PUB4, a CERK1-Interacting Ubiquitin Ligase, Positively Regulates MAMP-Triggered Immunity in Arabidopsis.

Lysin motif (LysM) receptor-like kinase CERK1 is a co-receptor essential for plant immune responses against carbohydrate microbe-associated molecular patterns (MAMPs). Concerning the immediate downstream signaling components of CERK1, receptor-like cytoplasmic kinases such as PBL27 and other RLCK VII members have been reported to regulate immune responses positively. In this study, we report that a novel CERK1-interacting E3 ubiquitin ligase, PUB4, is also involved in the regulation of MAMP-triggered immune responses. Knockout of PUB4 resulted in the alteration of chitin-induced defense responses, indicating that PUB4 positively regulates reactive oxygen species generation and callose deposition but negatively regulates MAPK activation and defense gene expression. On the other hand, detailed analyses of a double knockout mutant of pub4 and sid2, a mutant of salicylic acid (SA) synthesis pathway, showed that the contradictory phenotype of the pub4 mutant was actually caused by abnormal accumulation of SA in this mutant and that PUB4 is a positive regulator of immune responses. The present and recent findings on the role of PUB4 indicate that PUB4 is a unique E3 ubiquitin ligase involved in the regulation of both plant immunity and growth/development.

Selective regulation of the chitin-induced defense response by the Arabidopsis receptor-like cytoplasmic kinase PBL27.

Recognition of microbe-associated molecular patterns (MAMPs) initiates pattern-triggered immunity in host plants. Pattern recognition receptors (PRRs) and receptor-like cytoplasmic kinases (RLCKs) are the major components required for sensing and transduction of these molecular patterns. However, the regulation of RLCKs by PRRs and their specificity remain obscure. In this study we show that PBL27, an Arabidopsis ortholog of OsRLCK185, is an immediate downstream component of the chitin receptor CERK1 and contributes to the regulation of chitin-induced immunity in Arabidopsis. Knockout of PBL27 resulted in the suppression of several chitin-induced defense responses, including the activation of MPK3/6 and callose deposition as well as in disease resistance against fungal and bacterial infections. On the other hand, the contribution of PBL27 to flg22 signaling appears to be very limited, suggesting that PBL27 selectively regulates defense signaling downstream of specific PRR complexes. In vitro phosphorylation experiments showed that CERK1 preferentially phosphorylated PBL27 in comparison to BIK1, whereas phosphorylation of PBL27 by BAK1 was very low compared with that of BIK1. Thus, the substrate specificity of the signaling receptor-like kinases, CERK1 and BAK1, may determine the preference of downstream RLCKs.

Metabolomic Characterization of Knockout Mutants in Arabidopsis: Development of a Metabolite Profiling Database for Knockout Mutants in Arabidopsis.

Despite recent intensive research efforts in functional genomics, the functions of only a limited number of Arabidopsis (Arabidopsis thaliana) genes have been determined experimentally, and improving gene annotation remains a major challenge in plant science. As metabolite profiling can characterize the metabolomic phenotype of a genetic perturbation in the plant metabolism, it provides clues to the function(s) of genes of interest. We chose 50 Arabidopsis mutants, including a set of characterized and uncharacterized mutants, that resemble wild-type plants. We performed metabolite profiling of the plants using gas chromatography-mass spectrometry. To make the data set available as an efficient public functional genomics tool for hypothesis generation, we developed the Metabolite Profiling Database for Knock-Out Mutants in Arabidopsis (MeKO). It allows the evaluation of whether a mutation affects metabolism during normal plant growth and contains images of mutants, data on differences in metabolite accumulation, and interactive analysis tools. Nonprocessed data, including chromatograms, mass spectra, and experimental metadata, follow the guidelines set by the Metabolomics Standards Initiative and are freely downloadable. Proof-of-concept analysis suggests that MeKO is highly useful for the generation of hypotheses for genes of interest and for improving gene annotation. MeKO is publicly available at http://prime.psc.riken.jp/meko/.

Omics-based approaches to methionine side chain elongation in Arabidopsis: characterization of the genes encoding methylthioalkylmalate isomerase and methylthioalkylmalate dehydrogenase.

Glucosinolates (GSLs) are secondary metabolites in Brassicaceae plants synthesized from amino acids. Methionine-derived GSLs (Met-GSLs) with diverse side chains of various lengths are the major GSLs in Arabidopsis. Methionine chain elongation enzymes are responsible for variations in chain length in Met-GSL biosynthesis. The genes encoding methionine chain elongation enzymes are considered to have been recruited from the leucine biosynthetic pathway in the course of evolution. Among them, the genes encoding methylthioalkylmalate synthases and aminotransferases have been identified; however, the remaining genes that encode methylthioalkylmalate isomerase (MAM-I) and methylthioalkylmalate dehydro-genase (MAM-D) remain to be identified. In a previous study based on transcriptome co-expression analysis, we identified candidate genes for the large subunit of MAM-I and MAM-D. In this study, we confirmed their predicted functions by targeted GSL analysis of the knockout mutants, and named the respective genes MAM-IL1/AtleuC1 and MAM-D1/AtIMD1. Metabolic profiling of the knockout mutants of methionine chain elongation enzymes, conducted by means of widely targeted metabolomics, implied that these enzymes have roles in controlling metabolism from methionine to primary and methionine-related secondary metabolites. As shown here, an omics-based approach is an efficient strategy for the functional elucidation of genes involved in metabolism.

A new class of plant lipid is essential for protection against phosphorus depletion.

