from FA-CLIP and m 6 A-seq showed that the binding sites of ECT8 on transcripts of AT5G13570 and AT1G79440 contain m 6 A modifications, while the other 2 transcripts lack m 6 A modifications ( Fig. 5F ; Supplementary Fig. S8F ). The mRNA lifetime assays revealed that the mRNA half-lives of AT5G13570 and AT1G79440 are longer in ect8-1 than those in WT, but this difference is not observed in AT3G45970 and AT3G45970 ( Fig. 5G ; Supplementary Fig. S8G ). These results collectively demonstrate that ECT8, leveraging its m 6 A-binding capability, promotes RNA decay through its interaction with DCP5 in P-bodies.

ECT8 destabilizes negative regulators of salt stress response for enhancing salt stress tolerance

Building on the function of ECT8 that promotes m 6 A-modified mRNA decay through its interaction with DCP5 in P-bodies, we dedicated efforts into salt stress hypersensitivity that is observed in the ect8-1 mutant ( Fig. 2, D to G ; Supplementary Fig. S4, D to F ). Consistent with our observation that the common ECT8- and m 6 A-targeted genes are significantly enriched in the salt stress pathway ( Fig. 3I ), we also found that ECT8- and m 6 A-targeted genes, specifically those upregulated in ect8-1 from poly(A) + RNA sequencing data, are also enriched in the salt stress response pathway ( Supplementary Fig. S9, A and B ). Therefore, we selected 4 negative regulators of salt stress response: PROTEIN WITH THE RING DOMAIN AND TMEMB_185A 1 ( PPRT1 ), ARABIDOPSIS MULTICOPY SUPPRESSOR OF IRA1 ( MSI1 ), BIN2-LIKE 1 ( BIL1 ), and GTPASE/GTP-BINDING PROTEIN ( ENGD-1 ) ( Alexandre et al. 2009 ; Sui et al. 2019 ; Li et al. 2020 ; Liu et al. 2020 ). Notably, all these transcripts are targeted by ECT8 within their m 6 A regions, as illustrated in FA-CLIP and m 6 A-seq ( Fig. 6A ). Interestingly, we observed that the binding affinity of ECT8 on each transcript was much more enhanced under salt stress condition compared with normal condition, despite no significant change in m 6 A levels between these 2 conditions.

ECT8 amplifies the degradation of the negative salt stress regulators under salt stress condition. A) Integrative genomics viewer showing the sequencing results on PPRT1 , MSI1 , BIL1 , and ENGD-1 transcripts. FA-CLIP, formaldehyde crosslinking and immunoprecipitation. B) m 6 A-IP-qPCR validation of the m 6 A enrichment level in PPRT1 , MSI1 , BIL1 , and ENGD-1 in 12-d-old WT seedlings under mock and salt conditions. IgG-IP was used for negative control, and external m 6 A spike-in was used for calibration. IP, immunoprecipitation. Data are presented as means ± Se , n = 3 independent experiments, each with 3 technical replicates. **** P < 0.0001 by 2-way ANOVA. C) FA-RIP-qPCR validation of the binding affinity of ECT8 toward PPRT1 , MSI1 , BIL1 , and ENGD-1 in 12-d-old ECT8/ect8-1 and ECT8m/ect8-1 seedlings under both mock and salt stress conditions. ECT8m/ect8-1 was used as negative control. AT2G07689 was used as internal control. IP, immunoprecipitation. Data are presented as means ± Se , n = 3 independent experiments, each with 3 technical replicates. ** P < 0.01 and **** P < 0.0001 by 2-way ANOVA. D) Relative mRNA expression levels of PPRT1 , MSI1 , BIL1 , and ENGD-1 in 12-d-old WT, ect8-1 , ECT8/ect8-1 , and ECT8m/ect8-1 seedlings under mock and salt stress. TUB8 was used as the internal control. Data are presented as means ± Se , n = 3 independent experiments, each with 3 technical replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by 2-way ANOVA. E, F) The mRNA half-lives of PPRT1 , MSI1 , BIL1 , and ENGD-1 in 7-d-old WT and ect8-1 seedlings under mock E) and salt stress F) conditions. External spike-ins were used as internal control. Data are presented as means ± Se , n = 2 independent experiments, each with 3 technical replicates.

To confirm the results observed above, we performed m 6 A-IP-qPCR and FA-RIP-qPCR under normal and salt stress conditions. It showed that the transcripts of PPRT1 , MSI1 , BIL1 , and ENGD-1 were indeed modified with m 6 A and directly bound by ECT8 under both conditions ( Fig. 6, B and C ). Moreover, the expression levels of these 4 transcripts were elevated in the ect8-1 mutant compared with WT, with a more pronounced increase observed under salt stress ( Fig. 6D ). These results align with the finding from our sequencing data that the elevated ECT8 protein levels induced by salt stress lead to elevated binding capability. The increased expression levels of PPRT1 , MSI1 , BIL1 , and ENGD-1 in the ect8-1 mutant can be recovered by complementing with ECT8, but not with the m 6 A-binding impaired ECT8m ( Fig. 6D ). This provides evidence that the m 6 A-binding capacity of ECT8 is required for gene regulation and, therefore, the salt stress response.

In addition to our previous findings, we performed RT-qPCR analysis and observed no significant increase in the pre-mRNA levels of these targeted genes ( Supplementary Fig. S10 ). Coupled with our discovery that ECT8 primarily localizes in the cytoplasm ( Supplementary Fig. S3C ), these results collectively suggest that ECT8 participates in the degradation process of these representative transcripts but not in the regulation of transcription. Considering that ECT8 promotes the degradation of m 6 A-modified mRNA, we performed mRNA lifetime assays to measure half-lives of these 4 negative regulators of salt stress response. The results showed that their transcripts underwent slower degradation in ect8-1 compared with WT under both normal and salt stress conditions ( Fig. 6, E and F ).

Collectively, we demonstrated that ECT8 functions as a sensor for responding to abiotic stresses, facilitating the accelerated degradation of its bound m 6 A-modified mRNA through interacting and collaborating with DCP5 in P-bodies ( Fig. 7 ). The abiotic stresses, including salt stress, result in an increase in the transcription and expression levels of ECT8 . Considering that the number of P-bodies increases in Arabidopsis under salt stress condition ( Steffens et al. 2015 ), we hypothesize that the increased abundance of ECT8, induced by salt stress, facilitates the recruitment of more m 6 A-modified mRNAs into P-bodies. As a consequence, this amplifies the degradation of ECT8-bound mRNAs, including negative regulators of salt stress response, ultimately enhancing salt stress tolerance.

A model for ECT8 serves as a salt stress sensor in Arabidopsis. ECT8 functions as an abiotic stress sensor, promoting the degradation of targeted mRNAs in P-bodies by binding to m 6 A and interacting with the decapping protein DCP5. Using salt stress as an example, the transcription and expression levels of ECT8 are significantly increased. We hypothesize that the increased abundance of ECT8 induced by salt stress recruits more m 6 A-modified mRNAs to enter more P-bodies for the degradation of ECT8-bound mRNAs, including negative regulators of salt stress response, ultimately enhancing salt stress tolerance. LLPS, liquid–liquid phase separation; P-bodies, processing bodies.

Rapid climate changes and environmental stressors present great challenges to crop production. Epitranscriptomic modifications hold the promise of responding rapidly to environmental stresses, as evidenced by the alteration of the epitranscriptomic modification m 6 A in mammalian cells and plants under different stimuli ( Zhou et al. 2015 ; Fu and Zhuang 2020 ; Hu et al. 2021 ; Govindan et al. 2022 ; Zhang et al. 2022b ; Ries et al. 2023 ; Wang et al. 2023 ; Song et al. 2024 ). However, it remains unclear how m 6 A responds to these environmental stresses in detail. In this study, we identified ECT8 as an m 6 A reader in Arabidopsis and showed that it accelerates the degradation of m 6 A-modified mRNA by directly interacting with DCP5. Importantly, we discovered that ECT8 serves as a sensor for responding to abiotic stresses and enhances salt stress tolerance. Abiotic stresses lead to increased transcription of ECT8 and result in a higher abundance of ECT8 protein. The increased abundance of ECT8 not only enhances its binding capability to a larger set of m 6 A-modified mRNAs but also amplifies the regulatory functions under abiotic stresses.

The regulation of m 6 A modification involves m 6 A writers, erasers, and reader proteins. While m 6 A modification holds the promise of responding rapidly to environmental stresses, there has been no conclusive evidence or findings indicating which m 6 A regulatory protein facilitates a quick response to these stresses. Theoretically, the m 6 A reader-mediated pathway is the fastest way for m 6 A to respond to environmental stress, as only m 6 A readers sense the stimuli and control RNA fate. In contrast, the m 6 A writer or eraser-meditated pathway involves 3 steps: the m 6 A writer or eraser senses stimuli and adds or removes m 6 A, and then, m 6 A is recognized by m 6 A reader proteins to control RNA fate. It was indeed observed that the expression levels of m 6 A writer subunits increased by about 50% after 6 h of exposure to 150 m m NaCl treatment, while the total m 6 A level in mRNA significantly increased after 12 h of treatment with 150 m m NaCl ( Hu et al. 2021 ). This indicates that m 6 A alteration takes a longer time to respond to salt stress. In contrast, we found that both m 6 A writers and m 6 A modifications remained unchanged at 4 h of exposure to 150 m m NaCl treatment, whereas the expression level of ECT8 increased by 3-fold after 2 h of treatment with 150 m m NaCl ( Fig. 2A ; Supplementary Fig. S4C ). These results support our hypothesis that plants naturally employ the m 6 A reader-mediated pathway as the initial step to quickly respond to environmental stresses.

We have demonstrated that ECT8 rapidly responds to stress and amplifies the degradation of its bound m 6 A-modified mRNAs to regulate stress tolerance. However, the mechanism underlying how abiotic stresses induce increased transcription of ECT8 remains unclear. We suspect that abiotic stresses might manipulate the transcription activity of transcription factors through posttranslation modifications. Further investigation is required to understand the mechanism by which the transcription of ECT8 senses abiotic stresses.

Disrupting m 6 A writers or erasers revealed that m 6 A plays a role in mRNA degradation in plants ( Duan et al. 2017 ; Wang et al. 2022 ). However, the precise mechanism by which m 6 A regulates mRNA degradation remains elusive. We discovered that Arabidopsis ECT8, as an m 6 A reader, accelerates the degradation of its bound m 6 A-modified mRNA through its direct interaction with DCP5 within P-bodies ( Fig. 7 ). DCP5 facilitates the decapping of ECT8-bound m 6 A-modified mRNAs, followed by 5′-to-3′ exoribonucleolytic cleavage ( Xu and Chua 2009 ). We confirmed that disruption of DCP5 inhibits the degradation of ECT8-bound m 6 A-modified mRNA ( Fig. 5, D and E ). Human m 6 A reader protein YTHDF2 promotes mRNA degradation through 3 pathways: (i) YTHDF2 directly interacts with CNOT1, a component of the deadenylase complex (CCR4–NOT complex), for deadenylation ( Zheng et al. 2008 ; Du et al. 2016 ); (ii) YTHDF2 interacts with HRSP12 to recruit endoribonuclease RNase P/MRP complex for internal cleavage ( Park et al. 2019 ); and (iii) YTHDF2 interacts with UPF1 to recruit a decapping-promoting factor PNRC2 for decapping ( Boo et al. 2022 ). In contrast to human YTHDF2, ECT8 does not directly interact with NOT1, the components of the deadenylase complex (CCR4–NOT complex) in Arabidopsis ( Supplementary Fig. S8A ), indicating ECT8 may not follow the deadenylation pathway for mRNA decay ( Pereira et al. 2020 ). Our results also showed that 60% of ECT8- and m 6 A-targeted genes overlap with DCP5-regulated genes ( Supplementary Fig. S8E ), suggesting that other proteins, such as VCS ( Supplementary Fig. S8D ), might interact with ECT8 and facilitate ECT8-mediated m 6 A-modified mRNA decay.

It was previously observed that the number of P-bodies increases in Arabidopsis under salt stress condition ( Steffens et al. 2015 ). We hypothesize that the increased abundance of ECT8 induced by salt stress recruits more m 6 A-modified mRNAs to enter more P-bodies for the degradation of ECT8-bound mRNAs. During the revision process, it was reported that under high-concentration ABA treatment, ECT8 translocates to stress granules (SGs) to suppress protein translation ( Wu et al. 2024 ). Additionally, thus study showed that ECT8 undergoes LLPS under salt stress. As well as the similarity of the formation events of P-bodies and SGs, certain components of P-bodies can dynamically shuttle with SGs under stress conditions ( Kearly et al. 2024 ), indicating the coexistence of P-bodies and SGs under stress, such as salt stress. These findings suggest that ECT8 might have dual functions: promoting mRNA degradation in P-bodies and halting mRNA translation in SGs under stress.

Analysis of the evolutionary tree of the YTH family proteins in plants has revealed that ECT8 shares the same evolutionary branch with ECT6 and ECT7 ( Yin et al. 2021 ), suggesting potential functional redundancy among them. ECT8, along with the characterized m 6 A readers ECT2/3/4, localizes in the cytoplasm. In contrast to ECT8's function, the function of ECT2/3/4 promotes the stabilization of m 6 A-modified mRNA ( Song et al. 2023 ). We postulate that ECT8 and ECT2/3/4 may engage in antagonistic regulatory functions, collectively participating in maintaining transcript homeostasis.

In summary, our study demonstrated that (i) ECT8 functions as an m 6 A reader and accelerates the degradation of its bound m 6 A-modified mRNA through a direct interaction with DCP5, leading to decapping, and (ii) ECT8 serves as a rapid sensor of stress responses and the increased abundance of ECT8 protein amplifies the degradation of m 6 A-modified mRNA under environmental stresses. These findings uncover how plants utilize the m 6 A reader-mediated pathway to swiftly respond to stresses, offering potential opportunities for breeding more resilient crops.

Plant materials and cultivation condition

Arabidopsis ( A. thaliana ) T-DNA insertion mutant ect8-1 (SALK_206710) with Col-0 ecotype background was obtained from the Arabidopsis Biological Resource Center (ABRC). All seeds were sterilized and kept at 4 °C in darkness for 3 d then were grown on half-strength MS (1/2 MS) medium for 12 d before being transferred to greenhouse under same condition (22 °C, 16 h light/8 h dark using cool-white fluorescent tubes) with a light intensity by region from 90 to 120  μ mol m −2  s −1 . N. benthamiana was grown under the same lighting conditions at a temperature of 28 °C for 4 wks. In instances where the plants were subjected to transformation experiments, the apical meristem was pinched to induce the formation of larger leaves.

Plasmid and transgenic plant constructs

To construct proECT8:ECT8-FLAG for transgenic plant transformation, the ECT8 expression cassette, including the 2 kb 5′ upstream sequence and the entire ECT8 open reading frame without the stop codon, was amplified and cloned into the pCAMBIA1305-3   ×   FLAG vector. For proECT8:ECT8m-FLAG for transgenic plant transformation, the mutation was constructed using Mut Express MultiS Fast Mutagenesis kit V2 (C215, Vazyme). In detail, ECT8m was generated by mutagenesis PCR to change the 2 tryptophan (W)-encoding TGG sequences into alanine (A)-encoding GCC sequences. To construct proECT8:GUS , the 2 kb promoter region of ECT8 was cloned into pCAMBIA1305-GUS . All of the Agrobacterium tumefaciens -mediated genetic transformation used the floral dip method. To construct GST-ECT8 and GST-ECT8m for recombinant protein purification, the full-length or mutated coding sequence (CDS) of ECT8 was cloned into pGEX-6P-1 . To construct the plasmids used for Y2H bait expression, all the CDS of ECT8 , NOT1 , DCP1 , DCP2 , DCP5 , VCS , XRN4 , and AGO1 without the start codon were cloned into pGADT7 and pGBKT7 . To construct the 35S:ECT8-GFP , 35S:DCP1-mCherry , and 35S:DCP5-mCherry for PEG-mediated Arabidopsis protoplast transfection, CDS without the stop codon was cloned into pBSK-GFP and pBSK-mCherry , respectively. To construct the 35S:ECT8-NYFP and 35S:DCP5-CYFP for BiFC assay, the entire coding region without the stop codon was cloned into pBI121-NYFP or pBI121-CYFP , respectively. All these coding region were amplified from complementary DNA (cDNA), which was transcribed from WT poly(A) + RNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (RR047A) from TaKaRa. All primers used are listed in Supplementary Data Set 9 .

