Search

Significance Statement This study indicates that CIPK23 is a phototropin‐interacting protein kinase that promotes blue light‐ and phototropin‐dependent stomatal opening in Arabidopsis thaliana. We propose that CIPK23 does not mediate plasma membrane H+‐ATPase activation but mediates inward‐rectifying K+ channel activation in stomatal opening., SUMMARY Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL‐interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull‐down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin‐mediated responses. We further found that blue light activation of inward‐rectifying K+ (K+ in) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H+‐ATPase was not. The blue light activation of K+ in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K+ in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H+‐ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed.

Summary Metabolites, phytohormones, and genes involved in dehydration responses/tolerance have been predicted in several plants. However, metabolite/phytohormone–gene regulatory networks in soybean organs under dehydration conditions remain unclear. Here, we analyzed the organ specificity of metabolites, phytohormones, and gene transcripts and revealed the characteristics of their regulatory networks in dehydration‐treated soybeans. Our metabolite/phytohormone analysis revealed the accumulation of raffinose, trehalose, and cis‐zeatin (cZ) specifically in dehydration‐treated roots. In dehydration‐treated soybeans, raffinose, and trehalose might have additional roles not directly involved in protecting the photosynthetic apparatus; cZ might contribute to root elongation for water uptake from the moisture region in soil. Our integration analysis of metabolites–genes indicated that galactinol, raffinose, and trehalose levels were correlated with transcript levels for key enzymes (galactinol synthase, raffinose synthase, trehalose 6‐phosphate synthase, trehalose 6‐phosphate phosphatase) at the level of individual plants but not at the organ level under dehydration. Genes encoding these key enzymes were expressed in mainly the aerial parts of dehydration‐treated soybeans. These results suggested that raffinose and trehalose are transported from aerial plant parts to the roots in dehydration‐treated soybeans. Our integration analysis of phytohormones–genes indicated that cZ and abscisic acid (ABA) levels were correlated with transcript levels for key enzymes (cytokinin nucleoside 5′‐monophosphate phosphoribohydrolase, cytokinin oxidases/dehydrogenases, 9‐cis‐epoxycarotenoid dioxygenase) at the level of individual plants but not at the organ level under dehydration conditions. Therefore, processes such as ABA and cZ transport, among others, are important for the organ specificity of ABA and cZ production under dehydration conditions., Significance Statement We analyzed the characteristics of metabolite/phytohormone–gene regulatory networks in dehydration‐treated soybean plants. Our analysis revealed the accumulation of raffinose, trehalose, and cZ specifically in dehydration‐treated roots, and our integration analysis of metabolite/phytohormone–genes indicated that several metabolite/phytohormone levels, including those of raffinose, trehalose, and cZ, were correlated with transcript levels for these key enzymes at the level of individual plants but not at the organ level under dehydration conditions.

The molecular breeding of drought stress-tolerant crops is imperative for stable food and biomass production. However, a trade-off exists between plant growth and drought stress tolerance. Many drought stress-tolerant plants overexpressing stress-inducible genes, such as DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 1A (DREB1A), show severe growth retardation. Here, we demonstrate that the growth of DREB1A-overexpressing Arabidopsis plants could be improved by co-expressing growth-enhancing genes whose expression is repressed under drought stress conditions. We used Arabidopsis GA REQUIRING 5 (GA5), which encodes a rate-limiting gibberellin biosynthetic enzyme, and PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), which encodes a transcription factor regulating cell growth in response to light and temperature, for growth improvement. We observed an enhanced biomass and floral induction in the GA5 DREB1A and PIF4 DREB1A double overexpressors compared with those in the DREB1A overexpressors. Although the GA5 DREB1A double overexpressors continued to show high levels of drought stress tolerance, the PIF4 DREB1A double overexpressors showed lower levels of stress tolerance than the DREB1A overexpressors due to repressed expression of DREB1A. A multiomics analysis of the GA5 DREB1A double overexpressors showed that the co-expression of GA5 and DREB1A additively affected primary metabolism, gene expression and plant hormone profiles in the plants. These multidirectional analyses indicate that the inherent trade-off between growth and drought stress tolerance in plants can be overcome by appropriate gene-stacking approaches. Our study provides a basis for using genetic modification to improve the growth of drought stress-tolerant plants for the stable production of food and biomass.

