Arabidopsis Ecotypes Research Articles

Small RNA-Directed Epigenetic Natural Variation in Arabidopsis thaliana

Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA) is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of ∼24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler) could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC), a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col) with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of ∼24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42) were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes.

VERTICILLIUM DAHLIAE TOXINS INDUCE CHANGES IN EXPRESSION OF A PUTATIVE HISTONE H2B AND A PUMILIO/PUF RNA-BINDING PROTEIN IN ARABIDOPSIS THALIANA

To gain insight into the molecular mechanisms of plant defence response to Verticillium dahliae, we identified the genes whose expression patterns were altered during early response to V. dahliae toxins (VD-toxins) in Arabidopsis seedlings using the mRNA differential display approach. Four true positive bands (DD1, DD2, DD3 and DD4) were confirmed from the 30 differentially displayed bands. The deduced nucleotide sequence of the DD1 and DD2 clones showed strong homology with genes encoding a putative histone H2B and a pumilio /Puf RNA-binding protein, respectively. Quantitative real-time RT-PCR assays suggested that the expression pattern of genes of DD1 and DD2 in two ecotypes of Arabidopsis were clearly different. The results indicate that the putative histone H2B and pumilio /Puf RNA-binding protein encoded by DD1 and DD2 genes may be involved in regulation of the defence responses against VD-toxins in Arabidopsis and the pumilio/Puf RNA-binding protein plays a more important role in the defence responses.

Indole Glucosinolates and Camalexin do not Influence the Development of the Clubroot Disease in Arabidopsis thaliana

AbstractRoot galls of Brassicaceae caused by Plasmodiophora brassicae are dependent on increased auxin and cytokinin formation. In this study we investigated whether indole glucosinolates are involved in indole‐3‐acetic acid (IAA) biosynthesis in root galls, by using a genetic approach. The cytochrome P450 enzymes, CYP79B2 and CYP79B3, convert tryptophan to indole‐3‐acetaldoxime (IAOx), which is a precursor for indole glucosinolates and the phytoalexin camalexin in Arabidopsis thaliana. Root galls of the Arabidopsis ecotypes Wassilewskija (WS) and Columbia (Col) accumulated camalexin, WS at levels up to 320 μg/g dry weight. By contrast, camalexin was absent in root galls of cyp79b2/b3 double mutants. Infection rate and disease index as a measure of club development in mutant and wild‐type plants of the two ecotypes were investigated and no differences were found in gall formation. This demonstrates that camalexin is an ineffective inhibitor of P. brassicae and indole glucosinolates are not the source of elevated levels of IAA in galls, because free IAA levels in mutant galls were comparable with those in wild type.

CRT1, an Arabidopsis ATPase that Interacts with Diverse Resistance Proteins and Modulates Disease Resistance to Turnip Crinkle Virus

Plant immunity frequently involves the recognition of pathogen-encoded avirulence (avr) factors by their corresponding plant resistance (R) proteins. This triggers the hypersensitive response (HR) where necrotic lesions formed at the site(s) of infection help restrict pathogen spread. HRT is an Arabidopsis R protein required for resistance to turnip crinkle virus (TCV). In a genetic screen for mutants compromised in the recognition of TCV's avr factor, we identified crt1 (compromised recognition of TCV), a mutant that prematurely terminates an ATPase protein. Following TCV infection, crt1 developed a spreading HR and failed to control viral replication and spread. crt1 also suppressed HR-like cell death induced by ssi4, a constitutively active R protein, and by Pseudomonas syringae carrying avrRpt2. Furthermore, CRT1 interacts with HRT, SSI4, and two other R proteins, RPS2 and Rx. These data identify CRT1 as an important mediator of defense signaling triggered by distinct classes of R proteins.