Protein expression and purification

The plasmids for protein expression were transformed into Escherichia coli ( E. coli ) BL21 Gold (DE3) competent cells. Single colonies from the transformation were picked and cultured in 3 to 5 mL LB liquid medium containing 50  μ g/mL ampicillin at 37 °C and 220 rpm on a shaker for about 12 h. After that, all E. coli culture was then inoculated into 1 L LB liquid medium with 50  μ g/mL ampicillin and incubated at 37 °C and 200 rpm on a shaker. After about 1 h and a half, the optical density at 600 nm (OD 600 ) was monitored until it reached a range of 0.6 to 0.8. The temperature was then reduced to 16 °C, and isopropyl- β - d- thiogalactoside (IPTG) was added to a final concentration of 0.5 m m to induce protein expression overnight. The following day, the E. coli cells were harvested and resuspended in 30 mL lysis buffer (10 m m Tris-HCl, pH 8.0, 500 m m NaCl, 1 m m PMSF, 3 m m DTT, and 5% [ v / v ] glycerol) followed by sonication in an ice bath for 20 to 30 min (20 W power, with 10 s pulses and 10 s pauses). After centrifugation, the supernatant was filtered through a 0.45  μ m membrane to obtain the lysis fraction. This fraction was loaded into a GST affinity column (GE Healthcare) pre-equilibrated. The protein was eluted using elution buffer (10 m m Tris-HCl, pH 8.0, 500 m m NaCl, 10 m m reduced glutathione, and 3 m m DTT), and solution was collected. The protein solution was then concentrated to a volume of 2 to 5 mL and further purified using Superdex 75 exclusion column (GE Healthcare). All the fractions collected were then concentrated to a suitable volume and protein concentration was quantified using the BCA method. Finally, glycerol was added to 25% ( v / v ) of the final volume of the protein solution, thoroughly mixed, aliquoted based on the desired volume, flash-frozen in liquid nitrogen, and stored at −80 °C to maintain protein activity.

The precise concentrations of the recombinant proteins GST-ECT8 and GST-ECT8m (W404A/W417A) were determined using the BCA Protein Quantification Kit (Vazyme). Proteins were diluted into a range of concentrations (25, 12.5, 10, 5, and 2.5  μ m ) using 1× binding buffer (10 m m HEPES, pH 8.0, 50 m m KCl, 1 m m EDTA, 0.05% [ v / v ] Triton X-100, 5% [ v / v ] glycerol, and 10 mg/mL Salmon DNA [ZOMANBIO], 1 m m DTT, 40 U/mL RNase inhibitor [Thermo Fisher Scientific]). The EMSA reaction system was prepared by adding 1  μ L FAM-labeled RNA probe (4 n m as final concentration), 1  μ L recombinant protein for different concentration, 2  μ L 5× binding buffer, and 6  μ L nuclease-free water. After electrophoresis for 60 min at 90 V, samples with RNA loading buffer (250 m m Tris-HCl, pH 8.0, and 40% [ v / v ] glycerol) were loaded to Novex 4% to 20% ( w / v ) TBE gel (Thermo Fisher Scientific) in prechilled 0.5× TBE buffer (5 g/L Tris, 2.75 g/L boric acid, and 50 m m EDTA, pH 8.0) for electrophoresis separation for additional 90 min at 90 V in an ice bath. The results were visualized by ChemiDoc (Bio-Rad). All oligonucleotides used are listed in Supplementary Data Set 6 .

m 6 A quantification analysis by UPLC-MS/MS

One hundred to 200 ng of RNA was digested using 1 U of Nuclease P1 (Wako) in a 17  μ L volume, which included 10 m m NH 4 Ac (pH 5.3), at 42 °C for 4 h. Subsequently, 1 U of shrimp alkaline phosphatase (NEB) and 2  μ L of rCutsmart buffer (NEB) were added for an additional 4 h incubation at 37 °C. The digested nucleotides were then centrifuged at 21,000 × g for 30 min, and supernatant was extracted prior to sample injection. The nucleotides underwent separation through reverse phase ultra-performance liquid chromatography (Shimadzu) using a ZORBAX SB-Aq column (Agilent) and were detected by 5,500 triple–quadrupole mass spectrometer (AB SCIEX) operating in positive electrospray ionization mode with Multiple Reaction Monitoring (MRM) feature. The quantification of nucleosides was carried out by comparing the nucleoside's mass transitions to their respective base ions: m/z 282.0 to 150.1 (m 6 A), m/z 268.0 to 136.0 (A), m/z 245.0 to 113.1 (U), m/z 244.0 to 112.0 (C), and m/z 284.0 to 152.0 (G). Quantification was analyzed by referencing a standard curve created from a series of pure nucleoside standards (Sigma-Aldrich). The ratios of m 6 A to 4 kinds of nucleosides (A, U, C, and G) were then calculated using the fitting curve above.

In vitro RIP-UPLC-MS/MS

Total RNA was extracted from 12-d-old seedlings using TRIzol Reagent (Invitrogen). Subsequently, ∼75  μ g of this total RNA was employed to isolate poly(A) + RNA using oligo(dT) 25 Dynabeads (Thermo Fisher Scientific) following the manufacturer's procedure. The poly(A) + RNA (0.8  μ g, while an additional 0.2  μ g poly(A) + RNA is referred to as the Input fraction) and recombinant proteins with a GST-Tag (final concentration 500 n m ) were incubated at 4 °C in 200  μ L of IPP buffer (150 m m NaCl, 0.1% [ v / v ] NP-40, 10 m m Tris-HCl, pH 7.5, 4 U/μL RNase inhibitor [Thermo Fisher Scientific], and 0.5 m m DTT) for 2 h with gentle rotation. Pre-equilibrated GST-Tag affinity magnetic beads (Pierce) were added, followed by an additional 2 h incubation at 4 °C. The supernatant was collected, and the RNA was extracted through precipitation using ethanol, referred to as the flow-through fraction. Subsequently, the GST-Tag beads were washed 3 times with 200  μ L of IPP buffer, and RNA bound to the beads was extracted using TRIzol Reagent (Invitrogen) and recovered through precipitation using isopropanol, referred to as the protein-bound fraction. The abundance of m 6 A in the input, flow-through, and protein-bound fractions was measured using UPLC-MS/MS. All oligonucleotides used are listed in Supplementary Data Set 6 .

GUS staining assay

The homozygous ProECT8:GUS transgenic plants were planted following previous procedures. During the growth of Arabidopsis, the required tissue samples were collected and subjected to GUS staining (Coolaber, SL7160) for incubation at 37 °C overnight. Samples were washed with 75% ( v / v ) ethanol until the negative control (such as WT) appeared colorless, and observations were made using a stereomicroscope.

Separation of nuclear and cytoplasmic fractions

One gram of 12-d-old Arabidopsis WT seedlings grown on 1/2 MS medium was collected, flash-frozen in liquid nitrogen, and subjected to grinding using a TissueLyser II (Qiagen) at 30 Hz for 1 min and 45 s. The resulting powdered samples were mixed with 10 mL of Honda buffer (comprising 440 m m sucrose, 1.25% [ v / v ] Ficoll, 2.5% [ w / v ] Dextran T40, 20 m m HEPES, pH 7.4, 10 m m MgCl 2 , 0.5% [ v / v ] Triton X-100, 1 m m DTT, and 1× protease inhibitor cocktail [Roche]) and rotated at 4 °C until the samples were completely homogenized (about 20 min). After filtering the samples twice through a single layer of Miracloth, a total of 300  μ L of the sample was retained as the total RNA component, and 50  μ L of the sample was preserved as the total protein component. The remaining sample was centrifuged at 4 °C and 2,000 × g for 5 min, and 400  μ L of the supernatant was collected as the cytoplasmic RNA component, with 50  μ L of the sample preserved as the cytoplasmic protein component. To completely remove interference from nuclear components, 4  μ L of RNase inhibitor (Thermo Fisher Scientific) was added to the 400  μ L cytoplasmic RNA component, followed by centrifugation at 4 °C and 14,000 × g for 15 min. The pellet from the previous centrifugation step was resuspended in 3 to 5 mL of Honda buffer and subjected to centrifugation at 4 °C and 2,000 × g for 5 min, repeated 3 or more times, to completely eliminate any remaining cytoplasmic components. The final pellet was resuspended in 1 mL of Honda buffer and centrifuged at 4 °C and 8,000 × g for additional 1 min. The pellet collected in this step was named as nuclear RNA component as well as a portion should also be reserved for the nuclear protein fraction.

Salt stress phenotypic analysis and high-concentration NaCl treatment

Plants of WT and other different genotypes plants were cultivated under identical conditions as described before, and their seeds were collected at the same time. The mature seeds were carefully dried and stored at room temperature in darkness. The salt stress phenotypic experiments were conducted with a minimum of 3 repetitions, each consisting of 4 biological replicates (with no less than 35 seeds per genotype). As for the detail, using 1/2 MS culture medium containing varying NaCl concentrations (0, 100, 150 m m ), we periodically assessed the germination rate, using the appearance of the radicle as the criterion, and the rate of green cotyledons.

For root length measurements, we used ∼10 seedlings per replicate. This procedure was replicated 3 times with 3-d-old seedlings. Initially, these seedlings were grown on regular 1/2 MS medium. They were then transferred to 1/2 MS medium containing either no NaCl, 100 m m NaCl, or 150 m m NaCl. They were then cultivated vertically for 4 or 5 d, and their root lengths were meticulously recorded from ImageJ software (version 1.54d).

The high-concentration NaCl treatment procedure was as follows: a specific number of 12-d-old seedlings were placed in liquid 1/2 MS culture medium with 150 m m NaCl (salt condition) and in liquid 1/2 MS culture medium without NaCl (mock condition) for all kinds of seedlings needed. After a 4 h treatment, the liquid medium was promptly blotted dry with blotting paper and all the samples were stored by freezing quickly in liquid N 2 then stored at −80 °C.

Total RNA of 12-d-old or 7-d-old seedlings was extracted using TRIzol Reagent (Invitrogen). RNA was subjected to reverse transcription using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (RR047A) from TaKaRa. The procedure involved an initial step of gRNA erasure, followed by reverse transcription. Subsequently, the transcribed cDNAs were appropriately diluted and employed as templates for PCR reactions with Hieff qPCR SYBR Green Master Mix (Low Rox) (Yeasen). These PCR reactions were then analyzed using the ViiATM7 instrument (Applied Biosystems) following the provided instructions. To ensure result accuracy, TUB8 , ACTIN2 , or other appropriate internal controls, such as the external spike-ins ( GLuc and CLuc ) for mRNA stability assay, were utilized to normalize the results, and each independent sample included 3 biological replicates and 3 technical replicates, with the exception of the RNA stability assay, which included 2 biological replicates. All primers used are listed in Supplementary Data Set 6 .

The purified recombinant pGBKT7 or pGADT7 plasmid described before was cotransfected into yeast ( Saccharomyces cerevisiae ) AH109 chemically competent cells (ZOMANBIO, ZC1604) following the manufacturer’s instructions. Transformed cells were cultured on double dropout medium YSD-Leu-Trp, and protein interactions were evaluated on quadruple dropout medium YSD-Leu-Trp-Ade-His under 28 °C for 2 d.

The resulting construct pBI121-DCP5-CYFP and pBI121-ECT8-NYFP was introduced into A. tumefaciens GV3101 (pSoup-p19) chemically competent cell (ZOMANBIO, ZC1410). GV3101 containing plasmids above were coinfiltrated into 4-wk-old N. benthamiana leaves (p19 was employed to suppress transgenic silencing). The infiltrated leaves were initially incubated in darkness at 28 °C for 24 to 48 h. Subsequently, imaging was performed using an LSM700 AxioObserver (Zeiss) confocal laser scanning microscope with a Plan-Apochromat 20×/0.8 objective. Specifically, a 488 nm wavelength laser was utilized to excite the YFP, with the emission signal collected from 500 to 550 nm. All images were collected under a pinhole setting of 1.0 AU with a 2.0% laser intensity and digital gain of 1.0.

Protoplast transient expression

Healthy leaves were selected and cut into fine shreds of ∼1 mm in size. These shreds were transferred into a culture dish containing the 10 mL digestion buffer (12.5 mg/mL Cellulase R10 (Yakult), 3 mg/mL Macerozyme R10 (Yakult), 400 m m mannitol, 20 m m KCl, 20 m m MES, pH 5.7, 10 m m CaCl 2 , 10 mg BSA, and 1× protease inhibitor [Roche]) and vacuum infiltrate in the dark for 15 min. Culture dish with the shredded leaves were put on a horizontal shaker at 22 °C for slow and light-protected enzymatic digestion, lasting ∼4 h. The digested solution was filtered through a Miracloth filter, followed by centrifugation for 3 min at 100  g to collect protoplast cells. The supernatant was removed and 20 mL of W5 solution (154 m m NaCl, 125 m m CaCl 2 , 5 m m KCl, and 2 m m MES, pH 5.7) was added to resuspend the cells and repeat again. After that, 200  μ L of prechilled MMg solution (400 m m mannitol, 4 m m MES, pH 5.7, and 15 m m MgCl 2 ) was added on ice. In particular, 20  μ g plasmids were added into the bottom of the tube and gently mixed. Then, an equal volume of PEG solution (400 mg/mL PEG 4000, 200 m m mannitol, and 40 m m CaCl 2 ) was added and incubated at room temperature for 5 min. The protoplasts were collected and diluted with W5 solution to achieve a final concentration of 5% ( v / v ) serum. Next, the protoplast solution was then transferred into a 6-well plate, kept in the dark, and cultured at 22 °C overnight. Imaging was performed using an LSM700 AxioObserver (Zeiss) confocal laser scanning microscope with a Plan-Apochromat 63×/1.40 oil objective. Specifically, a 488 nm wavelength laser was utilized to excite the GFP, with the emission signal collected from 480 to 530 nm. Meanwhile, a 561 nm wavelength laser was used to excite mCherry, with the emission signal collected from 600 to 650 nm. All images were collected under a pinhole setting of 1.0 AU with a 2.0% laser intensity and digital gain of 1.0.

Poly(A) + RNA-seq

Total RNA was extracted using TRIzol Reagent (Invitrogen). The quality and integrity of the RNA were assessed by determining the RNA integrity number (RIN) through Agilent 2100 system analysis, following the manufacturer’s instructions. For each sample, 5 μg of intact total RNA with External RNA Controls Consortium (ERCC) spike-in was utilized to isolate poly(A) + RNA, employing oligo(dT) 25 Dynabeads (Thermo Fisher Scientific). The library construction was conducted using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB). The sizes of RNA fragments and the generated libraries were measured using Agilent 4150 TapeStation system. The libraries were sequenced on the Illumina NovaSeq 6000 platform with a paired-end model (PE150).

