Model Grass Species Research Articles

Genome-wide identification and expression analysis of SBP-like transcription factor genes in Moso Bamboo (Phyllostachys edulis)

BackgroundThe SQUAMOSA promoter binding protein-like (SPL) proteins are plant-specific transcription factors (TFs) that function in a variety of developmental processes including growth, flower development, and signal transduction. SPL proteins are encoded by a gene family, and these genes have been characterized in two model grass species, Zea mays and Oryza sativa. The SPL gene family has not been well studied in moso bamboo (Phyllostachys edulis), a woody grass species.ResultsWe identified 32 putative PeSPL genes in the P. edulis genome. Phylogenetic analysis arranged the PeSPL protein sequences in eight groups. Similarly, phylogenetic analysis of the SBP-like and SBP proteins from rice and maize clustered them into eight groups analogous to those from P. edulis. Furthermore, the deduced PeSPL proteins in each group contained very similar conserved sequence motifs. Our analyses indicate that the PeSPL genes experienced a large-scale duplication event ~15 million years ago (MYA), and that divergence between the PeSPL and OsSPL genes occurred 34 MYA. The stress-response expression profiles and tissue-specificity of the putative PeSPL gene promoter regions showed that SPL genes in moso bamboo have potential biological functions in stress resistance as well as in growth and development. We therefore examined PeSPL gene expression in response to different plant hormone and drought (polyethylene glycol-6000; PEG) treatments to mimic biotic and abiotic stresses. Expression of three (PeSPL10, −12, −17), six (PeSPL1, −10, −12, −17, −20, −31), and nine (PeSPL5, −8, −9, −14, −15, −19, −20, −31, −32) genes remained relatively stable after treating with salicylic acid (SA), gibberellic acid (GA), and PEG, respectively, while the expression patterns of other genes changed. In addition, analysis of tissue-specific expression of the moso bamboo SPL genes during development showed differences in their spatiotemporal expression patterns, and many were expressed at high levels in flowers and leaves.ConclusionsThe PeSPL genes play important roles in plant growth and development, including responses to stresses, and most of the genes are expressed in different tissues. Our study provides a comprehensive understanding of the PeSPL gene family and may enable future studies on the function and evolution of SPL genes in moso bamboo.

The Brachypodium distachyon methionine sulfoxide reductase gene family

The methionine sulfoxide reductases (MSRs) are a group of thiol-dependent enzymes able to catalyze the conversion of methionine sulfoxide to methionine. Although some plant MSRs are known to act as protectants against various abiotic stresses, their activity in the model grass species Brachypodium distachyon has not been characterized as yet. Here, six B. distachyon MSR (BdMSR) genes have been isolated; they generate eight distinct cDNAs, since two of them (BdMSRB1 and -B5) produce a pair of alternatively spliced messages. The genes were transcribed in the root, culm, leaf and during various stages of caryopsis development. Those induced by abiotic stress (salinity, drought, low temperature, CdCl2, H2O2 and abscisic acid) harbored known stress-responsive cis elements in their promoter sequences. The heterologous expression of five of the BdMSRs (-A2, -A4, -B1.1, -B3 and -B5.1) in yeast revealed that their products gave a measure of protection against salinity, mannitol and oxidative stress. Substrate specificity analysis revealed that BdMSRB1.1 could reduce free Met-R-SO to Met. The enzymatic activities of BdMSRA4, -B1.1 and -B5.1 in transformed yeast under salt treatment have checked and increased obviously resulting in reducing more Met-SO to Met including the peptide and the free types under salt stress than those in control.

Brachypodium distachyon - A Useful Model in the Qualification of Mutagen-Induced Micronuclei Using Multicolor FISH.

Brachypodium distachyon (Brachypodium) is now intensively utilized as a model grass species in various biological studies. Its favorable cytological features create a unique foundation for a convenient system in mutagenesis, thereby potentially enabling the ‘hot spots’ and ‘cold spots’ of DNA damage in its genome to be analyzed. The aim of this study was to analyze the involvement of 5S rDNA, 25S rDNA, the Arabidopsis-type (TTTAGGG)n telomeric sequence and the Brachypodium-originated centromeric BAC clone CB33J12 in the micronuclei formation in Brachypodium root tip cells that were subjected to the chemical clastogenic agent maleic hydrazide (MH). To the best of our knowledge, this is the first use of a multicolor fluorescence in situ hybridization (mFISH) with four different DNA probes being used simultaneously to study plant mutagenesis. A quantitative analysis allowed ten types of micronuclei, which were characterized by the presence or absence of specific FISH signal(s), to be distinguished, thus enabling some specific rules governing the composition of the MH-induced micronuclei with the majority of them originating from the terminal regions of chromosomes, to be identified. The application of rDNA sequences as probes showed that 5S rDNA-bearing chromosomes are involved in micronuclei formation more frequently than the 25S rDNA-bearing chromosomes. These findings demonstrate the promising potential of Brachypodium to be a useful model organism to analyze the effects of various genotoxic agents on the plant nuclear genome stability, especially when the complex FISH-based and chromosome-specific approaches such as chromosome barcoding and chromosome painting will be applied in future studies.