Characterization of Natural and Induced Variation in the LOV1 Gene, a CC-NB-LRR Gene Conferring Victorin Sensitivity and Disease Susceptibility in Arabidopsis

The fungus Cochliobolus victoriae, the causal agent of Victoria blight, produces a compound called victorin that is required for pathogenicity of the fungus. Victorin alone reproduces disease symptoms on sensitive plants. Victorin sensitivity and susceptibility to C. victoriae were originally described on oats but have since been identified on Arabidopsis thaliana. Victorin sensitivity and disease susceptibility in Arabidopsis are conferred by LOV1, a coiled-coil-nucleotide-binding-leucine-rich repeat (CC-NB-LRR) protein. We sequenced the LOV1 gene from 59 victorin-insensitive mutants and found that the spectrum of mutations causing LOV1 loss of function was similar to that found to cause loss of function of RPM1, a CC-NB-LRR resistance protein. Also, many of the mutated residues in LOV1 are in conserved motifs required for resistance protein function. These data indicate that LOV1 may have a mechanism of action similar to resistance proteins. Victorin sensitivity was found to be the prevalent phenotype in a survey of 30 Arabidopsis ecotypes, and we found very little genetic variation among LOV1 alleles. As selection would not be expected to preserve a functional LOV1 gene to confer victorin sensitivity and disease susceptibility, we propose that LOV1 may function as a resistance gene to a naturally-occurring pathogen of Arabidopsis.

Salinity tolerance of Arabidopsis: a good model for cereals?

Arabidopsis is a glycophyte species that is sensitive to moderate levels of NaCl. Arabidopsis offers unique benefits to genetic and molecular research and has provided much information about both Na(+) transport processes and Na(+) tolerance. A compilation of data available on Na(+) accumulation and Na(+) tolerance in Arabidopsis is presented, and comparisons are made with several crop plant species. The relationship between Na(+) tolerance and Na(+) accumulation is different in Arabidopsis and cereals, with an inverse relationship often found within cereal species that is not as evident in Arabidopsis ecotypes. Results on salinity tolerance obtained in Arabidopsis should therefore be extrapolated to cereals with caution. Arabidopsis remains a useful model to study and discover plant Na(+) transport processes.

Defense AgainstSclerotinia sclerotioruminArabidopsisIs Dependent on Jasmonic Acid, Salicylic Acid, and Ethylene Signaling

Genotypic differences in susceptibility of Arabidopsis thaliana to Sclerotinia sclerotiorum have not been reported due to the extreme susceptibility of this cruciferous plant. To overcome this limitation, we have established inoculation conditions that enable evaluation of differences in susceptibility to S. sclerotiorum among Arabidopsis mutants and ecotypes. Two coil mutant alleles conferred hypersusceptibility to S. sclerotiorum. The plant defensin gene PDF1.2 was no longer induced after challenging the coi1-2 mutant with S. sclerotiorum. Hypersusceptibility of the coi1-2 mutant to S. sclerotiorum was not correlated with oxalate sensitivity. The mutants npr1 and ein2 were also hypersusceptible to S. sclerotiorum. Induction of PDF1.2 and the pathogenesis-related gene PR1 was reduced in ein2 and npr1 mutants, respectively. Actigard, a commercial formulation of the systemic acquired resistance inducer benzothiadiazole, reduced susceptibility to S. sclerotiorum. Based on histochemical analysis of oxalate-deficient and wild-type strains of S. sclerotiorum, oxalate caused a decrease in hydrogen peroxide production but no detectable changes in plant superoxide production or gene expression.

Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis

BackgroundNucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes.ResultsWe analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing was found for at least 12 genes, 11 of which encode TIR-NBS-LRR proteins. There was no obvious correlation between expression pattern, phylogenetic relationship or genomic location of the NBS-LRR-encoding and related genes studied.ConclusionTranscripts of many NBS-LRR-encoding and related genes were defined. Most were present at low levels and exhibited tissue-specific expression patterns. Expression data are consistent with most Arabidopsis NBS-LRR-encoding and related genes functioning in plant defense responses but do not preclude other biological roles.