We conducted m 6 A-seq following established protocols. Initially, 4  μ g of poly(A) + RNA with 2  μ L m 6 A/A control RNA (1:1,000) was fragmented into 100 to 150 nt fragments using the magnesium RNA fragmentation module (NEB) and m 6 A-modified RNA was enriched using procedures described above. Libraries were prepared from both the input and RNA enriched with m 6 A (IP group) using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB) After the size selection using DNA clean beads (Vazyme, N411), the libraries were sequenced on the Illumina NovaSeq 6000 platform with a paired-end model (PE150).

Starting with about 3 g of formaldehyde-crosslinked 12-d-old Arabidopsis seedlings ( ECT8/ect8-1 and ECT8m/ect8-1 ), we conducted the grinding using TissueLyser II (Qiagen) at 30 Hz for 1 min and 45 s. Next, 3 mL of lysis solution (containing 150 m m KCl, 50 m m HEPES, pH 7.5, 2 m m EDTA, 0.5% [ v / v ] NP-40, 0.5 m m DTT, 1× cocktail protease inhibitor, and 40 U/mL RNase Inhibitor [Thermo Fisher Scientific]) was added. The mixture was incubated at 4 °C with gentle rotation. Afterward, the solution was centrifuged at 18,000 × g for 15 min at 4 °C, and the supernatant was filtered using a 0.22  µ m membrane filter. A total of 3  μ L of Turbo DNase (Thermo Fisher Scientific) and 3,000 U of RNase T1 (Thermo Fisher Scientific) were added, followed by a 15 min incubation at 22 °C. At the same time, 50  µ L Anti-Flag M2 Magnetic beads (Sigma-Aldrich) per sample were washed 4 times with 600  µ L low-salt wash buffer (300 m m KCl, 50 m m HEPES, pH 7.5, 0.05% [ v / v ] NP-40, 0.5 m m DTT, and 1× protease inhibitor [Roche]). The washed beads and the sample solution were incubated at 4 °C for 3 h. The beads were collected and washed 3 times with low-salt wash buffer (300 m m KCl, 50 m m HEPES, pH 7.5, 0.05% [ v / v ] NP-40, 0.5 m m DTT, and 1× protease inhibitor [Roche]). The beads were resuspended in 396  µ L of wash buffer with 4  µ L of RNase T1 (Thermo Fisher Scientific), followed by a 15 min incubation at 22 °C. After 4 washes with 500  µ L high-salt wash buffer (500 m m KCl, 50 m m HEPES, pH 7.5, 0.05% [ v / v ] NP-40, 0.5 m m DTT, and 1× protease inhibitor [Roche]), the beads were resuspended in 200  µ L 1× T4 PNK buffer (70 m m Tris-HCl, pH 7.6, 10 m m MgCl 2 ) and 10  µ L T4 PNK (NEB) was added followed by a 1 h reaction at 37 °C. Afterwards, 2  µ L of 10 m m ATP (NEB) and additional 3  µ L T4 PNK (NEB) were added followed by a 30 min reaction at 37 °C. A total of 800  µ L 1× T4 PNK buffer was used to remove residual ATPs during washing procedure. Finally, the beads were resuspended in 180  µ L of 1× proteinase K reaction buffer (10 m m Tris-HCl, pH 8.0, 50 m m NaCl, 5 m m EDTA, 0.5% SDS) and 20  µ L of proteinase K (Thermo Fisher Scientific, 10 mg/mL) and incubated at 50 °C for 30 min. After using phenol-chloroform extraction method, 10 ng RNA extracted was used for library preparation using NEBNext Small RNA Library Prep Set for Illumina (NEB) and sequenced on the Illumina NovaSeq 6000 platform with a paired-end model (PE150).

Nuclear run-on assay

The 12-d-old WT and ect8-1 seedlings were ground into a fine powder and then mixed with 5 mL of cold Honda buffer. After filtering the mixture through 2 layers of Miracloth and spinning it at 2,000 × g for 10 min at 4 °C, the nuclei were washed for 2 or 3 times with Honda buffer. Then, all the nuclei were resuspended in 50  µ L storage buffer (50 m m Tris-HCl, pH 7.8, 1 m m DTT, 20% [ v / v ] glycerol, 5 m m MgCl 2 and 0.44  m sucrose). Next, run-on assay was performed in a mixture that contained 10  µ L of a 10× transcription assay buffer, 50  µ L of nuclei in storage buffer, 5  µ L of an NTP mixture (100 m m ATP, 100 m m CTP, 100 m m GTP, and 100 m m BrUTP [Sigma-Aldrich]), and 35  µ L diethyl pyrocarbonate (DEPC)-treated H 2 O. This run-on reaction was carried out at 30 °C for 30 min. To stop the reaction, 900  µ L TRIzol Reagent (Invitrogen) was added and RNA was extracted using the Direct-zol RNA Miniprep Plus kit (ZYMO). The purified RNA was then diluted in 500  µ L incubation buffer (20 m m Tris-HCl, pH 7.5, 4 m m MgCl 2 , 0.2% [ v / v ] NP-40) and mixed with 60  μ L anti-BrdU beads (Santa Cruz) at 4 °C for 2 h. Finally, the precipitated RNA was extracted with TRIzol reagent and used for RT-qPCR analysis.

mRNA lifetime sequencing

RNA stability was determined using cordycepin to inhibit transcription. In brief, we organized this experiment into 5 groups, each representing a different time point. Each group consisted 7-d-old WT and ect8-1 seedlings (10 plants for each genotype). They were transferred to 2 mL incubation buffer (15 m m sucrose, 1 m m KCl, 1 m m PIPES, pH 6.25, and 1 m m sodium citrate). After 15 min of incubation at 80 rpm on a shaker, samples were collected at the zero time point, and the timing was started. Subsequently, the remaining samples were placed in incubation buffer containing 1 m m cordycepin (Macklin) and subjected to 3 vacuum infiltrations (0.6 Mpa, each lasting 1 min, with 1 min intervals in between). Samples were then collected at 15, 30, 60, and 120 min for further RNA extraction and subsequent experiments. Quantitative assays included the addition of the same amount of ERCC spike-in controls in same amount total RNA extracted before, which offered better correction effects for mRNA degradation-induced expression level differences compared with other endogenous genes. For library construction, poly(A) + RNA of these samples was extracted using oligo(dT) 25 Dynabeads (Thermo Fisher Scientific) followed by RNA library construction procedure. All degradation curves were fitted using the equation y = exp(−A × x ), with the expression level at the initial time set as 1 to calculate the relative expression levels and half-lives of each gene at other time points.

One gram of 12-d-old Arabidopsis seedlings was collected and rapidly frozen in liquid N 2 to halt translation. Subsequently, the seedlings were thoroughly ground, and 1 mL of prechilled polysome extraction buffer (200 m m Tris-HCl, pH 8.0, 50 m m KCl, 25 m m MgCl 2 , 2% [ v / v ] polyoxyethylene (10) tridecyl ether, 1% [ w / v ] deoxycholic acid, 2 m m DTT, 100  μ g/mL cycloheximide, and 10 U/mL DNase I [Thermo Fisher Scientific]) was added. The mixture was rotated at 4 °C for 30 min and then centrifuged at 15,000 rpm at 4 °C for 30 min. 200  μ L samples were set aside as the Input fraction, and all remained samples were treated with MNase with 1× MNase reaction buffer for 15 min at 22 °C then quenched by adding 20 U SUPERase-In (Thermo Fisher Scientific). The MNase-digested samples were loaded onto a prechilled 10% to 50% ( w / v ) sucrose gradient buffer (40 m m Tris-HCl, pH 8.4, 20 m m KCl, 10 m m MgCl 2 , and 5  μ g/mL cycloheximide). They were centrifuged at 27,500 rpm, 4 °C for 4 h using a Beckmann SW-40Ti rotor in an ultracentrifuge, and then, the 80S monosome was separated using Bio-Rad EM-1 Econo UV monitor. RNA was extracted using TRIzol Reagent (Invitrogen), and protein was identified using SDS–PAGE. The isolated RNA was separated through a 15% ( w / v ) TBE-urea PAGE (Thermo Fisher Scientific), and the gel slice containing fragments of 21 to 40 nt was excised. The recovered gel was dissolved in gel recovery solution (300 m m NaAc, pH 5.5, 1 m m EDTA, and 0.1 U/mL SUPERase-In [Thermo Fisher Scientific]) and rotated at 65 °C for 10 min. The gel was filtered out using Spin-X columns (Costar), and ethanol was used to precipitate and purify RNA. Subsequently, 3′ dephosphorylation and 5′ phosphorylation were performed before library construction using NEBNext Small RNA Library Prep Set for Illumina (NEB) and sequenced on the Illumina NovaSeq 6000 platform with a paired-end model (PE150).

m 6 A-IP-qPCR

The RNA fragmentation was performed by taking 18  μ L of RNA (including 1  μ L m 6 A/A control RNA, 1:1,000 diluted) and mixing it with 2  μ L of RNA fragmentation reagent. The mixture was then heated at 94 °C for 3 min. Afterward, it was immediately cooled, and 2  μ L of 10× RNA Fragmentation Stop Solution was added for termination. A certain volume was reserved for the input group, and the remaining samples were recovered using Oligo Clean & Concentrator kit (Zymo) following the manufacturer's instructions. A total of 25  μ L of protein G magnetic beads (Invitrogen) were taken and washed with 200  μ L of reaction buffer (150 m m NaCl, 10 m m Tris-HCl, pH 7.5, and 0.1% [ v / v ] NP-40). This mixture was then resuspended in 250  μ L of reaction buffer. Next, 1  μ L of m 6 A antibody (500 ng/μg, SySy) was added to it and allowed to incubate at 4 °C for 30 min. Subsequently, the beads were washed twice with reaction buffer and resuspended in 250  μ L of reaction buffer. An appropriate volume of RNA was added along with 2  μ L of RiboLock RNase inhibitor (Thermo Fisher Scientific). This mixture was then incubated at 4 °C for 1 h. The beads were washed twice with reaction buffer, low-salt buffer (50 m m NaCl, 10 m m Tris-HCl, pH 7.5, and 0.1% [ v / v ] NP-40), and high-salt buffer (500 m m NaCl, 10 m m Tris-HCl, pH 7.5, and 0.1% [ v / v ] NP-40). Finally, RNA was eluted in 50  μ L RLT buffer (Qiagen, #79216) at room temperature for 5 min and purified with Oligo Clean & Concentrator kit (Zymo) following procedure. All primers used are listed in Supplementary Data Set 9 .

In vivo FA-RIP

FA-RIP is based on the FA-CLIP method described previously, with the exclusion of the RNase T1 (Thermo Fisher Scientific) digestion step. In brief, seedlings crosslinked with formaldehyde were ground into a powder and incubated with lysis buffer at 4 °C for 30 min. The supernatant was collected and subjected to incubation with Anti-Flag M2 magnetic beads (Sigma-Aldrich) at 4 °C for 3 h. After washing, proteinase K (Thermo Fisher Scientific) digestion, and ethanol precipitation, the recovered RNA was reverse-transcribed to generate the first-strand cDNA. Relative enrichment levels were determined through RT-qPCR and AT2G07689 was used as negative control. Besides that, these RNAs could also be used for UPLC-MS/MS to detect m 6 A level. All primers used are listed in Supplementary Data Set 9 .

Statistical analysis

One-way or 2-way ANOVA followed by LSD post hoc tests, Wilcoxon rank sum test (Wilcoxon test), and 2-tailed Student’s t test were applied for statistical analysis ( Supplementary Data Set 10 ).

Data analysis for poly(A) + RNA-seq

The library sequencing results were initially processed by cutadapt (v4.4) for adapter trimming and then aligned to the TAIR10 reference genome using hisat2 ( Kim et al. 2015 ) (v2.2.1). PCR duplicates were removed using SAMtools ( Li et al. 2009 ) and Picard. Gene expression quantification was performed using featureCounts ( Liao et al. 2014 ) (v2.0.1) and edgeR ( Chen et al. 2016 ) (v3.36.0) to calculate counts, counts per million (CPM), and identify differential gene expression. The ERCC spike-in was used to normalize the results by using the R package RUVseq ( Risso et al. 2014 ) (v1.28.0). GO enrichment analysis of differentially expressed genes was conducted by gene annotation with the R package org.At.tair.db ( Yu et al. 2012 ) (v3.14.0) and gene set enrichment analysis (GSEA) pathway analysis using the R package clusterProfiler ( Yu et al. 2012 ) (v4.2.2).

Data analysis for m 6 A-seq

The analysis for the input group refers to poly(A) + RNA-seq pipeline. Adapter trimming and size selection were performed using cutadapt (v4.4) to retain fragments with a length of 50 nt or greater. The sequences were then mapped to the reference genome (TAIR10) with hisat2 ( Kim et al. 2015 ) (v2.2.1) using default parameters. PCR duplicates were removed using SAMtools ( Li et al. 2009 ) and Picard. Subsequently, region enrichment analysis was conducted using macs2 callpeak ( Zhang et al. 2008 ) (2.2.7.1) or the R package exomePeak ( Meng et al. 2013 ) (v2.13). Regions showing enrichment with a fold change of 2 or higher and false discovery rates (FDR) < 0.05 were considered for the analysis of strand-specific m 6 A-enriched regions. We calculated the m 6 A ratio of the m 6 A-modified spike-in from the NEB EpiMark N 6 -Methyladenosine Enrichment Kit ( r spike-in ) as follows ( Wei et al. 2022 ): (CPM IP + 0.01)/(CPM Input + 0.01). We defined the m 6 A normalization factor ( nf ) for each sample as the r spike-in divided by the average r spike-in of all WT samples. This factor represents the overall m 6 A level of each sample. Subsequently, we calculated the m 6 A level for every transcript or peak using the formula: (CPM IP + 0.01)/(CPM Input + 0.01) × nf .

Data analysis for FA-CLIP

We initiated the analysis by employing cutadapt (v4.4) for adapter removal and filtering, retaining fragments with a length of 20 nt or longer. Subsequently, we employed hisat2 ( Kim et al. 2015 ) (v2.2.1) to map the sequences to the reference genome (TAIR10) using default parameters. SAMtools ( Li et al. 2009 ) and Picard were used to remove PCR duplicates. For the identification of binding sites, we used macs2 callpeak ( Zhang et al. 2008 ) (2.2.7.1) using the --nomodel option with the criteria of enrichment ≥2 and FDR < 0.05. To evaluate the binding ability of ECT8, we calculated the enrichment fold of ECT8's binding peaks divided by the enrichment fold of ECT8m's binding peaks. Additionally, we applied a criterion based on at least a 1 nt overlap, consistent with both FA-CLIP and m 6 A-seq, to identify ECT8- and m 6 A-targeted genes using IntersectBed ( Quinlan and Hall 2010 ).

Data analysis for Ribo-seq

To commence the analysis, we utilized cutadapt (v4.4) for adapter trimming and filtered the fragments, retaining those with a length of 20 nt or longer. Then, we applied hisat2 ( Kim et al. 2015 ) (v2.2.1) with default parameters to map these sequences to the reference genome (TAIR10). To quantify transcript counts, we used featureCounts ( Liao et al. 2014 ) (v2.0.1), focusing specifically on CDS annotated in Araport11 ( Cheng et al. 2017 ). For calculations of CPM, we employed the R package edgeR ( Chen et al. 2016 ) (v3.36.0). The formula for calculating TE is as follows: TE = CPM Ribo /CPM Input .

