Masakazu Satou

Leucine-Rich Repeat Receptor-Like Kinase1 Is a Key Membrane-Bound Regulator of Abscisic Acid Early Signaling in Arabidopsis

Abscisic acid (ABA) is important in seed maturation, seed dormancy, stomatal closure, and stress response. Many genes that function in ABA signal transduction pathways have been identified. However, most important signaling molecules involved in the perception of the ABA signal or with ABA receptors have not been identified yet. Receptor-like kinase1 (RPK1), a Leu-rich repeat (LRR) receptor kinase in the plasma membrane, is upregulated by ABA in Arabidopsis thaliana. Here, we show the phenotypes of T-DNA insertion mutants and RPK1-antisense plants. Repression of RPK1 expression in Arabidopsis decreased sensitivity to ABA during germination, growth, and stomatal closure; microarray and RNA gel analysis showed that many ABA-inducible genes are downregulated in these plants. Furthermore, overexpression of the RPK1 LRR domain alone or fused with the Brassinosteroid-insensitive1 kinase domain in plants resulted in phenotypes indicating ABA sensitivity. RPK1 is involved in the main ABA signaling pathway and in early ABA perception in Arabidopsis.

Comparative Genomics in Salt Tolerance between Arabidopsis and Arabidopsis-Related Halophyte Salt Cress Using Arabidopsis Microarray

Salt cress (Thellungiella halophila), a halophyte, is a genetic model system with a small plant size, short life cycle, copious seed production, small genome size, and an efficient transformation. Its genes have a high sequence identity (90%–95% at cDNA level) to genes of its close relative, Arabidopsis. These qualities are advantageous not only in genetics but also in genomics, such as gene expression profiling using Arabidopsis cDNA microarrays. Although salt cress plants are salt tolerant and can grow in 500 mm NaCl medium, they do not have salt glands or other morphological alterations either before or after salt adaptation. This suggests that the salt tolerance in salt cress results from mechanisms that are similar to those operating in glycophytes. To elucidate the differences in the regulation of salt tolerance between salt cress and Arabidopsis, we analyzed the gene expression profiles in salt cress by using a full-length Arabidopsis cDNA microarray. In salt cress, only a few genes were induced by 250 mm NaCl stress in contrast to Arabidopsis. Notably a large number of known abiotic- and biotic-stress inducible genes, including Fe-SOD, P5CS, PDF1.2, AtNCED, P-protein, β-glucosidase, and SOS1, were expressed in salt cress at high levels even in the absence of stress. Under normal growing conditions, salt cress accumulated Pro at much higher levels than did Arabidopsis, and this corresponded to a higher expression of AtP5CS in salt cress, a key enzyme of Pro biosynthesis. Furthermore, salt cress was more tolerant to oxidative stress than Arabidopsis. Stress tolerance of salt cress may be due to constitutive overexpression of many genes that function in stress tolerance and that are stress inducible in Arabidopsis.

Monitoring the expression pattern of around 7,000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray.

Full-length cDNAs are essential for functional analysis of plant genes. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. Microarray technology is a powerful tool for identifying genes induced by environmental stimuli or stress and for analyzing their expression profiles in response to environmental signals. We prepared an Arabidopsis full-length cDNA microarray containing around 7,000 independent full-length cDNA groups and analyzed the expression profiles of genes. The transcripts of 245, 54, 299 and 213 genes increased after abscisic acid (ABA), drought-, cold-, and salt-stress treatments, respectively, with inducibilities more than fivefold compared with those of control genes. The cDNA microarray analysis showed that many ABA-inducible genes were induced after drought- and high-salinity-stress treatments, and that there is more crosstalk between drought and ABA responses than between ABA and cold responses. Among the ABA-inducible genes identified, we identified 22 transcription factor genes, suggesting that many transcriptional regulatory mechanisms exist in the ABA signal transduction pathways. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s10142-002-0070-6 or from http://www.gsc.riken.go.jp/Plant/index.html.Abstract. Full-length cDNAs are essential for functional analysis of plant genes. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. Microarray technology is a powerful tool for identifying genes induced by environmental stimuli or stress and for analyzing their expression profiles in response to environmental signals. We prepared an Arabidopsis full-length cDNA microarray containing around 7,000 independent full-length cDNA groups and analyzed the expression profiles of genes. The transcripts of 245, 54, 299 and 213 genes increased after abscisic acid (ABA), drought-, cold-, and salt-stress treatments, respectively, with inducibilities more than fivefold compared with those of control genes. The cDNA microarray analysis showed that many ABA-inducible genes were induced after drought- and high-salinity-stress treatments, and that there is more crosstalk between drought and ABA responses than between ABA and cold responses. Among the ABA-inducible genes identified, we identified 22 transcription factor genes, suggesting that many transcriptional regulatory mechanisms exist in the ABA signal transduction pathways. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s10142-002-0070-6 or from http://www.gsc.riken.go.jp/Plant/index.html.