The rules of engagement

The rules of engagement

Identification and Mutational Analysis of Arabidopsis FLS2 Leucine-Rich Repeat Domain Residues That Contribute to Flagellin Perception

Mutational, phylogenetic, and structural modeling approaches were combined to develop a general method to study leucine-rich repeat (LRR) domains and were used to identify residues within the Arabidopsis thaliana FLAGELLIN-SENSING2 (FLS2) LRR that contribute to flagellin perception. FLS2 is a transmembrane receptor kinase that binds bacterial flagellin or a flagellin-based flg22 peptide through a presumed physical interaction within the FLS2 extracellular domain. Double-Ala scanning mutagenesis of solvent-exposed beta-strand/beta-turn residues across the FLS2 LRR domain identified LRRs 9 to 15 as contributors to flagellin responsiveness. FLS2 LRR-encoding domains from 15 Arabidopsis ecotypes and 20 diverse Brassicaceae accessions were isolated and sequenced. FLS2 is highly conserved across most Arabidopsis ecotypes, whereas more diversified functional FLS2 homologs were found in many but not all Brassicaceae accessions. flg22 responsiveness was correlated with conserved LRR regions using Conserved Functional Group software to analyze structural models of the LRR for diverse FLS2 proteins. This identified conserved spatial clusters of residues across the beta-strand/beta-turn residues of LRRs 12 to 14, the same area identified by the Ala scan, as well as other conserved sites. Site-directed randomizing mutagenesis of solvent-exposed beta-strand/beta-turn residues across LRRs 9 to 15 identified mutations that disrupt flg22 binding and showed that flagellin perception is dependent on a limited number of tightly constrained residues of LRRs 9 to 15 that make quantitative contributions to the overall phenotypic response.

Transcriptional Regulation of NADP-Dependent Malate Dehydrogenase: Comparative Genetics and Identification of DNA-Binding Proteins

The transcriptional regulation of NADP-malate dehydrogenase (NADP-MDH) was analyzed in Arabidopsis ecotypes and other Brassicaceae. The amount of transcript increased twofold after transfer into low temperature (12 degrees C) or high light (750 microE) in all species. Analysis of the genomic DNA reveals that the NADP-MDH gene (At5g58330 in A. thaliana) in Brassicaceae is located between two other genes (At5g58320 and At5g58340 in Arabidopsis), both encoded on the opposite DNA strand. No promoter elements were identified in 5' direction of the NADP-MDH gene, and the expression of NADP-MDH was not affected in knock-out plants carrying a DNA insert in the 5' region. A yeast-one hybrid approach yielded only three DNA-binding proteins for the 500-bp fragment located upstream of the ATG sequence, but 34 proteins for its coding region. However, in Chlamydomonas and in some Poaceae, which do not possess any genes within the 1200 bp upstream region, typical promoter elements were identified. Alignments of genomic DNA reveal that, in contrast to Poaceae, the introns are highly conserved within Brassicaceae. We conclude that in Brassicaceae the majority of regulatory elements are located within the coding region. The NADP-MDH gene of both families evolved from a common precursor, similar to the gene in Chlamydomonas. Changes in the selection pressure allowed the insertion of At5g58340 into the promoter region of a common ancestor. When the demand for transcriptional regulation increased, At5g58340 disappeared in Poaceae, and a promoter developed in the 5' region. In contrast, Brassicaceae maintained At5g58340 and shifted all regulatory elements into the coding region of NADP-MDH.

Arabidopsis Ecotypes:A Model for Course Projects in Organismal Plant Biology & Evolution