Accession numbers

Sequence information of the genes studied in this article can be found in the Arabidopsis TAIR database ( https://www.arabidopsis.org ) under the following accession numbers: ECT1 (AT3G03950), ECT2 (AT3G13460), ECT3 (AT5G61020), ECT4 (AT1G55500), ECT5 (AT3G13060), ECT6 (AT3G17330), ECT7 (AT1G48110), ECT8 (AT1G79270), ECT9 (AT1G27960), ECT10 (AT5G58190), ECT11 (AT1G09810), ECT12 (AT4G11970), CPSF30-L (AT1G30460), MTA (AT4G10760), MTB (AT4G09980), FIP37 (AT3G54170), VIR (AT3G05680), HAKAI (AT5G01160), HIZ1 (AT1G32360), HIZ2 (AT5G53440), ALKBH9B (AT2G17970), ALKBH10B (AT4G02940), DCP1 (AT1G08370), DCP2 (AT5G13570), DCP5 (AT1G26110), NOT1 (AT1G02080), AGO1 (AT1G48410), VCS (AT3G13300), XRN4 (AT1G54490), PPRT1 (AT1G68820), MSI1 (AT5G58230), BIL1 (AT2G30980), and ENGD-1 (AT1G30580). The raw sequencing data of poly(A) + RNA-seq, FA-CLIP, m 6 A-seq, Ribo-seq, and mRNA lifetime sequencing reported in this paper have been deposited in the BIG Data Center ( http://bigd.big.ac.cn ) under the project number PRJCA020765.

We thank S. Tayier and M. Cui for their assistance and valuable suggestions regarding the experimental procedures. We thank Z. Chen and X. Chen for their help on sequencing data analysis. We also thank S. Chen for his expert advice on plant cultivation.

G.J. conceived the project; Z.C., Q.T., and P.S. performed the experiments; Z.C. and J. Y. analyzed the sequencing data; E.T. conducted protein structure modeling; G.J., Z.C, and Q.T. designed the experiments, interpreted the results, and wrote the manuscript.

The following materials are available in the online version of this article.

Supplementary Figure S1 . ECT8 is an m 6 A-binding protein, sharing sequence similarity to other YTH family proteins.

Supplementary Figure S2 . Material characterization for ect8-1 T-DNA mutant and transgenic plants.

Supplementary Figure S3 . The expression pattern and subcellular localization of ECT8.

Supplementary Figure S4 . ECT8 is highly expressed and quickly responds to salt stress in an m 6 A-dependent manner.

Supplementary Figure S5 . ECT8 consistently binds to the 3′-UTR of mRNA under normal and salt stress conditions.

Supplementary Figure S6 . Transcriptomic analysis indicates that ECT8 enhances m 6 A-modified mRNA decay.

Supplementary Figure S7 . ECT8 does not globally influence TE.

Supplementary Figure S8 . ECT8 collaborates with DCP5 and VCS rather than interacting with other P-body components.

Supplementary Figure S9 . GO analysis of upregulated ECT8- and m 6 A-targeted genes in ect8-1 under different conditions.

Supplementary Figure S10 . ECT8 does not influence the transcription of negative regulators of the salt stress response.

Supplementary Data Set 1 . Statistical analysis of m 6 A-seq under mock condition.

Supplementary Data Set 2 . Statistical analysis of m 6 A-seq under salt condition.

Supplementary Data Set 3 . Statistical analysis of ECT8-binding sites under mock condition.

Supplementary Data Set 4 . Statistical analysis of ECT8-binding sites under salt condition.

Supplementary Data Set 5 . Statistical analysis of RNA-seq between WT and ect8-1 under mock condition.

Supplementary Data Set 6 . Statistical analysis of RNA-seq between WT and ect8-1 under salt condition.

Supplementary Data Set 7 . Statistical analysis of RNA lifetime between WT and ect8-1 .

Supplementary Data Set 8 . Statistical analysis of Ribo-seq between WT and ect8-1 .

Supplementary Data Set 9 . List of primers and oligonucleotides used in this study.

Supplementary Data Set 10 . Statistical analysis in this study.

This work was supported by the National Natural Science Foundation of China (nos. 22225704 and 22321005), the National Key R&D Program of China (2023ZD04073), and the Beijing Natural Science Foundation (Z200010).

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

PAB4 Gramene: AT2G23350

PAB4 Araport: AT2G23350

SDS CHEBI: CHEBI:8984

AT1G79440 Gramene: AT1G79440

AT1G79440 Araport: AT1G79440

AT2G07689 Gramene: AT2G07689

AT2G07689 Araport: AT2G07689

HEPES CHEBI: CHEBI:46756

PAB2 Gramene: At4G34110

PAB2 Araport: At4G34110

ABA CHEBI: CHEBI:2365

GSEA Gramene: Gene set enrichment analysis

GSEA Araport: Gene set enrichment analysis

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• Published: 19 June 2024

A new biostimulant derived from soybean by-products enhances plant tolerance to abiotic stress triggered by ozone

• Angel Orts   ORCID: orcid.org/0000-0003-2343-914X 1 ,
• Salvadora Navarro-Torre   ORCID: orcid.org/0000-0002-1037-2057 2 ,
• Sandra Macías-Benítez   ORCID: orcid.org/0000-0002-2738-9656 1 ,
• José M. Orts   ORCID: orcid.org/0000-0003-0186-2107 1 ,
• Emilia Naranjo   ORCID: orcid.org/0000-0002-1525-5714 1 ,
• Angélica Castaño   ORCID: orcid.org/0000-0002-4853-8980 1 &
• Juan Parrado   ORCID: orcid.org/0000-0002-1462-408X 1  

BMC Plant Biology volume  24 , Article number:  580 ( 2024 ) Cite this article

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Tropospheric ozone is an air pollutant that causes negative effects on vegetation, leading to significant losses in crop productivity. It is generated by chemical reactions in the presence of sunlight between primary pollutants resulting from human activity, such as nitrogen oxides and volatile organic compounds. Due to the constantly increasing emission of ozone precursors, together with the influence of a warming climate on ozone levels, crop losses may be aggravated in the future. Therefore, the search for solutions to mitigate these losses becomes a priority.

Ozone-induced abiotic stress is mainly due to reactive oxygen species generated by the spontaneous decomposition of ozone once it reaches the apoplast. In this regard, compounds with antioxidant activity offer a viable option to alleviate ozone-induced damage. Using enzymatic technology, we have developed a process that enables the production of an extract with biostimulant properties from okara, an industrial soybean byproduct. The biostimulant, named as OEE (Okara Enzymatic Extract), is water-soluble and is enriched in bioactive compounds present in okara, such as isoflavones. Additionally, it contains a significant fraction of protein hydrolysates contributing to its functional effect.

Given its antioxidant capacity, we aimed to investigate whether OEE could alleviate ozone-induced damage in plants. For that, pepper plants ( Capsicum annuum ) exposed to ozone were treated with a foliar application of OEE.

OEE mitigated ozone-induced damage, as evidenced by the net photosynthetic rate, electron transport rate, effective quantum yield of PSII, and delayed fluorescence. This protection was confirmed by the level of expression of genes associated with photosystem II. The beneficial effect was primarily due to its antioxidant activity, as evidenced by the lipid peroxidation rate measured through malondialdehyde content. Additionally, OEE triggered a mild oxidative response, indicated by increased activities of antioxidant enzymes in leaves (catalase, superoxide dismutase, and guaiacol peroxidase) and the oxidative stress index, providing further protection against ozone-induced stress.

Conclusions

The present results support that OEE protects plants from ozone exposure. Taking into consideration that the promotion of plant resistance against abiotic damage is an important goal of biostimulants, we assume that its use as a new biostimulant could be considered.

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Introduction

Tropospheric ozone (O 3 ) is a major air pollutant that induces abiotic stress in plants causing negative effects on growth and crop productivity [ 1 , 2 , 3 ]. Differently from other air pollutants, tropospheric ozone is not emitted directly but is generated from primary pollutants resulting from human activity, such as methane, carbon monoxide, nitrogen oxides and volatile organic compounds, through chemical reactions in the presence of sunlight [ 4 ]. As a result, the tropospheric ozone levels are constantly increasing due to increased human activity. Despite the authorities' interest in controlling pollutant emissions, global warming is likely to contribute to increasing concentrations of tropospheric ozone over time, leading to a greater loss of agricultural productivity [ 5 , 6 , 7 ].

The damage induced by ozone in living organisms is due to its high oxidizing power. The adverse impacts of O 3 on plants encompass a reduction in photosynthesis, increased water loss, and the development of chlorotic and necrotic spots on leaves [ 2 , 8 ]. Ozone-induced damage primarily arises from the generation of reactive oxygen species (ROS) through the spontaneous decomposition of O 3 upon entering the apoplastic space or through direct interactions with various cellular components. This process triggers oxidative damage to biomolecules, potentially affecting crucial cellular functions [ 9 ].

The loss in crop productivity and its impact on global food supply [ 3 , 10 , 11 , 12 ] have heightened the interest in finding strategic solutions to minimize this loss in areas with elevated concentrations of tropospheric O 3 . While one possible solution is the use of genetically modified organisms, social rejection of their use drives the search for alternative solutions [ 13 ]. An alternative approach involves the application of exogenous compounds that protect plants against O 3 .

Compounds of various natures, including antioxidants, herbicides, pesticides, plant growth regulators or mechanical barriers have been used to protect plants from ozone damage, either alone or in combination. However, despite achieving a certain degree of protection in some cases, the side effects render their use in the field unacceptable [ 14 ]. To date, the antioxidant EDU (ethylene diurea– (N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N0 phenylurea), has been considered the most effective antiozonant. However, its primary mechanism of action remains unclear, and the use of EDU is advised primarily as a research tool for evaluating the phytotoxic effects of O 3 on crops but it has not yet been implemented at the commercial level [ 14 ].

Therefore, it is essential to seek new alternatives that allow overcoming these inconveniences. In this context, bio-stimulants derived from plant extracts emerge as a good option since they do not generate environmental toxicity. According to the European Biostimulant Industry Council (EBIC), biostimulants are defined as "substances and/or microorganisms that, when applied to plants or the rhizosphere, stimulate natural processes, enhancing nutrient absorption, nutritional efficiency, abiotic stress tolerance, and crop quality.

Through the use of enzymatic technology, our group has already developed a rice bran extract with bio-stimulant properties that protected pepper plants against acute exposure to O 3 [ 15 ]. Thus, we have extended this technology to other agricultural by-products such as the soy pulp, also called okara.

Okara is a byproduct generated during the production of soy milk and tofu. Despite having a rich composition of nutrients, such as proteins of high-quality, fiber, fats, and carbohydrates, as well as containing bioactive compounds like isoflavones, two inconveniences contribute to its underutilization. First, the susceptibility to rapid deterioration due to high moisture content, and second its low water solubility [ 16 , 17 ]. Through the application of enzymatic technology, our team has developed a process that facilitates the creation of a stable, water-soluble enzymatic extract from okara, OEE. This extract retains most of the isoflavones from the original okara while being rich in bioactive peptides, all of which contribute to its high antioxidant potency [ 18 ].

The elevated level of bioactive compounds present in OEE and its potent antioxidant activity has prompted us to explore its potential as plant biostimulant. Similar to our previous work [ 15 ], we opted for pepper plants ( Capsicum annuum ) for this research due to the global significance of the pepper crop. Capsicum pepper is among the most widely cultivated vegetable crops internationally, experiencing a substantial increase in total production in recent years, reaching around 40 million tons in 2020 [ 19 ]. These plants are predominantly grown in subtropical regions globally, with major producers situated in developing countries like China, India, or Mexico, where O 3 concentrations are anticipated to rise more significantly than in other nations. Due to these factors, capsicum peppers serve as a compelling subject for studying the adverse effects of O 3 .

To evaluate OEE biostimulant capacity, pepper plants exposed to O 3 were treated with a foliar spray of an aqueous solution of OEE. The protective effect was analyzed through the assessment of biochemical parameters such as antioxidant enzymes in leaves (catalase, superoxide dismutase, and guaiacol peroxidase) or lipid peroxidation rate (MDA) as well as key plants physiological parameters such as net photosynthetic rate (A N ), electron transport rate (ETR), effective quantum yield of PSII (PhiPSII), and delayed fluorescence (DF). Additionally, genes related to photosystem II was also evaluated.

Material and methods

Okara extract preparation.

Okara, obtained from Soria Natural S.L. (Garray, Soria, Spain), was prepared by washing soybeans, soaking them in cold water (33.3% w/v) for 30 h, and then heating them at 95 °C for 5 min to deactivate trypsin and lipoxygenase inhibitors. The soaked soybeans were ground with hot water in a 1:1 (w/v) ratio to produce soy milk through pressure and filtration, leaving behind soy pulp known as okara.

The enzymatic hydrolysis of okara was carried out using a liquid enzyme serine-endoprotease subtilisin (EC 3.4.21.62) from enterprise Biocon (Spain). The preparation of okara was a 10% concentration in water (dry w/v), and protein hydrolysis was conducted at pH 10.0 using the pH–stat method. The process included sequential incubation with subtilisin (0.3% v/v) for 2 h at 55 °C without shaking. After centrifugation for 40 min at 4 °C and 10,000 g, the soluble phase (OEE) was heat-dried and analyzed, while the sediment was weighed and discarded.

The nutritional composition of hydrolyzed okara was characterized for macro- and micronutrients as described in previous work by our group [ 18 ].

The protein content of the soluble portion of okara was analyzed by size-exclusion chromatography on an ÄKTA-purifier FPLC system (GE Healthcare), filtration chromatography and a Superdex Peptide 10/300GL column. This column has an exclusion range of 700 to 10,000 Da, which separates free peptides and amino acids. After centrifugation, the supernatant underwent filtration through a 0.2 μm filter and was then loaded into a 0.1 mL loop connected to an ÄKTA-purifier system. The column was equilibrated and eluted with 0.25 M Tris–HCl buffer (pH 7.0) using isocratic mode at a flow rate of 0.5 mL/min. Protein and peptide detection occurred at 280 and 215 nm, respectively, using a GE Healthcare UV900 module coupled to the column elution.

Plant Treatment

The selected plants and the treatment applied were carried out according to previous work by the group [ 15 ] . Capsicum annum L. var. grossum (pepper) plants were raised from seeds in plastic pots containing an organic commercial substrate (Gramoflor GmbH und Co. KG.) and Osmocote® (NPK 15: 9: 12), and grown inside the University of Seville Glasshouse General Services on a phytoclimatic chamber, Fitoclima 18,000 PHL (Aralab-Spain), with a controlled temperature of 18 − 22 °C, 50% relative humidity, adequate irrigation with tap water and a photoperiod of 16 h light/8 h darkness, being the maximum photosynthetic photon flux density level incident on leaves of 1200 μmol m −2  s −1 .

After eight days of transplantation, 20 pepper plants were selected and divided in 4 groups (5 plants for group): control plants (Ct), control plants under O 3 exposition (Ct + O 3 ), plants treated with OEE (OEE) and plants treated with OEE under O 3 exposition (OEE + O 3 ). Following protocol previously describe by us [ 15 ], to evaluate the protection capacity of the treatment with OEE, plants were foliar sprayed a total of 4 times at five-day intervals, with an aqueous solution of OEE at 0.1% (groups OEE and OEE + O 3 ) or distilled water (groups Ct and Ct + O 3 ). After 5 days of the last spray treatment, Ct + O 3 and OEE + O 3 plants were transferred to another phytoclimatic chamber with an ozone generator (ZONOSISTEM GM 5000 O 3 Generator) attached and exposed to 3 consecutive fumigations with 100 ppb of O 3 for 6 h (from 10:00 am to 4:00 pm). After ozone fumigation all the test plants were sprayed again with the corresponding solution (OEE 0.1% or distilled water).

Finally, 24 h after the last exposure to ozone, foliar samples were taken from each plant and the analyses described below were carried out.

Plants status after the ozone exposition

Analyses of photosynthetic parameters.