Role of bifunctional ammonia-lyase in grass cell wall biosynthesis.

L-Phenylalanine ammonia-lyase (PAL) is the first enzyme in the biosynthesis of phenylpropanoid-derived plant compounds such as flavonoids, coumarins and the cell wall polymer lignin. The cell walls of grasses possess higher proportions of syringyl (S)-rich lignins and high levels of esterified coumaric acid compared with those of dicotyledonous plants, and PAL from grasses can also possess tyrosine ammonia-lyase (TAL) activity, the reason for which has remained unclear. Using phylogenetic, transcriptomic and in vitro biochemical analyses, we identified a single homotetrameric bifunctional ammonia-lyase (PTAL) among eight BdPAL enzymes in the model grass species Brachypodium distachyon. (13)C isotope labelling experiments along with BdPTAL1-downregulation in transgenic plants showed that the TAL activity of BdPTAL1 can provide nearly half of the total lignin deposited in Brachypodium, with a preference for S-lignin and wall-bound coumarate biosynthesis, indicating that PTAL function is linked to the characteristic features of grass cell walls. Furthermore, isotope dilution experiments suggest that the pathways to lignin from L-phenylalanine and L-tyrosine are distinct beyond the formation of 4-coumarate, supporting the organization of lignin synthesis enzymes in one or more metabolons.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana. However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium.

The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots.

Quantitative trait loci associated with natural diversity in water-use efficiency and response to soil drying in Brachypodium distachyon

All plants must optimize their growth with finite resources. Water use efficiency (WUE) measures the relationship between biomass acquisition and transpired water. In the present study, we performed two experiments to understand the genetic basis of WUE and other parameters of plant-water interaction under control and water-limited conditions. Our study used two inbred natural accessions of Brachypodium distachyon, a model grass species with close phylogenetic affinity to temperate forage and cereal crops. First, we identify the soil water content which causes a reduction in leaf relative water content and an increase in WUE. Second, we present results from a large phenotyping experiment utilizing a recombinant inbred line mapping population derived from these same two natural accessions. We identify QTLs associated with environmentally-insensitive genetic variation in WUE, including a pair of epistatically interacting loci. We also identify QTLs associated with constitutive differences in biomass and a QTL describing an environmentally-sensitive difference in leaf carbon content. Finally, we present a new linkage map for this mapping population based on new SNP markers as well as updated genomic positions for previously described markers. Our studies provide an initial characterization of plant-water relations in B. distachyon and identify candidate genomic regions involved in WUE.

CG Methylation Covaries with Differential Gene Expression between Leaf and Floral Bud Tissues of Brachypodium distachyon.

DNA methylation has the potential to influence plant growth and development through its influence on gene expression. To date, however, the evidence from plant systems is mixed as to whether patterns of DNA methylation vary significantly among tissues and, if so, whether these differences affect tissue-specific gene expression. To address these questions, we analyzed both bisulfite sequence (BSseq) and transcriptomic sequence data from three biological replicates of two tissues (leaf and floral bud) from the model grass species Brachypodium distachyon. Our first goal was to determine whether tissues were more differentiated in DNA methylation than explained by variation among biological replicates. Tissues were more differentiated than biological replicates, but the analysis of replicated data revealed high (>50%) false positive rates for the inference of differentially methylated sites (DMSs) and differentially methylated regions (DMRs). Comparing methylation to gene expression, we found that differential CG methylation consistently covaried negatively with gene expression, regardless as to whether methylation was within genes, within their promoters or even within their closest transposable element. The relationship between gene expression and either CHG or CHH methylation was less consistent. In total, CG methylation in promoters explained 9% of the variation in tissue-specific expression across genes, suggesting that CG methylation is a minor but appreciable factor in tissue differentiation.