Here we provide a how-to-do-it model for exploring how a single species can adapt to a variety of climates and, potentially, become a new species. The project involves the use of ecotypes of Arabidopsis (Arabidopsis thaliana) and integrates biology, geography, earth sciences, and mathematics. Students will grow different ecotypes of Arabidopsis under different environmental conditions and measure fitness of the ecotype to each treatment. They are allowed to select all experimental conditions themselves including the ecotypes, the environmental conditions, and which growth parameters to measure. Students own the experiments and, therefore, are more excited about the outcomes. The project can be modified to use from middle school through college and fits nicely into modules on plant growth and development and/or evolution and speciation. It is driven by studentled experimentation and provides an opportunity for in-depth discussion of scientific methods and implementation of an experimental design. It also allows students to critically evaluate how environmental factors direct plant growth, development and reproduction and might lead to speciation. Arabidopsis is a member of the mustard family, a wild relative of such species as cabbage and radish. It is not a major agronomic plant species but offers important advantages to the research community. It is small plant that is easy to grow, with limited resources. Hundreds of plants can be grown in a 4 x 4 pot at room temperature with no special lights or growth conditions and can go from seed germination to seed harvest within six weeks. Because of its genetics characteristics, it has become the plant research model system (Somerville et al., 1985; Ausubel, 2000; Federspiel, 2000). It has the smallest genome of any known plant and was the first plant genome to be fully sequenced (Arabidopsis Genome Initiative, 2000). Arabidopsis self pollinates naturally but is easy to cross pollinate, produces thousands of seed per plant, and is easy to genetically modify. Literally, thousands of researchers around the world work with Arabidopsis on research relating to gene discovery, plant physiology, growth and development, and biotechnology. More recently, because of the variety of natural populations, it is gaining popularity with ecologists. Arabidopsis is also gaining popularity among teachers because of the vast resources and information available publicly. Most important for this project are the ecotypes available from around the world through the Arabidopsis Biological Resource Center (ABRC, Columbus, OH). Over 750 natural accessions (commonly referred to as ecotypes) of Arabidopsis have been collected from around the world, and information about seed purchase and growth conditions is freely available at the TAIR Web site (http://www. arabidopsis.org/). The ecotypes vary in traits such as leaf shape, flowering time, disease resistance, seed dormancy, etc. and are used by researchers around the world to uncover the genetic interactions such as those that underlie plant responses to the environment and evolution of morphological traits. The project exploits these ecotypes and variations to give students an opportunity to explore ecotypic differentiation and speciation as a scientific hypothesis. Ecotypic Differentiation as a Testable Hypothesis in Plant Speciation Speciation is the process of developing new species through evolution; plants or animals producing progeny that evolve over time to a point that they can no longer interbreed. Ecotypes, however, are subgroups within a species that have unique physical characteristics genetically adapted to particular environmental conditions. The development of ecotypes (ecotypic differentiation) could be viewed as an initial step toward speciation. The principle of ecotypic differentiation as a stage of speciation was first set forth by Turesson (1922) and later promoted by Clausen, Keck and Hiesey (1948). …

High‐throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE

Bioluminescent strains of the Arabidopsis thaliana pathogens Pseudomonas syringae pathovar (pv.) tomato and pv. maculicola were made by insertion of the luxCDABE operon from Photorhabdus luminescens into the P. syringae chromosome under the control of a constitutive promoter. Stable integration of luxCDABE did not affect bacterial fitness, growth in planta or disease outcome. Luminescence accurately and reliably reported bacterial growth in infected Arabidopsis leaves both with a fixed inoculum followed over time and with varying inocula assayed at a single time point. Furthermore, the bioluminescence assay could detect a small (1.3-fold) difference in bacterial growth between different plant genotypes with a precision comparable to that of the standard plate assay. Luminescence of luxCDABE-tagged P. syringae allows rapid and convenient quantification of bacterial growth without the tissue extraction, serial dilution, plating and manual scoring involved in standard assays of bacterial growth by colony formation in plate culture of samples from infected tissue. The utility of the bioluminescence assay was illustrated by surveying the 500-fold variation in growth of the universally virulent P. syringae pv. maculicola ES4326 among more than 100 Arabidopsis ecotypes and identification of two quantitative trait loci accounting for 48% and 16%, respectively, of the variance of basal resistance to P. syringae pv. tomato DC3000 in the Col-0 x Fl-1 F(2) population. Luminescence assay of bacteria chromosomally tagged with luxCDABE should greatly facilitate the genetic dissection of quantitative differences in gene-for-gene, basal and acquired disease resistance and other aspects of plant interactions with bacterial pathogens requiring high-throughput assays or large-scale quantitative screens.