Twenty-four hours after the last ozone treatment, net photosynthetic rate (A N ), electron transport rate (ETR) and effective quantum yield of PSII (PhiPS2) were determined in plants using an IRGA (LI-6400XT, LI-COR Inc., Nev., EEUU) with a light chamber for the leaf (Li-6400-02B, Li-Cor Inc.) according to Macias-Benitez et al. [ 15 ]. Briefly, measurements ( n  = 20) were performed between 10 a.m. and 2 p.m. hours under a photosynthetic photon flux density of 1500 \upmu mol.m −2 . s −1 , a deficit of vapor pressure of 2–3 kPa, a temperature around 25ºC, and a CO 2 concentration environment of 400 \upmu mol.mol −1 air. Each measurement was recorded after the stabilization of the exchange of gases was equilibrated (120 s).

Delayed fluorescence determination

Delayed fluorescence (DF) was recorded at the end of the experiment in a random leaf from each plant. For that, the collected leaves were analyzed using a NightShade LB 985 (Berthold Technologies, Germany) equipped with a deeply cooled CCD camera [ 20 ]. The recorded data were converted to counts per second (cps) and normalized to the leaf area.

Sample Collection

The sample collection was carried out following the protocol provided by the company Corning.

Extraction, purification of Samples and library Preparation

The extraction and purification of the input RNA was performed by GENEWIZ Multiomics & Synthesis Solutions from Azenta Life Sciences.

Mapping sequence reads to the reference genome

Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the capsicum_annuum reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated because of this step.

Extracting gene hit counts

Unique gene hit counts were calculated by using feature counts from the Subread package v.1.5.2. The hit counts were summarized and reported using the gene_id feature in the annotation file. Only unique reads that fell within exon regions were counted.

Differential gene expression analysis

After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the customer-defined groups of samples was performed. The Wald test was used to generate p -values and log2 fold changes. Genes with an adjusted p -value < 0.05 and absolute log2 fold change > 1 were called as differentially expressed genes for each comparison.

Bioinformatics tools for functional analysis

To verify the annotation, and thus the function of the overexpressed genes and proteins, the gene ontology provided by UniprotKB, annotations from NCBI, PATRIC, and Ecogene were consulted, as well as the gene ontology assigned by the JCVI Microbial Resource Center. Additionally, these genes and proteins were sorted according to the orthologous classification provided by KEGG [ 21 ], incorporating into this classification those genes and proteins reviewed by the various annotations and ontologies mentioned earlier. As the first functional analysis, the different functional categories described in the clusters of orthologous groups (COG) associated with each overexpressed gene or protein were consulted.

Oxidative stress level in plants after the ozone exposition

Antioxidant enzyme analysis.

In the same way of the determination of lipid peroxidation, a pool of leaves was created and collected in liquid nitrogen and stored at -80˚C until the analysis.

Extraction was carried out using a 50 mM sodium phosphate buffer (pH 7.6). After samples homogenization and centrifugation at 4˚C, the total protein content in extracts was determined according to Bradford protocol [ 22 ].

The analysis of the antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), and guaiacol peroxidase (GPX)) was performed according to Duarte et al. [ 23 ]. Basically, for CAT activity, the disappearance of H 2 O 2 was recorded at 240 nm after the addition of the vegetal extract. SOD activity was determined by oxidation of the pyrogallol at 325 nm after the addition of vegetal extract. Finally, guaiacol oxidation was measured at 470 nm after the addition of vegetal extract to determine the activity of the GPX. In the three assays, the auto-oxidation of the respective substrates was also recorded in absence of the vegetal extract.

Lipid peroxidation analysis

Random leaves for each plant were collected creating a pool of leaves from the same treatment using liquid nitrogen and stored until analysis at -80˚C.

To determine lipid peroxidation, MDA concentration was measured following the protocol suggested by [ 24 ]. Briefly, samples were incubated with 20% TCA containing 0.5% TBA at 95˚C for 1 h and then, samples were measured at 532 and 600 nm using a spectrophotometer to determine the MDA concentration (extinction coefficient of 156 mM −1  cm −1 ).

Oxidative stress index

The oxidative stress index (OSI) was calculated based on the results of the lipid peroxidation and the activities of the antioxidant enzymes to express the global stress in pepper plants after the experiments. This parameter was calculated following the formula described [ 25 ].

In the context of this statement, [SOD], [CAT], [GPX], and [MDA] represent the respective enzyme values under different treatments applied, while [SOD]0, [CAT]0, [GPX]0, and [MDA]0 signify the control values. An index exceeding 1 suggests that the leaves experienced stress, while values below 1 suggest an absence of oxidative stress in the leaves.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 8.4.0.671. Normality was assessed using the Kolmogorov–Smirnov test. The means of the different treatments were compared using two-way ANOVA, and statistical differences were determined using the Tukey multiple comparison test.

Okara Enzymatic Extract Preparation

Okara is a solid organic byproduct, insoluble in water, derived from the aqueous industrial extraction of soybeans. Okara is a potential source of bioactive molecules such as peptides, isoflavones and soluble fiber but due to the insolubility it must be consider treatments that facilitate the release of its useful components. One approach to this process is the use of hydrolytic enzymes, such as proteases, from which a new soluble product, the OEE, has been obtained.

The basic chemical composition of the enzyme extract OEE is outlined in Table  1 , highlighting that the primary constituent is the protein fraction at 63.4%, with carbohydrates following at 24.4%. This includes soluble fiber at 8% and insoluble fiber at 2%.

The protein fraction of the enzyme extract is composed of peptides < 5 kDa (Fig.  1 ; Table  2 ). Peptides are low molecular weight protein fractions with high bioactive potential.

Chromatography profile of the soluble protein content of OEE according to its molecular weight using a Superdex Peptide 10/300 GL column

Physiological Status in Plants

The physiological state of the plants was determined through various photosynthetic parameters such as A N , ETR and PhiPS2, as well as DF.

After O 3 exposure, A N , ETR and PhiPS2 were significantly affected (Fig.  2 A-C), showing decreases of 75%, 58% and 57.8% respectively, compared to the control. Treatment with OEE didn’t significantly modify these parameters but clearly protect the decrease induced by O 3 in all of them (51% in A N ; 29% in ETR; 38.4% in PhiPS2).

Physiological parameters. A A N ; B  ETR and C  PhiPSII in response to O 3 (0 and 100 ppm) under a treatment without and with OEE. Values represent mean ± SD, n  = 5. Different letters indicate means that are significantly different from each other (two-way ANOVA, O 3 exposition × OEE treatment; HSD test, P  < 0.05). O 3 exposition and OEE treatment in the corner of the panel indicate main or interaction significant effects (* P  < 0.05; ** P  < 0.01; *** P  < 0.0005; **** P  < 0.0001)

We also evaluated DF, closely link to photosynthesis reactions and thus an indicator of plant stress status. In fact DF has been used as a direct indicator of the chlorophyll content [ 26 ]. As could be visualized by the imaging and graph of Figs.  3 A and B, the Ct and OEE groups showed similar DF values which indicates that OEE did not induce stress in the plants. O 3 exposure significantly decrease DF values (26% compared to Ct; Fig.  3 A) and this decreased was completely prevented by OEE (Fig.  3 A).

Delayed fluorescence in leaves of pepper plants in response to ozone (O3) (0 and 100 ppm) under a treatment without and with OEE). A Counts per second (cps) values for each treatment. Values represent mean ± SD, n  = 5. Different letters indicate means that are significantly different from each other (two-way ANOVA, O 3 exposition × OEE treatment; HSD test, P  < 0.05). O 3 exposition and OEE treatment in the corner of the panel indicate main or interaction significant effects (* P  < 0.05; ** P  < 0.01; *** P  < 0.0005; **** P  < 0.0001). B photographs taken by the plant imaging system NightShade LB 985. Delayed fluorescence was used as a direct indicator of the chlorophyll content. The color scale reflects the detected counts per second (cps) of delayed fluorescence emission in leaves. Red colour indicates high intensities representing high chlorophyll content, blue colour indicated low intensities of fluorescence, indicating low amounts of chlorophyll

These results suggest that OEE does not affect physiological status in plants but protect them against photosynthetic damage induced by ozone helping to maintain physiological status under this abiotic stress.

To further investigate the impact of ozone on the photosynthetic machinery, the RNA expression levels of various components of photosystem II were analyzed.

As seen in Fig.  4 , in the presence of ozone, genes related to photosystem II are repressed compared to the control group. However, plants exposed to ozone and treated with OEE show lower levels of inhibition of these genes. For example, the 5 KDa protein of photosystem II is three times less inhibited, and the W protein of the reaction center is twice as inhibited compared to plants only treated with ozone. Therefore, the OEE treatment appears to offer protection against ozone-induced damage in photosynthetic system.

Fold-change of differentially expressed genes related to Photosystem II. Genes differentially expressed when ozone is applied to the plant are shown in blue, and in yellow when ozone plus the treatment with OEE was applied

Oxidative Stress Level in Plants

To evaluate oxidative stress induced by O 3 , antioxidant enzyme activities, CAT, SOD and GPX were measured. As expected, ozone significantly induced the enzymatic activities (Fig.  5 A-C), being SOD activity specially affected (increase was more than threefold). Interestingly OEE treatment also induced CAT and SOD activities in absence of ozone (87% and 121% respectively), but completely prevented the increased induced by ozone. Finally, OEE did not induce significantly the GPX activity compared with control group (Fig.  5 C) although after OEE treatment, the increased induced by ozone (122%, Ct + O 3 compared to Ct) was completely reverted.

Antioxidant enzyme activities. A CAT B  SOD and C  GPX in response to O 3 (0 and 100 ppm) under a treatment without and with OEE. Values represent mean ± SD, n  = 5. Different letters indicate means that are significantly different from each other (two-way ANOVA, O 3 exposition × OEE treatment; HSD test, P  < 0.05). O 3 exposition and OEE treatment in the corner of the panel indicate main or interaction significant effects (* P  < 0.05; ** P  < 0.01; *** P  < 0.0005; **** P  < 0.0001)

MDA was measured as an indicator of lipid peroxidation due to oxidative stress [ 27 ]. As showing Fig.  6 A, OEE treatment avoid the increase in MDA induced by ozone with no effect in MDA values in absence of ozone, which again underline that OEE did not induce stress in the plants.

MDA content ( A ) and OSI ( B ) in leaves of pepper plants in response to ozone (O 3 ) (0 and 100 ppm) under a treatment without and with OEE. Values represent mean ± SD, n  = 5. Different letters indicate means that are significantly different from each other (two-way ANOVA, O 3 exposition × OEE treatment; HSD test, P  < 0.05). O 3 exposition and OEE treatment in the corner of the panel indicate main or interaction significant effects (* P  < 0.05; ** P  < 0.01; *** P  < 0.0005; **** P  < 0.0001)

The protective role of OEE against oxidative damage was also reflected in the OSI values (Fig.  6 B). OSI values are in accordance with activities values, that show an increase after OEE treatment (1.58) but clearly protection after ozone exposition compared with control group (0.9 vs 2.37, respectively).

The present work demonstrates the protective effect of a biostimulant (OEE) from okara against O 3 -induced damage in peppers plants. We selected pepper plants because pepper is a vegetable crop of significant agricultural and economic importance, ranking as the second most traded spice globally. Substantial losses in pepper production often result from abiotic stresses, including ozone exposure. In fact, capsicum pepper cultivation is predominantly situated in regions where ozone concentrations are escalating to phytotoxic levels [ 28 ]. OEE protects against ozone-induced changes in photosynthetic parameters including A N , ETR and PhiPS2 as well as in DF while also protecting against lipid peroxidation. Given that the relief of abiotic stress is often highlighted as a prominent benefit of biostimulants [ 29 ] we propose that OEE possesses biostimulant capacity.

The chemical composition of okara is described in [ 18 ], highlighting the high level of protein and fibers. Okara is a potential source of bioactive molecules such as peptides, isoflavones and soluble fiber that are currently of great interest due to their countless benefits for both humans and agriculture [ 30 , 31 ]. However the high content of insoluble biomolecules in okara can limit its direct effectiveness as a biostimulant. To employ okara in a way that improves its usefulness as a bio-stimulant, it is important to consider treatments that increase its solubility or facilitate the release of its useful components, more soluble and accessible to plants and microorganisms. As show in Fig.  1 , protein hydrolysates contain peptides and amino acids resulting from enzymatic hydrolysis. They currently have great interest in agronomy due to their positive influence on growth, improvement in N absorption and assimilation, and their direct involvement in numerous metabolic processes and defense against oxidative stress by plants [ 32 ].

The adverse impacts of O 3 on plants are extensively documented and include, among other effects, a decline in photosynthesis and the manifestation of chlorotic and necrotic spots on leaves [ 2 , 8 ]. Accordingly, the present data show that the acute O 3 -exposure experimental design by us induced changes in physiological status of pepper plants, as evidenced by the decrease in parameters such as A N , ET and PhiPS2 (Fig.  2 A-C), which is in agreement with previous report by our group [ 15 ]. Interestingly, foliar application of OEE partly reverted the O 3 -induced decreased in those parameters.

The damage inflicted on plants by O 3 is primarily attributed to the elevated production of ROS upon its entry into the apoplastic space [ 33 ]. ROS generated by exposure to O 3 are responsible for direct oxidative damage to various molecules involved in the photosynthetic process, such as chlorophylls a and b, and Rubisco, whose activity and content have been shown to decrease under such stress conditions [ 34 ]. Accordingly, our results show that DF was also affected (Fig.  3 ). Delayed fluorescence has been used as a direct indicator of the chlorophyll content. Low signals of delayed fluorescence has been described previously after biotic ozone-induced stress [ 15 ] and also in wheat leaves after biotic stress, such as infection with S. graminum , which suggested the occurrence of chlorophyll degradation [ 26 ]. Additionally, the harmful effects of O 3 on photosynthetic electron transport, particularly on the function of photosystem II, have been demonstrated [ 35 , 36 ]. In this context, our RNAseq results revealed that ozone represses genes related to photosystem II (Fig.  4 ). Interestingly OEE partly reverted ozone-induced effect on DF and gene expression.

In the cell, ROS interacts directly or indirectly with biomolecules, damaging them. Thus, ROS induces lipid peroxidation of cell membranes, denaturation of proteins, oxidation of carbohydrates, or fragmentation of pigments [ 37 ]. To counteract ROS, which are produced not only under stress but also during the normal metabolism of plant cells in processes such as mitochondrial respiration, photosynthesis and the activity of flavin-oxidoreductases, cells possess enzymatic and non-enzymatic systems that protect them from ROS. Indeed, the increase in ROS production is associated with certain antioxidant enzymes [ 38 , 39 ], which are stimulated by the upregulation of antioxidant genes [ 38 ].

Principio del formulario

To analyse the protective role of OEE against O 3 -induced damage, we examined antioxidant enzymes activities. The results presented here demonstrate that the antioxidant enzyme activities assayed (CAT, GPX, and SOD) were upregulated after ozone exposition, and this induction was significantly reversed by foliar treatment with OEE (Fig.  5 ). Surprisingly, OEE also stimulated enzymatic activities even in the absence of ozone. It's crucial to highlight that the production of ROS is a prevalent plant response to various stresses, encompassing both biotic and abiotic factors (as reviewed by Sewelan et al. [ 40 ]). ROS can serve as a convergence point for different signalling pathways. Within this context, it's plausible to speculate that OEE induced a mild response activating signalling pathways that contribute to coping with subsequent stress. This could be considered a hormetic-like effect. In fact, in previous work, we have already speculated that enzymatic extracts of plant origin may exert a hormetic effect by inducing antioxidant enzyme activity [ 15 ]. Interestingly, both ROS and reactive nitrogen species are frequently linked to dose–response hormesis in both plants and animals [ 41 , 42 , 43 ].