Genome-wide analysis of response to low sulfur (LSU) genes in grass species and expression profiling of model grass species Brachypodium distachyon under S deficiency

Sulfur (S) affects the plant life cycle and crop yield and has nutritional importance for human and animal diet. Its deficiency is one of the major problems in agriculture. However, the plant-specific LSU (response to Low Sulfur) gene family has not been extensively analyzed in major plant species such as grasses. In this study, we have performed in silico genome-wide analysis of LSU genes in 6 grass species, including Brachypodium distachyon, Sorghum bicolor, Oryza sativa, Zea mays, Triticum aestivum, and Panicum virgatum. All identified LSU genes contained one exon encoding proteins of acidic character with cytoplasmic localization. In silico analysis of cis-elements revealed that sulfur-responsive elements (SURE boxes, Sulfur Response Element, GAGAC motif) were present in all LSU genes. In phylogenetic analysis, dicot and monocot LSU genes were separated. Expression profiles of B. Distachyon BdLSU1 and BdLSU2 genes were analyzed by qRT-PCR method. Two Brachypodium LSU genes demonstrated different expression patterns when subjected to 48 h of S-depletion treatment. In roots, the BdLSU2 gene was upregulated, while BdLSU1 was downregulated. In leaves, expression levels were decreased for both genes. Analysis of the BdLSU expression under drought, cold, salt, and heat stresses was carried out based on the Brachypodium stress atlas. Results showed that BdLSU genes are not specific to S limitation; indeed, they may be involved in different stress conditions by cooperating with their interacting partner proteins. The results of this study could significantly contribute to the understanding of LSU genes in plants, particularly in grass species. These results may also support plant molecular studies by aiding the understanding of the sulfur assimilation pathway.

The cytoplasmic localization of the catalytic site of CSLF6 supports a channeling model for the biosynthesis of mixed-linkage glucan.

Mixed-linkage glucan (MLG) is a significant cell wall carbohydrate in grasses and an important carbon source for human consumption and biofuel production. MLG biosynthesis depends on the biochemical activity of membrane spanning glucan synthases encoded by the CSLH and CSLF cellulose synthase-like gene families. CSLF proteins are the best characterized to date but relatively little information is known about their topology with respect to the biosynthetic membranes. In this study, we report on the topology of CSLF6 protein derived from the model grass species Brachypodium distachyon (BdCSLF6) when it is expressed in heterologous systems. Using live cell imaging and immuno-electron microscopy analyses of tobacco epidermal cells expressing BdCSLF6, we demonstrate that a functional yellow fluorescent protein (YFP) fusion of BdCSLF6 is localized to the Golgi apparatus and that the Golgi localization of BdCSLF6 is sufficient for MLG biosynthesis. By implementing protease protection assays of BdCSLF6 expressed in the yeast Pichia pastoris, we also demonstrate that the catalytic domain, the N-terminus and the C- terminus of the protein are exposed in the cytosol. Furthermore, we found that BdCSLF6 is capable of producing MLG not only in tobacco cells but also in Pichia, which generally does not produce MLG. Together, these results support the conclusion that BdCSLF6 can produce both of the linkages present in the (1,3;1,4)-β-d-glucan chain of MLG and that the product is channelled at the Golgi into the secretory pathway for deposition into the cell wall.

Gene functional analysis using protoplast transient assays.

The protoplast transient assay system has been widely used for rapid functional analyses of genes using cellular and biochemical approaches. This system has been increasingly employed for functional genetic studies using double-stranded (ds) RNA interference (RNAi). Here, we describe a modified procedure for the isolation of protoplasts from leaf mesophyll cells of 14-day-old Arabidopsis thaliana. This modification significantly simplifies and speeds up functional studies without compromising the yield and the viability of protoplasts. We also present the procedure for the isolation and transfection of protoplasts from mesophyll cells of an emerging model grass species, Brachypodium distachyon. Further, we detail procedures for RNAi-based functional studies of genes using transient expression of in vitro synthesized dsRNA in protoplasts.

Methods to promote germination of dormant Setaria viridis seeds.

Setaria viridis has recently emerged as a promising genetic model system to study diverse aspects of monocot biology. While the post-germination life cycle of S. viridis is approximately 8 weeks long, the prolonged dormancy of freshly harvested seeds can more than double the total time required between successive generations. Here we describe methods that promote seed germination in S. viridis. Our results demonstrate that treating S. viridis seeds with liquid smoke or a GA3 and KNO3 solution improves germination rates to 90% or higher even in seeds that are 6 days post-harvest with similar results obtained whether seeds are planted in soil or on gel-based media. Importantly, we show that these treatments have no significant effect on the growth of the adult plant. We have tested these treatments on diverse S. viridis accessions and show variation in their response. The methods described here will help advance research using this model grass species by increasing the pace at which successive generations of plants can be analyzed.