The Silver Lining of a Viral Agent: Increasing Seed Yield and Harvest Index in Arabidopsis by Ectopic Expression of the Potato Leaf Roll Virus Movement Protein

Ectopic expression of viral movement proteins (MPs) has previously been shown to alter plasmodesmata (PD) function and carbon partitioning in transgenic plants, giving rise to the view of PD being dynamic and highly regulated structures that allow resource allocation to be adapted to environmental and developmental needs. However, most work has been restricted to solanaceous species and the potential use of MP expression to improve biomass and yield parameters has not been addressed in detail. Here we demonstrate that MP-mediated modification of PD function can substantially alter assimilate allocation, biomass production, and reproductive growth in Arabidopsis (Arabidopsis thaliana). These effects were achieved by constitutive expression of the potato leaf roll virus 17-kD MP (MP17) fused to green fluorescent protein (GFP) in different Arabidopsis ecotypes. The resulting transgenic plants were analyzed for PD localization of the MP17:GFP fusion protein and different lines with low to high expression levels were selected for further analysis. Low-level accumulation of MP17 resulted in enhanced sucrose efflux from source leaves and a considerably increased vegetative biomass production. In contrast, high MP17 levels impaired sucrose export, resulting in source leaf-specific carbohydrate accumulation and a strongly reduced vegetative growth. Surprisingly, later during development the MP17-mediated inhibition of resource allocation was reversed, and final seed yield increased in average up to 30% in different transgenic lines as compared to wild-type plants. This resulted in a strongly improved harvest index. The release of the assimilate export block was paralleled by a reduced PD binding of MP17 in senescing leaves, indicating major structural changes of PD during leaf senescence.

Senescence-associated genes induced during compatible viral interactions with grapevine and Arabidopsis

The senescence process is the last stage in leaf development and is characterized by dramatic changes in cellular metabolism and the degeneration of cellular structures. Several reports of senescence-associated genes (SAGs) have appeared, and an overlap in some of the genes induced during senescence and pathogen infections has been observed. For example, the enhanced expression of SAGs in response to diseases caused by fungi, bacteria, and viruses that trigger the hypersensitive response (HR) or during infections induced by virulent fungi and bacteria that elicit necrotic symptoms has been observed. The present work broadens the search for SAGs induced during compatible viral interactions with both the model plant Arabidopsis thaliana and a commercially important grapevine cultivar. The transcript profiles of Arabidopsis ecotype Uk-4 infected with tobacco mosaic virus strain Cg (TMV-Cg) and Vitis vinifera cv. Carménère infected with grapevine leafroll-associated virus strain 3 (GLRaV-3) were analysed using microarray slides of the reference species Arabidopsis. A large number of SAGs exhibited altered expression during these two compatible interactions. Among the SAGs were genes that encode proteins such as proteases, lipases, proteins involved in the mobilization of nutrients and minerals, transporters, transcription factors, proteins related to translation and antioxidant enzymes, among others. Thus, part of the plant's response to virus infection appears to be the activation of the senescence programme. Finally, it was demonstrated that several virus-induced genes are also expressed at elevated levels during natural senescence in healthy plants.

Functional Characterization of AtATM1, AtATM2, and AtATM3, a Subfamily of Arabidopsis Half-molecule ATP-binding Cassette Transporters Implicated in Iron Homeostasis

The functional capabilities of one of the smallest subfamilies of ATP-binding cassette transporters from Arabidopsis thaliana, the AtATMs, are described. Designated AtATM1, AtAATM2, and AtATM3, these half-molecule ABC proteins are homologous to the yeast mitochondrial membrane protein ATM1 (ScATM1), which is clearly implicated in the export of mitochondrially synthesized iron/sulfur clusters. Yeast ATM1-deficient (atm1) mutants grow very slowly (have a petite phenotype), are respiration-deficient, accumulate toxic levels of iron in their mitochondria, and show enhanced compensatory high affinity iron uptake. Of the three Arabidopsis ATMs, AtATM3 bears the closest functional resemblance to ScATM1. Heterologously expressed AtATM3 is not only able to complement the yeast atm1 petite phenotype but is also able to suppress the constitutively high capacity for high affinity iron uptake associated with loss of the chromosomal copy of ScATM1, abrogate intra-mitochondrial iron hyperaccumulation, and restore mitochondrial respiratory function and cytochrome c levels. By comparison, AtATM1 only weakly suppresses the atm1 phenotype, and AtATM2 exerts little or no suppressive action but instead is toxic when expressed in this system. The differences between AtATM3 and AtATM1 are maintained after exchanging their target peptides, and these proteins as well as AtATM2 colocalize with the mitochondrial fluor MitoTracker Red when expressed in yeast as GFP fusions. Although its toxicity when heterologously expressed in yeast, except when fused with GFP, precludes the functional analysis of native AtATM2, a common function, mitochondrial export of Fe/S clusters or their precursors for the assembly of cytosolic Fe/S proteins, is inferred for AtATM3 and AtATM1.