Hormesis is defined as “an adaptive response of biphasic dose where it responds to a stress determining factor, in which sub-doses induce stimulation and high doses induce inhibition” [ 44 ]. From a physiological standpoint, hormesis is an adaptive response activated in an organism when subjected to low levels of a stressor, accompanied by overcompensation, when the homeostasis readjustment has been interrupted [ 45 , 46 , 47 ]. In plants, hormesis has been elucidated through exposure to low levels of biotic or abiotic stressors, including temperature fluctuations or radiation. This exposure predisposes plants to respond to challenging conditions by activating cellular defense mechanisms [ 47 , 48 ].

The mechanism of hormesis in plants is not well-defined, although it has been proposed that the induction of ROS by weak stressors may play a central role through activation of antioxidant defense systems, stress-signaling hormones, or adaptive growth responses [ 49 ]. Accordingly, it has been described that the induction of low and sub-toxic concentrations of ROS by mild stressors, such as may occurs after foliar application of OEE, has the capacity to generate a hormetic response, activating antioxidative defense and adaptive responses [ 49 ].

Biostimulants are defined as substances that promote plant growth, increase the ability to tolerate biotic or abiotic stress, and improve crop quality [ 50 , 51 ]. Biostimulants are a broad group of compounds of diverse nature, including plant growth-promoting bacteria, beneficial fungi, humic acids, seaweeds, protein hydrolysates or amino acids [ 50 , 51 , 52 , 53 , 54 , 55 ]. In the context of induced hormesis, biostimulant activate secondary metabolism and induce genes expression to recover homeostasis [ 56 ] enabling plants to tolerate stresses [ 57 ]. So, when biostimulants are applied at right time can improved plant growth, and the simultaneous use of several biostimulants can effectively alleviate environmental impacts [ 58 ].

The enzymatic hydrolysis of okara yielding an extract rich in isoflavones [ 18 ]. Isoflavones engage in hydrophobic interactions with the proteins in which they are embedded, and treatment with proteases at alkaline pH solubilizes them. In this regard, a direct correlation has been identified between the solubilization of proteins and isoflavones in okara [ 18 , 59 ], indicating that protease treatment is effective for the recovery of isoflavones. Interestingly OEE contains most of the isoflavones found in okara, with a predominant presence of beta-glucosides such as genistin, which represents about 50% of the total isoflavone content [ 18 ]. Isoflavones are the most prominent functional component of soy. They exert antioxidant activity, protect plants from diseases (such as antimicrobial and antiherbivore activities) and have positive effects on the life quality of plants [ 60 ]. In soybean, it has been described that isoflavones act as phytoalexins [ 61 ]. Phytoalexins are plant metabolites that protect plants due to potent antibacterial, antiviral effects, antiherbivore effects, and even effects in abiotic stress situations such as ozone [ 62 , 63 , 64 , 65 ]. We propose that isoflavones present in OEE may act as elicitors, being metabolite-inducing factors that mimic stress conditions and contribute to the hormetic-like effect in pepper plants, allowing them to cope with further abiotic stress induced by ozone.

The hormetic-like effect was also evinced in the oxidative stress index. OSI values (Fig.  6 B) indicated an increase in oxidative stress after OEE treatment (1.58). However, there was a clear protection after ozone exposition when compared with control group (0.9 vs 2.37, respectively). This supports the hypothesis that OEE´s bioactive compounds induce a mild stress condition in pepper plants, which, in turn, protects them against further abiotic stress.

MDA resulting from the peroxidation of polyunsaturated fatty acids within cells is regarded as a dependable indicator for evaluating the degree of injury in stressed plants [ 66 ]. The higher the extent of damage to the plant, the greater the MDA content, as evidenced by studies on plant responses to both abiotic and biotic stresses [ 67 ]. In fact, when pepper plants are exposed to O 3 , it has been observed that biomolecules undergo damage from oxidation caused by reactive oxygen species (ROS) as well as direct interaction with O 3 . This results in a decrease in chlorophyll content and an increase in lipid peroxidation [ 68 ]. Consistent with the antioxidant activity of OEE [ 18 ] we observed that the foliar application of OEE prevented the lipid peroxidation induced by ozone, as evidenced by the MDA values (Fig.  6 A). Therefore, we can assume that OEE protects plants from ozone-induced abiotic damage both through its hormetic effect and, as expected, due to its antioxidant capacity.

OEE contains different bioactives compounds that contribute to its antioxidant capacity including isoflavones. Genistein and genistin, both prevalent in OEE, have been characterized as having the most substantial antioxidant activities among all soy isoflavones [ 69 ]. Regarding the antioxidant capacity, it is essential to highlight that the protein fraction in OEE primarily consists of peptides < 1 kDa, which could contribute to the extract's antioxidant activity [ 18 ]. Treatment with proteases release releases peptides from proteins, thereby converting them into their active form [ 70 ]. These protein hydrolysates (PHs) exert multiple bioactivities, with antioxidant activity being one of the first to be recognized among their bioactive properties (for review see [ 71 ]. The bioactivities of PHs have led to considering them as a novel approach to stimulate plants, as foliar application of protein hydrolysates to plants has the potential to alleviate the impact of abiotic stressors by enhancing antioxidant capability [ 72 ]. Several studies have shown that soybean hydrolysates have the capacity to effectively counteract free radicals [ 73 , 74 ]. Even more, it has been also reported antioxidant activity in soy protein derived peptides obtaining after treatment with different proteases including subtilisin from Bacillus subtilils [ 75 ]. The antioxidative properties of a peptide are influenced by its structure and amino acid sequence. In this regard, we have previously highlighted that the amino acid composition of OEE is characterized by a predominance of hydrophobic amino acids [ 18 ] which have been associated with the antioxidant activity of bioactive peptides [ 31 , 74 , 76 , 77 ]. Besides, the hydrolysis of proteins results in an elevation of the concentration of sulfur amino acids, namely methionine and cysteine, both of which possess significant antioxidant potential [ 78 ]. Furthermore, the peptides in OEE have a direct relationship with isoflavones. These isoflavones are converted into their bioactive form (aglycones) after hydrolysis with proteases, with a notable concentration of genistin, a highly antioxidant aglycone [ 18 ]

Moreover, it should be also taking into account that soybean seeds and its derived okara are good sources of dietary fibers [ 16 ]. In OEE, the carbohydrates fraction represents 24.4% of the dry matter including insoluble (2%) and soluble fibers (8%) (Table  1 ). Okara is enriched in cell wall polysaccharides, and it has been described that certain fractions of okara polysaccharides exert antioxidant activity, with pectins or solubilized simple saccharides playing a significant role [ 79 ]. Therefore, a characterization of OEE carbohydrate fraction would be interesting.

In this study, we have analyzed the protective role of an enzymatic extract obtained from okara against abiotic stress induced by O 3 . The chosen model is of interest as O 3 is responsible for significant losses in agriculture, and in the coming years, the increase in pollutants derived from human activity, coupled with global warming, is expected to worsen these losses. Additionally, for the study, pepper plants ( Capsicum annum ) were selected since they are predominantly grown in subtropical regions globally, with major producers situated in developing countries where O 3 concentrations are anticipated to rise more significantly than in other nations.

Consistent with its antioxidant activity, in line with the composition of bioactive compounds such as isoflavones and PHs, OEE treatment protects against ozone-induced damage, as evidenced by physiological parameters such as A N , ETR, PhiPS2, and DF, expression of genes related to photosystem II as well as levels of MDA. Interestingly, OEE induced a moderate oxidative stress that protected against subsequent ozone-induced damage, which can be interpreted as a hormetic effect.

Altogether, considering that promoting plant resistance against abiotic damage is a central feature of biostimulants, we propose that OEE possesses properties that make its use as a biostimulant feasible. Nevertheless, additional research is necessary to elucidate the mechanisms underlying hormetic and protective effects. Given our hypothesis that bioactive compounds in OEE, especially isoflavones, may function as elicitors, it would be valuable to explore the key pathways associated with the synthesis of secondary metabolites and defense mechanisms.

Availability of data and materials

All data generated or analysed during this study are included in this article.

Abbreviations

Reactive oxygen species

Okara Enzymatic Extract

Net photosynthetic rate

Electron transport rate

Effective quantum yield of PSII

Delayed fluorescence

Malondialdehyde

Ethylene diurea– (N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N0 phenyl urea

Superoxide dismutase

Guaiacol peroxidase

Protein hydrolysates

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Acknowledgements

We especially thank Luis Martín Presas for his technical assistance in plant treatment. We thank the University of Seville Greenhouse General Service (CITIUS) for their collaboration and providing the facilities.

This study was supported by Grant PID2021-124964OB-C21 and Grant TED2021-129822B-I00 funded by MCIN/AEI/ https://doi.org/10.13039/501100011033 and, as appropriate, by “ESF Investing in your future” or by “European Union Next Generation EU/PRTR”.

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Angel Orts, Sandra Macías-Benítez, José M. Orts, Emilia Naranjo, Angélica Castaño & Juan Parrado

Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla. C/Profesor García González, Nº2. 41012, Seville, Spain

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AO, SM-B, AC, and JP designed the study. AO, SN-T, EN and SM-B performed the research. AO, SN-T, JMO, EN, AC, and JP analyzed the data and wrote the manuscript. All authors reviewed the manuscript.

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Orts, A., Navarro-Torre, S., Macías-Benítez, S. et al. A new biostimulant derived from soybean by-products enhances plant tolerance to abiotic stress triggered by ozone. BMC Plant Biol 24 , 580 (2024). https://doi.org/10.1186/s12870-024-05290-3

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Root carboxylate release is common in phosphorus-limited forest ecosystems in China: using leaf manganese concentration as a proxy

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• Li Yan   ORCID: orcid.org/0000-0003-3937-2509 1 , 2 ,
• Dan Tang   ORCID: orcid.org/0009-0006-0861-6442 2 ,
• Jiayin Pang   ORCID: orcid.org/0000-0002-8127-645X 2 , 3 &
• Hans Lambers   ORCID: orcid.org/0000-0002-4118-2272 2 , 3  

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Background and aims

Certain plant species release root carboxylates in response to phosphorus (P) limitation; however, the prevalence of root exudate release in species in P-limited forest ecosystems remains unexplored due to challenges in field assessment.

Manganese (Mn) accumulation in mature leaves can indicate the presence of root carboxylate exudates in rhizosphere soil. To account for environmental factors such as soil pH, a negative reference species that does not release carboxylates is used for comparison. In this study, we assessed multiple forest stands across soil types and different levels of P availability in northern (Gansu) and southern (Guangxi) China. Leaf and soil samples were collected from 188 plant families representing various life forms, and leaf Mn concentration ([Mn]) was analyzed as a proxy for root carboxylate exudation patterns, using Dryopteridaceae as a negative reference.

The results supported our hypotheses that leaf [Mn] was higher in P-limited forests of southern China compared to P-richer forests of northern China, even though the soil [Mn] was higher in the forests of northern China. Additionally, we observed a higher prevalence of species with high leaf [Mn] across various plant families in Guangxi (82%) than in Gansu (42%).

Our findings suggest a potential common strategy among plants in Guangxi forests, where root exudates are released in response to P limitation, possibly due to ineffective mycorrhizal symbiosis for nutrient acquisition. The diverse forest systems in China exhibit varying soil P availability, leading to the evolution of plant species with distinct P-acquisition strategies.

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Introduction

Forestry ecosystems play a vital role in global carbon cycling, biodiversity conservation, and sustainable resource management (Miura et al. 2015 ; Watson et al. 2018 ; Migliavacca et al. 2021 ; Taye et al. 2021 ). Understanding nutrient dynamics within these ecosystems is crucial for effective management and conservation (Silver et al. 1996 ; Watson et al. 2018 ; Meier et al. 2019 ). Among essential nutrients, phosphorus (P) often limits plant growth, particularly in highly weathered and leached soils (Du et al. 2020 ; Hou et al. 2020 ), which are prevalent in southern China (Zhang et al. 2021 ; Yan et al. 2023 ). In response to P limitation, plants have evolved various adaptive strategies, such as the release of root exudates, especially carboxylates and phosphatases, to enhance P acquisition from P-impoverished soils (Lambers et al. 2015b ; Güsewell and Schroth 2017 ; Lambers 2022 ). Investigating and quantifying root-exudation patterns in P-limited forestry ecosystems is therefore essential for understanding nutrient cycling processes and optimizing forest management practices. However, due to the challenge of directly accessing and measuring plant-soil nutrient dynamics in forests (Oburger and Jones 2018 ; Escolà Casas and Matamoros 2021 ), root exudation patterns across different forest ecosystems are largely unknown.

Leaf manganese (Mn) concentration ([Mn]) has been proposed as a proxy to estimate the extent of root exudation for P acquisition in a number of plant species (Lambers et al. 2015a , 2021 ; Pang et al. 2018 ; Zhou et al. 2022 ; Yu et al. 2023b ). Some plants experiencing P limitation increase their root carboxylate exudation and, in turn, enhance Mn uptake from the soil (Shane and Lambers 2005 ; Lambers et al. 2015a ). Plants absorb and transfer soil Mn through a combination of passive and active processes, lacking dedicated transporters for Mn (shared with iron and other elements), and the poor control of Mn uptake makes it possible to indicate its availability in rhizosphere soil (Page and Feller 2005 ; Millaleo et al. 2010 ). Manganese is transported to leaf cells through various transporters present in the plasma membrane, and subsequently accumulates in mature leaves. Manganese uptake rates may exceed the plant’s requirement by over 100-fold in some species, resulting in hyperaccumulation (Millaleo et al. 2010 ; Losfeld et al. 2015 ). Excessive Mn can be toxic, promoting plants to develop various mechanisms to accumulate Mn without adverse effects. These mechanisms include redistributing and compartmentalizing Mn within different tissues to maintain optimal [Mn] in essential tissues while sequestering excess Mn in less sensitive compartments such as cell walls (oxidized Mn and phenolic compounds) (Wissemeier and Horst 1992 ). In addition, plants can chelate excess Mn within cells in vacuoles by binding it to organic compounds such as citrate, oxalate, and malate (Memon and Yatazawa 1984 ; Millaleo et al. 2010 ). Due to the involvement of Mn in redox reactions and generation of reactive oxygen species (ROS), plants possess antioxidant defense systems to scavenge and neutralize ROS (Sytar et al. 2021 ; Kosakivska et al. 2021 ; Zhao et al. 2022 ). These findings suggest that leaf [Mn] has the potential to serve as a reliable indicator of rhizosphere carboxylate concentrations and P-acquisition strategies across various ecosystems (Lambers et al. 2015a ; Huang et al. 2017 ).

Low soil P availability promotes leaf Mn accumulation in plants, and is often observed in P-deficient soils where plants adjust their nutrient-uptake and -allocation strategies to cope with the limited P availability (Millaleo et al. 2010 ; Lambers et al. 2015a , 2021 ). One of the mechanisms involves the exudation of carboxylates and phosphatases from their roots, enhancing the solubilization and mobilization of soil P. However, these carboxylates also influence the availability of other nutrients, including Mn (Shane and Lambers 2005 ). Under P-deficient conditions, increased secretion of carboxylates enhances the desorption of Mn from soil particles, making it more accessible to plant roots (Kochian et al. 2004 ). Subsequently, root uptake and shoot transport result in elevated [Mn] within mature leaves (Page and Feller 2005 ; Page et al. 2006 ; Losfeld et al. 2015 ).