Molecular Control of Grass Inflorescence Development

The grass family is one of the largest families in angiosperms and has evolved a characteristic inflorescence morphology, with complex branches and specialized spikelets. The origin and development of the highly divergent inflorescence architecture in grasses have recently received much attention. Increasing evidence has revealed that numerous factors, such as transcription factors and plant hormones, play key roles in determining reproductive meristem fate and inflorescence patterning in grasses. Moreover, some molecular switches that have been implicated in specifying inflorescence shapes contribute significantly to grain yields in cereals. Here, we review key genetic and molecular switches recently identified from two model grass species, rice (Oryza sativa) and maize (Zea mays), that regulate inflorescence morphology specification, including meristem identity, meristem size and maintenance, initiation and outgrowth of axillary meristems, and organogenesis. Furthermore, we summarize emerging networks of genes and pathways in grass inflorescence morphogenesis and emphasize their evolutionary divergence in comparison with the model eudicot Arabidopsis thaliana. We also discuss the agricultural application of genes controlling grass inflorescence development.

Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties

Fructans can act as cryoprotectants and contribute to freezing tolerance in plant species, such as in members of the grass subfamily Pooideae that includes Triticeae species and forage grasses. To elucidate the relationship of freezing tolerance, carbohydrate composition and degree of polymerization (DP) of fructans, we generated transgenic plants in the model grass species Brachypodium distachyon that expressed cDNAs for sucrose:fructan 6-fructosyltransferases (6-SFTs) with different enzymatic properties: one cDNA encoded PpFT1 from timothy grass (Phleum pratense), an enzyme that produces high-DP levans; a second cDNA encoded wft1 from wheat (Triticum aestivum), an enzyme that produces low-DP levans. Transgenic lines expressing PpFT1 and wft1 showed retarded growth; this effect was particularly notable in the PpFT1 transgenic lines. When grown at 22°C, both types of transgenic line showed little or no accumulation of fructans. However, after a cold treatment, wft1 transgenic plants accumulated fructans with DP=3-40, whereas PpFT1 transgenic plants accumulated fructans with higher DPs (20 to the separation limit). The different compositions of the accumulated fructans in the two types of transgenic line were correlated with the differences in the enzymatic properties of the overexpressed 6-SFTs. Transgenic lines expressing PpFT1 accumulated greater amounts of mono- and disaccharides than wild type and wft1 expressing lines. Examination of leaf blades showed that after cold acclimation, PpFT1 overexpression increased tolerance to freezing; by contrast, the freezing tolerance of the wft1 expressing lines was the same as that of wild type plants. These results provide new insights into the relationship of the composition of water-soluble carbohydrates and the DP of fructans to freezing tolerance in plants.

Green leaf volatiles, fire and nonanoic acid activate MAPkinases in the model grass species Lolium temulentum.

BackgroundPreviously it has been shown that mechanical wounding, salinity and heat activated a 46 kDa and 44 kDa mitogen-activated protein kinases (MAPKs) in forage related grasses. Forage and turf related grasses are utilized in diverse environments where they are routinely subjected to herbicides and exposed to fire and volatiles after cutting, however very little is known concerning the perception or molecular responses to these different stresses or compounds.ResultsIn the model grass species Lolium temulentum (Lt), a 46 kDa mitogen-activated protein kinase (MAPK) was activated in the leaves within 5 min and a 44 kDa MAPK 15 min after exposure to green leaf volatiles released from grass clippings. When the tips of leaves of Lt plants were scorched by fire, the 46 kDa MAPK and 44 kDa MAPK were rapidly activated within 5 min and 20 min respectively in the treated leaf, and 15 min systemically in an adjacent untreated tiller after exposure to fire. Nonanoic acid (pelargonic acid), a component in herbicides used on grasses, activated a 46 kDa MAPK in the treated leaves within 5 min of exposure and 15 min in systemic tissues. At concentrations normally used in the herbicides, nonanoic acid was found to only weakly activate the 44 kDa MAPK after an hour in treated leaves, but strongly activated it in the systemic tillers 30 min after treatment. Acetic acid, HCl and NaOH also were found to activate these MAPKs in treated tillers.ConclusionThe rapid activation of these MAPKs to a wide range of stress stimuli, suggest that these MAPKs play a role in the perception and response to these stresses and compounds. The activation of the MAPK by green leaf volatiles indicates a role for these compounds in wound signaling in grasses.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-807) contains supplementary material, which is available to authorized users.