Rhizobacteria-Induced Priming inArabidopsisIs Dependent on Ethylene, Jasmonic Acid, andNPR1

A nonpathogenic rhizobacterium, Pseudomonas putida LSW17S, elicited systemic protection against Fusarium wilt and pith necrosis caused by Fusarium oxysporum f. sp. lycopersici and P. corrugata in tomato (Lycopersicon esculentum L.). LSW17S also confers disease resistance against P. syringae pv. tomato DC3000 (DC3000) on Arabidopsis ecotype Col-0. To investigate mechanisms underlying disease protection, expression patterns of defense-related genes PR1, PR2, PR5, and PDF1.2 and cellular defense responses such as hydrogen peroxide accumulation and callose deposition were investigated. LSW17S treatment exhibited the typical phenomena of priming. Strong and faster transcription of defense-related genes was induced and hydrogen peroxide or callose were accumulated in Arabidopsis treated with LSW17S and infected with DC3000. In contrast, individual actions of LSW17S and DC3000 did not elicit rapid molecular and cellular defense responses. Priming by LSW17S was translocated systemically and retained for more than 10 days. Treatment with LSW17S reduced pathogen proliferation in Arabidopsis ecotype Col-0 expressing bacterial NahG; however, npr1, etr1, and jar1 mutations impaired inhibition of pathogen growth. Cellular and molecular priming responses support these results. In sum, LSW17S primes Arabidopsis for NPR1-, ethylene-, and jasmonic acid-dependent disease resistance, and efficient molecular and cellular defense responses.

Plastic and adaptive gene expression patterns associated with temperature stress in Arabidopsis thaliana

Transcriptional profiling using DNA microarrays has become a widely used approach for identifying genes with important roles in stress-regulatory networks. In previous studies, genes exhibiting a plastic expression pattern with respect to stress and control treatments have been identified as candidates with putative roles in stress-response pathways. This approach, however, often identifies numerous genes, and it is difficult to discern which genes have major effects that impact the fitness of individuals under stress. In this study, we investigated the impacts of temperature stress (cold and heat) on gene expression in the Arabidopsis thaliana model system. We identified genes exhibiting plastic patterns of gene expression with respect to temperature stress, but in contrast to previous studies, we also considered the adaptive significance of genes by examining their expression patterns among 10 Arabidopsis ecotypes indigenous to a range of latitudes. Our findings support a general association between plasticity of gene expression and adaptive value. In comparison to non-plastic genes, genes exhibiting plastic expression patterns were associated with greater among-ecotype variation in expression levels, and such variation was more strongly correlated with geographical temperature gradients. Surprisingly, while more than 16,000 genes were associated with plastic expression patterns, significant evidence of both expression plasticity and adaptive value was obtained for only 43 genes. These selected genes represent strong candidates for future experimental investigations into the molecular basis of temperature acclimation in the A. thaliana model system.

Multidimensional Protein Identification Technology (MudPIT) Analysis of Ubiquitinated Proteins in Plants

Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (MudPIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.

MAM3 catalyzes the formation of all aliphatic glucosinolate chain lengths in Arabidopsis.

Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of omega-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in the Arabidopsis ecotype Columbia: MAM1 and MAM3 (formerly known as MAM-L). Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. Underlining its broad substrate specificity, MAM3 also accepts a range of non-Met-derived 2-oxoacids, e.g. converting pyruvate to citramalate and 2-oxoisovalerate to isopropylmalate, a step in leucine biosynthesis. To investigate its role in vivo, we identified plant lines with mutations in MAM3 that resulted in a complete lack or greatly reduced levels of long-chain glucosinolates. This phenotype could be complemented by reintroduction of a MAM3 expression construct. Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. The localization of MAM3 in the chloroplast suggests that this organelle is the site of Met chain elongation.