There is a lack of comprehensive studies examining the correlation between root exudation, leaf [Mn], and P limitation in forestry ecosystems of southern China. Very few studies have explored the relationship between root exudates and P availability among different plant species and ecosystems in south China. For example, research conducted in subtropical rainforests has shown that root exudation is a major root functional trait (Sun et al. 2021 ), and root exudation is largely affected by soil P availability and mycorrhizal type (Jiang et al. 2022 ). However, how prevalent the release of root exudates is among different species in P-limited forest ecosystems remains unexplored (Wang and Lambers 2020 ). Are species in any plant family consistently exhibiting high levels of root exudation in both P-rich and P-limited environments? Does root carboxylate exudation demonstrate specific trends among plant life forms? Responses to these questions are essential to bridge the knowledge gap and provide valuable insights into the nutrient dynamics of significant forest ecosystems.

In the context of forestry ecosystems in southern China, characterized by acidic soils with low P availability, investigating the correlation between root exudation, leaf [Mn], and P limitation holds particular promise. This region also encompasses vast areas of plantation forests dominated by fast-growing tree species widely utilized for timber production and ecological restoration (Zhou et al. 2017 ; Sun et al. 2021 ; Yan et al. 2023 ). Understanding the mechanisms underlying nutrient acquisition and utilization in these ecosystems is crucial for sustainable forest management and ecosystem functioning. Additionally, using leaf [Mn] as a proxy can allow us to assess whether plants have the capacity to be hyperaccumulators and remove excessive soil Mn for soil restoration (Li et al. 2007 ; Liu et al. 2020 ; Wildová et al. 2021 ).

This study aimed to investigate the potential of using leaf [Mn] to proxy root exudation in different P-limited forestry ecosystems in southern China as well as in P-rich systems in northern China. Based on previous measurements (Lai et al. 2024 ), we assumed that the forest ecosystems in southern China likely experience P limitation due to their acidic soils and significant erosion. We hypothesized: 1) The leaf [Mn] would be higher in the forest ecosystems of southern China (low-P soils) than those in northern China (relatively P-rich soils). Therefore, we expected plants in the southern forest ecosystems to exhibit faster root carboxylate-exudation rates, leading to increased Mn uptake and consequently higher leaf [Mn] than those in the northern forest ecosystems. According to previous studies showing that root exudation is widespread across different plant families and plant forms, we further hypothesized: 2) high leaf [Mn] in P-limited southern China would be more common compared to that in the relatively P-rich ecosystems in northern China.

To test the above hypotheses, we assessed multiple forest stands across different soil types with different levels of P availability. Leaf and soil samples were collected from selected stands encompassing a range of plant families and life forms, and leaf [Mn] was analyzed to proxy root carboxylate-exudation patterns. Furthermore, we quantified soil P availability and characterized soil properties to assess their impact on the dynamics of root exudation.

Methods and materials

Study sites and species selection.

The study was conducted in 2020, assessing multiple forest stands across different soil types with different P availability in two distinct natural forest ecosystems (Fig.  1 ; this map was made using R package “ggmap”; the source is “stadia” with a type “stamen_terrain”). One ecosystem is located in the Maijishan Region (34°20′N, 106°01′E) of Gansu Province, northern China. Known for its rich biodiversity, the Maijishan ecosystem has a total of 2371 species of vascular plants, representing 218 families and 962 genera (Lu 2006 ). Located within the Qinling Mountains, it spans altitudes ranging from 1200 to 1600 m above sea level. It experiences a continental climate characterized by cool, humid, and semi-humid zones, with an average annual rainfall of 700 mm, primarily occurring between July and September. The mean temperature is 9 °C with a relative air humidity of approximately 73% (Yang et al. 2023 ). The dominant soils in this region are mountain brown loams (Tudi et al. 2022 ).

Map of the study sites in the relatively phosphorus-rich Maijishan region, Gansu, China and in the phosphorus-limited Damingshan region, Guangxi, China

Another ecosystem studied is situated in the Damingshan Region (23°24′N, 108°31′E), within the south-central Guangxi Zhuang Autonomous Region of southern China. Due to its unique geographic location, Damingshan exhibits rich biodiversity with a total of 2374 vascular plant species belonging to 918 genera across 234 families (Wen et al. 1998 ). This ecosystem lies between the northern tropics and southern subtropics at an average altitude of 1200 m, experiencing a southern subtropical monsoon climate. It receives an average precipitation of 2630 mm and an average temperature of 15.1℃ (Zhu et al. 2011 ).

According to the literature (Wen et al. 1998 ; Suo et al. 2008 ; Zhu et al. 2011 ; Yang et al. 2023 ) and recommendations from local botanists, we established a 200 m × 200 m study site in each ecosystem in 2020. Each site was subdivided into eight to 10 subsites based on the landscape for the purpose of investigating and selecting plant species. Taking into consideration factors such as abundance, life form, and endemism, a total of 188 species were collected from the two ecosystems: 38 plant families, 81 species from relatively P-rich soils of Gansu; 56 plant families, 107 species from low-P soils of Guangxi (Supplementary Table  1 ).

Reference species selection

The leaf [Mn] can serve as an indicator of root carboxylate release, implying that higher concentrations suggest plants release greater amount of carboxylates that mobilize Mn and enhance its uptake from the soil. To assess the relationship between different leaf [Mn] levels and the extent of root carboxylate release, a “negative reference” is necessary (Lambers et al. 2021 ; Lambers 2023 ). Therefore, we selected the family of Dryopteridaceae (ferns) species as the negative reference for several reasons: 1) Dryopteridaceae are adapted to efficiently utilize limited Mn in their habitats (Grosjean et al. 2019 ; Schmitt et al. 2017 ) which may not provide sufficient amounts of Mn for these ferns to accumulate high concentrations in their leaves (Cornara et al. 2007 ); these adaptations often involve conservative strategies for Mn uptake and allocation within the plant, resulting in lower leaf [Mn] (Bai et al. 2020 ; Zhou et al. 2022 ); 2) Dryopteridaceae typically exhibit slow growth rates compared with many vascular plant species (Pinson et al. 2017 ; Rünk and Zobel 2007 ), leading to reduced demand for Mn and consequently lower concentrations in their leaves (Reimann et al. 2007 ); 3) Dryopteridaceae occurred in both studied ecosystems. It is worth noting that leaf [Mn] in different fern families may vary due to their specific growth microhabitats, suggesting a discernible difference in leaf [Mn] among different fern families. In this study, when referring to “high” leaf [Mn], it indicates a comparison with the negative reference group of Dryopteridaceae.

Leaf and soil sampling

All samples were collected between August and October 2020. Five plants per species (except for a few species, which included three to four plants) were selected, and mature leaves were utilized for chemical analyses. The leaves were placed in envelopes for subsequent determination of dry weight (at 60℃ for seven days). Bulk soil samples were obtained by combining soil from three points within each plot of every subsite. All soil samples were collected in plastic bags, air-dried for one week, sieved to a particle size of 2 mm, and then ground.

Leaf and soil analyses

Aliquots of 80 mg sample were digested using concentrated HNO 3 and HClO 4 (v/v 3:1) and diluted to 10 ml with Milli-Q water, and leaf [Mn] was analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES) (Perkin Elmer, PE Avio 500, Shelton, CT, USA). Leaf P concentration ([P]) was analyzed using the molybdenum blue-based method (Ames 1966 ) with a microplate reader (Thermo Fisher Scientific, Varioskan Flash, Finland).

Aliquots of 0.5 g soil sample were weighed in a digestion tube and digested using concentrated H 2 SO 4 and HClO 4 at 360℃ for one hour. The total nitrogen concentration ([N]) and total [P] were then determined using an automatic chemical analyzer (AMS, Smartchem 450, Rome, Italy) and the molybdenum blue-based method (Olsen and Sommers 1982 ), respectively. Soil nitrate and ammonium were extracted with 2 M KCl and measured using a continuous segmented flow autoanalyzer (AA3, SEAL Analytical, Norderstedt, Germany). Anion exchange membranes (AEMs, VWR Chemical, Leuven, Belgium) were used to measure soil resin P (‘plant-available P’) (Bentley et al. 1999 ). Soil pH (water based) was measured using a pH meter.

Statistical analyses

All statistical analyses were performed using the R software platform (version 4.0.2, R Core Team 2020 ). Shapiro-Wilk normality was tested using ‘rstatix’ (version 0.7.2, Kassambara 2023 ), and homogeneity was tested using ‘stats’ (R Core Team 2020 ). Considering the varying sample sizes, such as three to five replicates for each species and one to 10 species from different families, the utilization of Welch t-test is more suitable for this study (Stewart-Oaten et al 1992 ; Ruxton 2006 ). The Welch t-test was used to compare the difference of mean concentrations between the “negative reference” and target families, and the difference between two locations using ‘stats’ (R Core Team 2020 ). All plant species from two locations were compared for significance (Figs.  2 , 3 and 5 ), excluding the top 50% of species with the highest leaf [Mn], as depicted in Fig.  4 . Appropriate generalised least square models (GLS) were selected based on the lowest Akaike's Information Criterion corrected (AICc) values and the Tukey's HSD post hoc test (Pinheiro & Bates 2000 ), to compare the significance among life forms at the same location ( p  < 0.05) in Fig.  5 .

Leaf phosphorus (P) ( a ) and manganese (Mn) ( b ) concentration in different locations of relatively P-rich soils in Gansu and low-P soils in Guangxi. Each point indicates an individual plant, with a total of 327 and 395 individuals in Gansu and Guangxi, respectively. Asterisks **** indicate significant difference between values in Gansu and Guangxi using the Welch t-test, p  < 0.0001

Leaf manganese (Mn) concentration of 38 families in relatively P-rich soils of Gansu ( a ) and 56 families in low-P soils of Guangxi ( b ). Asterisks * indicates significant difference compared with the negative reference (Dryopteridaceae, marked as R) using a Welch t-test, p  < 0.05

Top 50% plant species with the highest leaf manganese (Mn) concentration in relatively P-rich soils of Gansu ( a ) and low-P soils of Guangxi ( b )

Leaf manganese (Mn) concentration of different plant life forms in relatively P-rich soils Gansu ( a ) and low-P soils of Guangxi ( b ). Different letters indicate significant difference among life forms within the same location using appropriate generalized least square models based Tukey's HSD post hoc test, p  < 0.05

Soil characteristic in different ecosystems

The soil in Guangxi exhibited significantly lower pH than that in Gansu (Table  1 ), accompanied by lower concentrations of total P and resin P (less than 20% of the levels observed in Gansu). Conversely, the soil total N concentration was higher in Guangxi, although not statistically significant; the concentration of NH 4 + -N in Gansu was 70% higher than that in Guangxi ( p  < 0.05), whereas the soil in Guangxi exhibited a higher level of NO 3 − -N ( p  < 0.05). Although we did not measure soil [Mn], literature data indicates a 1.9-fold higher level of soil [Mn] in Gansu compared with that in Guangxi (Wang et al. 2012 , 2016 ).

Leaf P and Mn concentration in different ecosystems, families and species

In the ecosystem of low-P Guangxi, plants exhibited 79% lower leaf [P] (0.9 mg g −1 , p  < 0.0001, Fig.  2 a), but 51% higher leaf [Mn] (230 mg kg −1 ; p  < 0.0001, Fig.  2 b) compared with those in the ecosystem of relatively P-rich soils of Gansu (1.6 mg g −1 leaf [P], and 113 mg kg −1 leaf [Mn]; Fig.  2 ).

We examined 38 and 56 families in relatively P-rich soils of Maijishan (Gansu) and low-P soils of Damingshan (Guangxi), respectively (Fig.  3 ). Approximately half of the families from relatively P-rich soils (16 out of 38) exhibited significantly higher leaf [Mn] than the reference group, while a substantial majority of the families from low-P soils (46 out of 56) showed significantly elevated leaf [Mn] relative to the negative reference. Leaf [Mn] was generally higher in low-P soils than in relatively P-rich soils (Fig.  3 ). Notably, in low-P soils, the families Altingiaceae, Verbenaceae, Clethraceae, Chloranthaceae, Araliaceae, Melastomataceae, and Selaginellaceae all displayed average leaf [Mn] values exceeding 500 mg kg −1 , while in relatively P-rich soils, only the Fagaceae family exhibited an average leaf [Mn] above this threshold. When compared with the negative reference using a Welch t-test, the p -values were marginal for the Sapindaceae and Lauraceae families (0.052 and 0.051, respectively) in relatively P-rich soils, and for the Anacardiaceae, Juglandaceae, and Lindsaeeacea families (0.051, 0.056, and 0.05, respectively) in low-P soils.

Plant species with the highest leaf [Mn] in two ecosystems were presented in Fig.  4 . In Maijishan, which had a higher soil P concentration, the top 10 species with the highest leaf [Mn] were Quercus spinosa (881 mg kg −1 , Fagaceae), Taxus wallichiana var. chinensis (702 mg kg −1 , Taxaceae), Quercus mongolica (516 mg kg −1 , Fagaceae), Elaeagnus umbellate (442 mg kg −1 , Elaeagnaceae), Symplocos tanakana (344 mg kg −1 , Symplocaceae), Quercus aliena var. acutiserrata (310 mg kg −1 , Fagaceae), Schisandra chinensis (303 mg kg −1 , Schisandraceae), Deutzia grandiflora (247 mg kg −1 , Hydrangeaceae), Litsea pungens (236 mg kg −1 , Lauraceae) and Elaeagnus pungens (178 mg kg −1 , Elaeagnaceae). In Damingshan where soil P concentration was lower than that in Majishan, the top 10 species with the highest leaf [Mn] were Clerodendrum kwangtungense (1506 mg kg −1 , Verbenaceae), Altingia chinensis (1108 mg kg −1 , Altingiaceae), Dendropanax dentiger (897 mg kg −1 , Araliaceae), Selaginella doederleinii (852 mg kg −1 , Selaginellaceae), Clethra delavayi (812 mg kg −1 , Clethraceae), Blastus pauciflorus (756 mg kg −1 , Melastomataceae), Betula austrosinensis (629 mg kg −1 , Betulaceae), Elaeocarpus decipiens (628 mg kg −1 , Elaeocarpaceae), Sarcandra glabra (625 mg kg −1 , Chloranthaceae) and Lophatherum gracile (582 mg kg −1 , Poaceae).

Compared with the top 10 species with the highest leaf [Mn] in relatively P-rich soils of Gansu (with an average of 416 mg Mn kg −1 ) and low-P soils of Guangxi (840 mg Mn kg −1 ), both the maximum and minimum leaf [Mn] in low-P soils were significantly higher than those observed on relatively P-rich soils, respectively. Amongst those top 10 species with highest leaf [Mn], Fagaceae (three species) and Elaeagnaceae (two species) were families most represented in relatively P-rich soils; in contrast, the top 10 highest leaf [Mn] species belonged to 10 different families in low-P soils.

In low-P soils of Guangxi, within the Verbenaceae family, two species demonstrated significant differences in leaf [Mn]: Callicarpa bodinieri exhibited 170 mg Mn kg −1 , while Clerodendrum kwangtungense showed a value of 1500 mg Mn kg −1 . Similarly, among Araliaceae, three species displayed high distinct levels of leaf [Mn], namely Dendropanax dentiger (894 mg Mn kg −1 ), Aralia armata (410 mg Mn kg −1 ), and Heptapleurum delavayi (454 mg Mn kg −1 ). In the Altingiaceae family, only one species was included: Altingia chinensis , and all five replicates exhibited high leaf [Mn], although there was considerable variation, ranging from 826 to 1813 mg Mn kg −1 . Among the seven Poaceae there was considerable variation; Lophatherum gracile and Chimonobambusa damingshanensis had a leaf [Mn] of 582 mg Mn kg −1 and 360 mg Mn kg −1 , respectively. In contrast, Miscanthus floridulus and Setaria palmifolia exhibited low leaf [Mn], 12 mg Mn kg −1 and 24 mg Mn kg −1 , respectively. However, all five Poaceae in relatively P-rich soils of Gansu exhibited relatively low leaf [Mn], with an average concentration of 38 ± 7 (standard error of the average) mg Mn kg −1 .