Exploring the interaction between small RNAs and R genes during Brachypodium response to Fusarium culmorum infection

The present study aims to investigate small RNA interactions with putative disease response genes in the model grass species Brachypodium distachyon. The fungal pathogen Fusarium culmorum (Fusarium herein) and phytohormone salicylic acid treatment were used to induce the disease response in Brachypodium. Initially, 121 different putative disease response genes were identified using bioinformatic and homology based approaches. Computational prediction was used to identify 33 candidate new miRNA coding sequences, of which 9 were verified by analysis of small RNA sequence libraries. Putative Brachypodium miRNA target sites were identified in the disease response genes, and a subset of which were screened for expression and possible miRNA interactions in 5 different Brachypodium lines infected with Fusarium. An NBS-LRR family gene, 1g34430, was polymorphic among the lines, forming two major genotypes, one of which has its miRNA target sites deleted, resulting in altered gene expression during infection. There were siRNAs putatively involved in regulation of this gene, indicating a role of small RNAs in the B. distachyon disease response.

Gene transfer: A tool for the functional analysis of the rice genome

Rice (Oryza sativa L.) has the dual status of being a model grass species and the main cereal for human consumption. Elucidating the function of genes underlying agronomic traits in the first model crop will contribute to hastening plant breeding not only in rice but also in other cereals. Gene transfer is a tool that has the potential to accelerate both discovery and innovation. The first transgenic rice plant was produced in 1988, and the technologies have since then improved towards more efficiency and precision. We here present the main milestones in the evolution of the technologies and their applications for the functional analysis of rice genes. In particular, we show the prospects opened by on-going technological breakthroughs that include the targeted modification of genomes without integration of superfluous DNA.

The physical map of wheat chromosome 1BS provides insights into its gene space organization and evolution

BackgroundThe wheat genome sequence is an essential tool for advanced genomic research and improvements. The generation of a high-quality wheat genome sequence is challenging due to its complex 17 Gb polyploid genome. To overcome these difficulties, sequencing through the construction of BAC-based physical maps of individual chromosomes is employed by the wheat genomics community. Here, we present the construction of the first comprehensive physical map of chromosome 1BS, and illustrate its unique gene space organization and evolution.ResultsFingerprinted BAC clones were assembled into 57 long scaffolds, anchored and ordered with 2,438 markers, covering 83% of chromosome 1BS. The BAC-based chromosome 1BS physical map and gene order of the orthologous regions of model grass species were consistent, providing strong support for the reliability of the chromosome 1BS assembly. The gene space for chromosome 1BS spans the entire length of the chromosome arm, with 76% of the genes organized in small gene islands, accompanied by a two-fold increase in gene density from the centromere to the telomere.ConclusionsThis study provides new evidence on common and chromosome-specific features in the organization and evolution of the wheat genome, including a non-uniform distribution of gene density along the centromere-telomere axis, abundance of non-syntenic genes, the degree of colinearity with other grass genomes and a non-uniform size expansion along the centromere-telomere axis compared with other model cereal genomes. The high-quality physical map constructed in this study provides a solid basis for the assembly of a reference sequence of chromosome 1BS and for breeding applications.

The perennial ryegrass GenomeZipper: targeted use of genome resources for comparative grass genomics.

Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources have yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne) genome on the basis of conserved synteny to barley (Hordeum vulgare) and the model grass genome Brachypodium (Brachypodium distachyon) as well as rice (Oryza sativa) and sorghum (Sorghum bicolor). A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macrocollinearity and was then utilized to anchor a collection of perennial ryegrass genes in silico to their predicted genome positions. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci, which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species.

Abiotic stresses activate a MAPkinase in the model grass species Lolium temulentum

Forage and turf grasses are utilized in diverse environments that expose them to a variety of abiotic stresses, however very little is known concerning the perception or molecular responses to these various stresses. In the model grass species Lolium temulentum, a 46kDa mitogen-activated protein kinase (MAPK) was activated in the leaf within 10min of exposing the roots to salt stress. When plants were subjected cold stress, no significant activation of the MAPK was observed. However, the 46kDa MAPK was rapidly activated in the leaves of plants within 3min of exposure to heat stress. Previously, mechanical wounding has been shown to rapidly activate a 46kDa and a 44kDa MAPK in L. temulentum. The wound activation of the MAPKs was delayed and diminished in plants undergoing cold treatment. In plants subjected simultaneously to 40°C and wounding, the activation of the 46kDa MAPK was enhanced. However if plants were subjected to heat and cold stress for more than 2h or exposed to 300mM NaCl for 24h prior to wounding, the wound activation of the 46kDa and a 44kDa MAPKs were significantly inhibited. These results suggest that the 46kDa MAPK plays a role in the response to various environmental stimuli.