Identification of plant promoter constituents by analysis of local distribution of short sequences

BackgroundPlant promoter architecture is important for understanding regulation and evolution of the promoters, but our current knowledge about plant promoter structure, especially with respect to the core promoter, is insufficient. Several promoter elements including TATA box, and several types of transcriptional regulatory elements have been found to show local distribution within promoters, and this feature has been successfully utilized for extraction of promoter constituents from human genome.ResultsLDSS (Local Distribution of Short Sequences) profiles of short sequences along the plant promoter have been analyzed in silico, and hundreds of hexamer and octamer sequences have been identified as having localized distributions within promoters of Arabidopsis thaliana and rice. Based on their localization patterns, the identified sequences could be classified into three groups, pyrimidine patch (Y Patch), TATA box, and REG (Regulatory Element Group). Sequences of the TATA box group are consistent with the ones reported in previous studies. The REG group includes more than 200 sequences, and half of them correspond to known cis-elements. The other REG subgroups, together with about a hundred uncategorized sequences, are suggested to be novel cis-regulatory elements. Comparison of LDSS-positive sequences between Arabidopsis and rice has revealed moderate conservation of elements and common promoter architecture. In addition, a dimer motif named the YR Rule (C/T A/G) has been identified at the transcription start site (-1/+1). This rule also fits both Arabidopsis and rice promoters.ConclusionLDSS was successfully applied to plant genomes and hundreds of putative promoter elements have been extracted as LDSS-positive octamers. Identified promoter architecture of monocot and dicot are well conserved, but there are moderate variations in the utilized sequences.

Monitoring the expression profiles of genes induced by hyperosmotic, high salinity, and oxidative stress and abscisic acid treatment in Arabidopsis cell culture using a full-length cDNA microarray.

Transcriptional regulation in response to hyperosmotic, high-salinity and oxidative stress, and abscisic acid (ABA) treatment in Arabidopsis suspension-cultured cell line T87 was investigated with a cDNA microarray containing 7000 independent full-length Arabidopsis cDNAs. The transcripts of 102, 11, 84 and 73 genes were increased more than 5-fold within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50μM ABA and 10mM H2O2, respectively. On the other hand, the transcripts of 44, 57, 25 and 34 genes were down-regulated to less than one-third within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50μM ABA and 10mM H2O2, respectively. Venn diagram analysis revealed 11 genes were induced significantly by mannitol, NaCl, and ABA, indicating crosstalk among these signaling pathways. Comparison of the genes induced by each stress revealed that 32%, 17% and 33% of mannitol-, NaCl- and ABA-inducible genes were also induced by H2O2, indicating the crosstalk between the signaling pathways for osmotic stress and oxidative stress. Although the expression profiles revealed that the T87 cells had most of the regulatory systems seen in Arabidopsis seedlings, the T87 cells did not have one of ABA-dependent signaling pathways.

Monitoring expression profiles of Arabidopsis genes during cold acclimation and deacclimation using DNA microarrays

A comparative analysis of gene expression profiles during cold acclimation and deacclimation is necessary to elucidate the molecular mechanisms of cold stress responses in higher plants. We analyzed gene expression profiles in the process of cold acclimation and deacclimation (recovery from cold stress) using two microarray systems, the 7K RAFL cDNA microarray and the Agilent 22K oligonucleotide array. By both microarray analyses, we identified 292 genes up-regulated and 320 genes down-regulated during deacclimation, and 445 cold up-regulated genes and 341 cold down-regulated genes during cold acclimation. Many genes up-regulated during deacclimation were found to be down-regulated during cold acclimation, and vice versa. The genes up-regulated during deacclimation were classified into (1) regulatory proteins involved in further regulation of signal transduction and gene expression and (2) functional proteins involved in the recovery process from cold-stress-induced damages and plant growth. We also applied expression profiling studies to identify the key genes involved in the biosynthesis of carbohydrates and amino acids that are known to play important roles in cold acclimation. We compared genes that are regulated during deacclimation with those regulated during rehydration after dehydration to discuss the similarity and difference of each recovery process.