Leaf Mn concentration as dependent on life form

When plant species were classified into different life forms (Fig.  5 ), in relatively P-rich soils of Gansu, trees exhibited the highest leaf [Mn], while herbs and ferns presented the lowest leaf [Mn]; in contrast, in low-P soils of Guangxi, shrubs demonstrated the highest leaf [Mn], whereas lianas had the lowest leaf [Mn]. Notably, relatively P-rich soils of Gansu showed more pronounced variation among plant life forms compared with relatively smaller differences in low-P soils of Guangxi.

The present findings provide robust support for our hypotheses that leaf [Mn] in P-limited forests of southern China is higher than that in the forests of northern China with higher soil P, despite Gansu having a higher soil [Mn]. Additionally, we observed a more widespread distribution of high leaf [Mn] compared with the negative reference across various plant families in low-P soils than in relatively P-rich soils. This suggests a root carboxylate-release strategy commonly exhibited by plants in response to P limitation in Guangxi forests.

Soil P limitation promotes leaf Mn accumulation

Although soil [Mn] was higher in relatively P-rich soils of Gansu than in low-P soils of Guangxi (Table  1 ), Mn availability can vary. Soils that are more acidic generally exhibit higher Mn availability, while alkaline or highly weathered soils may have a very low Mn availability (Sims 1986 ). Additionally, a high availability of soil P can reduce the uptake and accumulation of Mn in leaves (Pedas et al. 2011 ). This is attributed to high levels of soil P that can induce alterations in soil pH and affect the availability of elements, including Mn. Furthermore, it is well-documented that P and Mn often exhibit antagonistic interactions within the soil environment, leading to mutual inhibition of their respective uptake (Barben et al. 2010 ). To mitigate the confounding effect of soil pH in different locations, the negative control is recommended to be used to standardize leaf [Mn] in response to soil P (Lambers et al. 2021 ). Leaf [Mn] was significantly higher (Fig.  2 ) across various plant families in P-limited Guangxi (Fig.  3 ), indicating enhanced mobilization and absorption of soil Mn by plants. The soil total P and resin P concentration both indicated a more severe P-deficiency in Guangxi than in Gansu (Table  1 ) which is consistent with previous studies (Zhang et al. 2005 ; Li et al. 2015 ; Liu et al. 2022 ). The combination of a lower soil pH, karst land, and higher precipitation in Guangxi exacerbates the severity of P deficiency when compared with that in relatively P-rich soils of Gansu (Zhang et al. 2021 ; Li et al. 2023 ).

Mycorrhizal fungi play a pivotal role in enhancing nutrient acquisition in plants through symbiotic interactions. However, the soil microbial diversity in low-P soils of Guangxi forests is comparatively lower than that at other locations (Zhao et al. 2020 ). Furthermore, the richness of arbuscular mycorrhizal fungi (AMF) exhibits a significant positive correlation with plant-available P and soil pH (Xiao et al. 2019 ). This symbiotic association and nutrient-uptake ability can be suppressed under severe P limitation (Abbott et al. 1984 ; Bolan et al. 1984 ; Treseder and Allen 2002 ; Albornoz et al. 2017 ), despite mycorrhizal symbiosis still playing a vital role in pathogen defense (Guillemin et al. 1994 ; Branzanti et al. 1999 ; Gille et al. 2024 ). In such scenarios, plants may rely on alternative strategies, such as root exudates, for P acquisition. This would partially explain the higher leaf [Mn] widely observed across various plant families in low-P soils of Guangxi.

A high leaf Mn concentration in mature leaves is a proxy for significant root exudation

Manganese accumulation in plants requires more study, as some species, such as Banksia (Proteaceae) and some Eucalyptus species (Myrtaceae) show stronger Mn enrichment characteristics than others in P-deficient conditions (Shane and Lambers 2005 ; Lambers et al. 2021 ; Zhou et al. 2022 ). Several families, including Fagaceae, Symplocaceae, Smilacaceae, Araliaceae, Pinaceae, Cupressaceae exhibited higher leaf [Mn] than the negative references in both relatively P-rich soils and low-P soils. Moreover, many of these species are known to release carboxylates from their roots (Table  2 ). For instance, Quercus species (Fagaceae) in temperate and tropical forests release significant amounts of oxalate from their fine roots (Sun et al. 2017 ; Wang et al. 2021 ; Nottingham et al. 2022 ); while Panax species (Araliaceae) release glycolate, propionate, glycerate and glutarate (Luo et al. 2022 ). Similarly, Picea species (Pinaceae) exude root carboxylates like oxalate and lactate into the rhizosphere (Sandnes et al. 2005 ). Monocarboxylates like lactate and glycolate do not chelate Mn, but they may affect Mn availability in soil when associated with a decrease in rhizosphere pH (Lambers et al. 2021 ).

Some plant families occurred at both locations but only showed significantly higher leaf [Mn] than the negative reference at the low-P location, such as Lauraceae, Asteraceae, Sapindaceae, Polypodiaceae, Fabaceae, Thelypteridaceae, Poaceae and Dennstaedtiaceae. Though previous studies found fern families in general have low leaf [Mn] (Grosjean et al. 2019 ; Schmitt et al. 2017 ), the present study observed that certain fern families (i.e. Thelypteridaceae and Dennstaedtiaceae) also responded to the soil P levels with the values at low-P soils of Guangxi being significantly higher than in relatively P-rich soils of Gansu (Fig.  5 ). This may be attributed to specific growth conditions, although such occurrences are not widespread (Reimann et al. 2007 ), which requires further investigation. The observation of high leaf [Mn] in some species aligns with the previous reports on the exudation of carboxylates from roots. For example, species belonging to Fabaceae and Poaceae are found to release root carboxylates such as citrate, oxalate and malate (Table  2 ). Additionally, species from Lauraceae release citrate (Aoki et al. 2012 ) and other root exudates (Sun et al. 2021 ); Asteraceae species release oxalate (Olivares et al. 2002 ). Interestingly, Cyperaceae showed higher leaf [Mn] than the negative reference in relatively P-rich soils of Gansu, but not in P-limited Guangxi ( p  = 0.07); many species of Cyperaceae produce dauciform roots and release root carboxylates and phosphatases (Playsted et al. 2006 ; Shane et al. 2006 ). The variation among Cyperaceae in P-limited Guangxi might be accounted for by cations other than protons accompanying the release of carboxylates (Roelofs et al. 2001 ; Zhu et al. 2005 ). Exudation of cations like K + and Na + , rather than H + , contributes to maintaining the charge balance during the release of carboxylate anions. However, this affects the rhizosphere pH, and therefore the Mn availability, and subsequent leaf Mn accumulation (Fig.  3 b) (Lambers et al. 2021 ). However, further investigation is required to fully comprehend these effects.

We observed significant variation in leaf [Mn] among certain families, such as Fagaceae, Taxaceae and Elaeagnaceae in relatively P-rich soils of Gansu. Moreover, a greater number of plant families exhibited significant variation in P-limited Guangxi, including Altingiaceae, Verbenaceae, Araliaceae, Melastomataceae, Selaginellaceae, Betulaceae, Zingiberaceae, Elaeocarpaceae, Polypodiaceae and Poaceae. These findings suggest that the capacity to accumulate leaf [Mn] varies within plant families. Additionally, some plants do not respond to low soil P by releasing root exudates and consequently maintain low leaf [Mn] (Lambers et al 2022 ).

Enhancing the perspectives on leaf manganese and root exudate research demands meticulous attention and focus

Investigating leaf [Mn] and root exudation of carboxylates is crucial for comprehending plant nutrient acquisition, rhizosphere dynamics, and plant-microbe interactions. Given the importance of further leaf Mn studies, it is important to note a number of factors affecting soil Mn availability and plant absorption and accumulation. It requires careful selection of the study sites. Climate conditions, soil moisture, and drainage can influence the availability and uptake of Mn by plants. Waterlogged or poorly-drained soils can enhance both Fe and Mn availability; however, plants down-regulate Fe uptake due to the tightly controlled Fe-acquisition mechanisms (Lambers et al. 2021 ), which consequently reduces Mn acquisition as Mn and Fe share the same transporter (Baxter et al. 2008 ). Conversely, dry soils may restrict root uptake and result in reduced leaf Mn accumulation. Soil characteristics such as pH, organic matter content, redox conditions, and [Mn] impact the availability of Mn for plant uptake.

Different plant species exhibit varying capacities for leaf [Mn]. Some plant species possess inherent adaptations for higher levels of [Mn], such as Schima superba (Theaceae), a Mn hyperaccumulator that can reach 10,000 mg Mn kg −1 in leaf in a pot experiment (Yang et al. 2008 ), Larix decidua (Pinaceae), Betula pendula (Betulaceae) and Vaccinium myrtillus (Ericaceae) accumulate 7,000 to 10,000 mg Mn kg −1 in leaf when growing in air-polluted mountain areas (Wildová et al. 2021 ). Certain plants form specialized roots such as cluster root in Proteaceae and many actinorhizal species and dauciform root in some Cyperaceae which enable them to release root carboxylates in an exudative burst thus efficiently mobilizing soil P to cope with P-impoverished environments in Western Australia, South Africa and South America (Neumann and Martinoia 2002 ; Shane et al. 2006 ; Lambers et al. 2015b ). Additionally, an increasing number of species without specialized root systems have been reported to release root exudates for soil P acquisition (Table  2 ); however, it remains largely unknown how widespread this strategy is, globally.

Ericaceae exhibit a symbiosis with ericoid mycorrhizal fungi which is expected to enhance their P-acquisition (Smith et al. 2015 ). Additionally, the mycorrhizal hyphae can intercept mobilized Mn and prevent its accumulation in leaves (Hashem 1995 ). Interestingly, we collected 10 Ericaceae species in P-limited Guangxi, eight of them exhibiting relatively high leaf [Mn] ranging from 200 to 360 mg Mn kg −1 compared with the negative references. Similarly, other Ericaceae species have also been observed to accumulate high leaf Mn, such as Vaccinium myrtillus (Wildová et al. 2021 ), and Leucopogon verticillatus (Zhou et al. 2022 ). Notably, previous research has shown that Vaccinium species possess the ability to release root carboxylates like oxalate and citrate (Millaleo et al. 2020 ). Therefore, our findings suggest that symbiotic fungi did not intercept Mn and that they did not occupy a dominant position in the low-P soils, indicating that the symbiosis was possibly ineffective in facilitating plant P uptake. Instead, we propose that these Ericaceae species in Guangxi, which are limited by soil P availability, instead, rely on root carboxylate exudation for their P acquisition, similar to how some Eucalyptus plants that associate with AM and ECM fungi release root carboxylates to acquire P in severely P-depleted habitats in southwestern Australia (Zhou et al. 2022 ). These findings suggest that further research on leaf Mn dynamics along with investigations on mycorrhizal symbioses and root exudation is warranted, globally.

By integrating field observations (Lambers et al. 2021 ; Zhou et al. 2022 ), controlled-environment experiments (Pang et al. 2018 ; Yu et al. 2023b ), and molecular analyses, we can gain deeper insights into the impact of environmental factors on leaf [Mn] and its correlation with root exudates. Long-term field studies offer valuable perspectives on the temporal and spatial patterns associated with these processes (Wildová et al. 2021 ). Molecular techniques such as transcriptomics, proteomics, and metabolomics can unveil the genetic and molecular mechanisms underlying leaf [Mn] and root carboxylate exudation (Sharma and Jha 2023 ; Yu et al. 2023a ). Implementing these strategies will advance our understanding of leaf [Mn] and root carboxylate exudation to acquire deeper insights into plant nutrient-acquisition strategies and their ecological significance.

This study investigated leaf [Mn] in two forest ecosystems characterized by contrasting soil P availability. We observed that plants growing in low-P soils of Guangxi exhibited significantly higher average leaf [Mn] compared with those in relatively P-rich soils of Gansu, despite the fact that Gansu had higher soil [Mn]. By utilizing the same plant family, i.e. Dryopteridaceae, as a negative reference, the results indicated a greater number of species with high leaf [Mn] in low-P soils than in relatively P-rich soils. This trend was further confirmed across different plant families and life forms, suggesting that high leaf [Mn] is more prevalent in low-P soils. Furthermore, we synthesized literature data to demonstrate that a substantial proportion of plants with high leaf [Mn] also release significant amounts of root carboxylates, thereby establishing mature leaf [Mn] as a reliable proxy for root carboxylate release under field conditions. The utilization of root carboxylates for soil P acquisition represents a common strategy exhibited by plants inhabiting P-limited environments.

Data availability

The data that support the findings of this study are available now in [Figshare]: https://figshare.com/s/3cfbdbc16874ab7e9af8 .

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Acknowledgements

We thank Gansu Forestry Technological College, Guangxi Damingshan National Nature Reserve Administration Bureau, Yanlin Zhang, Juyuan Wang, Keming Pan, Xiongwen Yu, Wei Wang and Jinqi Zhang for the assistance in plant identification and sample collection.

Open Access funding enabled and organized by CAUL and its Member Institutions. This study was financially supported by funds from Natural Science Foundation of Gansu Province, China (22JR5RA530) and the Fundamental Research Funds for the Central Universities (lzujbky-2021-pd07), Li Yan was supported by the International Postdoctoral Exchange Fellowship Program (PC2022030) by the Office of China Postdoctoral Council.

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Hans Lambers and Li Yan conceived the study. Li Yan and Dan Tang carried out the lab analyses. Li Yan finished statistical analyses and wrote the first draft of the manuscript, Hans Lambers, Jiayin Pang and Dan Tang contributed to revisions.

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Yan, L., Tang, D., Pang, J. et al. Root carboxylate release is common in phosphorus-limited forest ecosystems in China: using leaf manganese concentration as a proxy. Plant Soil (2024). https://doi.org/10.1007/s11104-024-06791-8

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Frumin AM, Nussbaum J, Esposito M. Functional asplenia: demonstration of splenic activity by bone marrow scan. Blood 1979;59 Suppl 1:26-32.

Book chapter, or an article within a book

Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. In: Bourne GH, Danielli JF, Jeon KW, editors. International review of cytology. London: Academic; 1980. p. 251-306.

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Saito Y, Hyuga H. Rate equation approaches to amplification of enantiomeric excess and chiral symmetry breaking. Top Curr Chem. 2007. doi:10.1007/128_2006_108.

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Blenkinsopp A, Paxton P. Symptoms in the pharmacy: a guide to the management of common illness. 3rd ed. Oxford: Blackwell Science; 1998.

Online document

Doe J. Title of subordinate document. In: The dictionary of substances and their effects. Royal Society of Chemistry. 1999. http://www.rsc.org/dose/title of subordinate document. Accessed 15 Jan 1999.

Online database

Healthwise Knowledgebase. US Pharmacopeia, Rockville. 1998. http://www.healthwise.org. Accessed 21 Sept 1998.

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Doe J. Title of supplementary material. 2000. http://www.privatehomepage.com. Accessed 22 Feb 2000.

University site

Doe, J: Title of preprint. http://www.uni-heidelberg.de/mydata.html (1999). Accessed 25 Dec 1999.

Doe, J: Trivial HTTP, RFC2169. ftp://ftp.isi.edu/in-notes/rfc2169.txt (1999). Accessed 12 Nov 1999.

Organization site

ISSN International Centre: The ISSN register. http://www.issn.org (2006). Accessed 20 Feb 2007.

Dataset with persistent identifier

Zheng L-Y, Guo X-S, He B, Sun L-J, Peng Y, Dong S-S, et al. Genome data from sweet and grain sorghum (Sorghum bicolor). GigaScience Database. 2011. http://dx.doi.org/10.5524/100012 .

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