Phosphorus supply is a major factor responsible for reduced crop yields. As a result, plants utilize various adaptive mechanisms against phosphorus depletion, including lipid remodelling. Here we report the involvement of a novel plant lipid, glucuronosyldiacylglycerol, against phosphorus depletion. Lipidomic analysis of Arabidopsis plants cultured in phosphorus-depleted conditions revealed inducible accumulation of glucuronosyldiacylglycerol. Investigation using a series of sulfolipid sulfoquinovosyldiacylglycerol synthesis-deficient mutants of Arabidopsis determined that the biosynthesis of glucuronosyldiacylglycerol shares the pathway of sulfoquinovosyldiacylglycerol synthesis in chloroplasts. Under phosphorus-depleted conditions, the Arabidopsis sqd2 mutant, which does not accumulate either sulfoquinovosyldiacylglycerol or glucuronosyldiacylglycerol, was the most severely damaged of three sulfoquinovosyldiacylglycerol-deficient mutants. As glucuronosyldiacylglycerol is still present in the other two mutants, this result indicates that glucuronosyldiacylglycerol has a role in the protection of plants against phosphorus limitation stress. Glucuronosyldiacylglycerol was also found in rice, and its concentration increased significantly following phosphorus limitation, suggesting a shared physiological significance of this novel lipid against phosphorus depletion in plants.

A chloroplastic UDP-glucose pyrophosphorylase from Arabidopsis is the committed enzyme for the first step of sulfolipid biosynthesis.

Plants synthesize a sulfur-containing lipid, sulfoquinovosyldiacylglycerol, which is one of three nonphosphorus glycerolipids that provide the bulk of the structural lipids in photosynthetic membranes. Here, the identification of a novel gene, UDP-glucose pyrophosphorylase3 (UGP3), required for sulfolipid biosynthesis is described. Transcriptome coexpression analysis demonstrated highly correlated expression of UGP3 with known genes for sulfolipid biosynthesis in Arabidopsis thaliana. Liquid chromatography-mass spectrometry analysis of leaf lipids in two Arabidopsis ugp3 mutants revealed that no sulfolipid was accumulated in these mutants, indicating the participation of UGP3 in sulfolipid biosynthesis. From the deduced amino acid sequence, UGP3 was presumed to be a UDP-glucose pyrophosphorylase (UGPase) involved in the generation of UDP-glucose, serving as the precursor of the polar head of sulfolipid. Recombinant UGP3 was able to catalyze the formation of UDP-glucose from glucose-1-phosphate and UTP. A transient assay using fluorescence fusion proteins and UGPase activity in isolated chloroplasts indicated chloroplastic localization of UGP3. The transcription level of UGP3 was increased by phosphate starvation. A comparative genomics study on UGP3 homologs across different plant species suggested the structural and functional conservation of the proteins and, thus, a committing role for UGP3 in sulfolipid synthesis.

Comprehensive analysis of glucan elicitor-regulated gene expression in tobacco BY-2 cells reveals a novel MYB transcription factor involved in the regulation of phenylpropanoid metabolism.

We previously demonstrated that a beta-1,3-, 1,6-oligoglucan (AaGlucan) from the fungus Alternaria alternata 102 shows strong elicitor activity in tobacco BY-2 cells. We have used cDNA microarray analysis to monitor global changes in gene expression in tobacco cells treated with this A. alternata fraction or with laminarin. In total, we identified 265 genes that were induced 1 h after treatment with an AaGlucan-enriched fraction or laminarin. Among them, we characterized in detail a novel tobacco R2R3 MYB-type transcription factor homolog (NtMYBGR1) and two DC1 domain-containing genes (NtDC1A and NtDC1B). Microarray data, together with overexpression and metabolic analyses, indicated that NtMYBGR1, but not the NtDC1 proteins, primarily targets the phenylpropanoid synthesis-related genes PAL and 4CL. These results suggest that NtMYBGR1 specifically regulates defense responses in BY-2 cells by enhancing phenylpropanoid metabolism in response to AaGlucan and laminarin elicitors.

A novel R2R3 MYB transcription factor NtMYBJS1 is a methyl jasmonate-dependent regulator of phenylpropanoid-conjugate biosynthesis in tobacco.

Target metabolic and large-scale transcriptomic analyses of tobacco (Nicotiana tabacum L.) Bright Yellow-2 (BY-2) cells were employed to identify novel gene(s) involved in methyl jasmonate (MJ)-dependent function in plants. At the metabolic level, we describe the specific accumulation of several phenylpropanoid-polyamine conjugates in MJ-treated BY-2 cells. Furthermore, global gene expression analysis of MJ-treated cells using a 16K cDNA microarray containing expressed sequence tags (ESTs) from BY-2 cells revealed 828 genes that were upregulated by MJ treatment within 48 h. Using time-course expression data we identified a novel MJ-inducible R2R3 MYB-type transcription factor (NtMYBJS1) that was co-expressed in a close temporal pattern with the core phenylpropanoid genes phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). Overexpression of NtMYBJS1 in tobacco BY-2 cells caused accumulation of specific phenylpropanoid conjugates in the cells. Subsequent microarray analysis of NtMYBJS1 transgenic lines revealed that a limited number of genes, including PAL and 4CL, were specifically induced in the presence of the NtMYBJS1 transgene. These results, together with results of both antisense expression analysis and of gel mobility shift assays, strongly indicate that the NtMYBJS1 protein functions in tobacco MJ signal transduction, inducing phenylpropanoid biosynthetic genes and the accumulation of phenylpropanoid-polyamine conjugates during stress.