RCH1, a Locus in Arabidopsis That Confers Resistance to the Hemibiotrophic Fungal Pathogen Colletotrichum higginsianum

When challenged with the crucifer pathogen Colletotrichum higginsianum, Arabidopsis thaliana ecotype Columbia (Col-0) was colonized by the fungus within 2 to 3 days, developing brown necrotic lesions surrounded by a yellow halo. Lesions spread from the inoculation site within 3 to 4 days, and subsequently continued to expand until they covered the entire leaf. Electron microscopy confirmed that C. higginsianum is a hemibiotroph on Arabidopsis, feeding initially on living cells as a biotroph before switching to a necrotrophic mode of growth. A collection of 37 ecotypes of Arabidopsis varied in their responses to infection by C. higginsianum. The ecotype Eil-0 was highly resistant, with symptoms limited to necrotic flecking and with only very limited fungal colonization. Analyses suggested that the hypersensitive response and reactive oxygen species may be important in this defense response. Expression analyses with cDNA microarrays indicated that the defense reaction depends primarily on the jasmonic acid- and ethylene-dependent signaling pathways and, to a lesser extent, on the salicylate-dependent pathway. Crosses between the Eil-0 and Col-0 ecotypes suggested that the resistance in Eil-0 was dominant and was conferred by a single locus, which we named RCH1. RCH1 is the first resistance locus to be identified from Arabidopsis against the hemibiotrophic fungus genus Colletotrichum.

RARGE: a large-scale database of RIKEN Arabidopsis resources ranging from transcriptome to phenome

The RIKEN Arabidopsis Genome Encyclopedia (RARGE) database houses information on biological resources ranging from transcriptome to phenome, including RIKEN Arabidopsis full-length (RAFL) complementary DNAs (cDNAs), their promoter regions, Dissociation (Ds) transposon-tagged lines and expression data from microarray experiments. RARGE provides tools for searching by resource code, sequence homology or keyword, and rapid access to detailed information on the resources. We have isolated 245 946 RAFL cDNA clones and collected 11 933 transposon-tagged lines, which are available from the RIKEN Bioresource Center and are stored in RARGE. The RARGE web interface can be accessed at http://rarge.gsc.riken.jp/. Additionally, we report 90 000 new RAFL cDNA clones here.

Chloroplast ribosome release factor 1 (AtcpRF1) is essential for chloroplast development

To study the functions of nuclear genes involved in chloroplast development, we systematically analyzed albino and pale green Arabidopsis thaliana mutants by use of the Activator/Dissociation (Ac/Ds) transposon tagging system. In this study, we focused on one of these albino mutants, designated apg3-1 (for albino or pale green mutant 3). A gene encoding a ribosome release factor 1 (RF1) homologue was disrupted by the insertion of a Ds transposon into the APG3 gene; a T-DNA insertion into the same gene caused a similar phenotype (apg3-2). The APG3 gene (At3g62910) has 15 exons and encodes a protein (422-aa) with a transit peptide that functions in targeting the protein to chloroplasts. The amino acid sequence of APG3 showed 40.6% homology with an RF1 of Escherichia coli, and complementation analysis using the E. colirf1 mutant revealed that APG3 functions as an RF1 in E. coli, although complementation was not successful in the RF2-deficient (rf2) mutants of E. coli. These results indicate that the APG3 protein is an orthologue of E. coli RF1, and is essential for chloroplast translation machinery; it was accordingly named AtcpRF1. Since the chloroplasts of apg3-1 plants contained few internal thylakoid membranes, and chloroplast proteins related to photosynthesis were not detected by immunoblot analysis, AtcpRF1 is thought to be essential for chloroplast development.

Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins

We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.