Summary To analyze the molecular mechanisms underlying plant responses to different levels of drought stress, we developed a soil matric potential (SMP)-based irrigation system that precisely controls soil moisture. Using this system, rice seedlings were grown under three different drought levels, denoted Md1, Md2 and Md3, with SMP values set to -9.8, -31.0, and -309.9 kPa, respectively. Although the Md1 treatment did not alter the visible phenotype, the Md2 treatment caused stomatal closure and shoot growth retardation (SGR). The Md3 treatment markedly induced SGR, without photosynthesis inhibition. More severe drought (Sds) treatment, under which irrigation was terminated, resulted in leaf wilting and photosynthesis inhibition. Metabolome analysis revealed the accumulation of primary sugars under Md3 and Sds and of most amino acids under Sds. The starch content was increased under Md3 and decreased under Sds. Transcriptome data showed that the expression profiles of associated genes supported the observed changes in photosynthesis and metabolites, suggesting that the time lag from SGR to photosynthesis inhibition might lead to accumulation of photosynthates under Md3, which can be used as osmolytes under Sds. To gain further insight into the observed SGR, transcriptome and hormonome analyses in specific tissues were performed. The results showed specific decreases in IAA and cytokinin levels in Md2-, Md3- and Sds-treated shoot bases, though the expression levels of hormone metabolism-related genes were not reflected in IAA and cytokinin contents. These observations suggest that drought stress affects the distribution or degradation of cytokinin and IAA molecules. This article is protected by copyright. All rights reserved.

Summary Soybean (Glycine max) is a globally important crop, and its growth and yield are severely reduced by abiotic stresses, such as drought, heat, and cold. The cis-acting element DRE (dehydration-responsive element)/CRT plays an important role in activating gene expression in response to these stresses. The Arabidopsis DREB1/CBF genes that encode DRE-binding proteins function as transcriptional activators in the cold stress responsive gene expression. In this study, we identified 14 DREB1-type transcription factors (GmDREB1s) from a soybean genome database. The expression of most GmDREB1 genes in soybean was strongly induced by a variety of abiotic stresses, such as cold, drought, high salt, and heat. The GmDREB1 proteins activated transcription via DREs (dehydration-responsive element) in Arabidopsis and soybean protoplasts. Transcriptome analyses using transgenic Arabidopsis plants overexpressing GmDREB1s indicated that many of the downstream genes are cold-inducible and overlap with those of Arabidopsis DREB1A. We then comprehensively analyzed the downstream genes of GmDREB1B;1, which is closely related to DREB1A, using a transient expression system in soybean protoplasts. The expression of numerous genes induced by various abiotic stresses were increased by overexpressing GmDREB1B;1 in soybean, and DREs were the most conserved element in the promoters of these genes. The downstream genes of GmDREB1B;1 included numerous soybean-specific stress-inducible genes that encode an ABA receptor family protein, GmPYL21, and translation-related genes, such as ribosomal proteins. We confirmed that GmDREB1B;1 directly activates GmPYL21 expression and enhances ABRE-mediated gene expression in an ABA-independent manner. These results suggest that GmDREB1 proteins activate the expression of numerous soybean-specific stress-responsive genes under diverse abiotic stress conditions.

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.

To identify candidate genes involved in Arabidopsis flavonoid biosynthesis, we applied transcriptome coexpression analysis and independent component analyses with 1388 microarray data from publicly available databases. Two glycosyltransferases, UGT79B1 and UGT84A2 were found to cluster with anthocyanin biosynthetic genes. Anthocyanin was drastically reduced in ugt79b1 knockout mutants. Recombinant UGT79B1 protein converted cyanidin 3-O-glucoside to cyanidin 3-O-xylosyl(1→2)glucoside. UGT79B1 recognized 3-O-glucosylated anthocyanidins/flavonols and uridine diphosphate (UDP)-xylose, but not 3,5-O-diglucosylated anthocyanidins, indicating that UGT79B1 encodes anthocyanin 3-O-glucoside: 2''-O-xylosyltransferase. UGT84A2 is known to encode sinapic acid: UDP-glucosyltransferase. In ugt84a2 knockout mutants, a major sinapoylated anthocyanin was drastically reduced. A comparison of anthocyanin profiles in ugt84a knockout mutants indicated that UGT84A2 plays a major role in sinapoylation of anthocyanin, and that other UGT84As contribute the production of 1-O-sinapoylglucose to a lesser extent. These data suggest major routes from cyanidin 3-O-glucoside to the most highly modified cyanidin in the potential intricate anthocyanin modification pathways in Arabidopsis.

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.

A myriad of drought stress-inducible genes have been reported, and many of these are activated by abscisic acid (ABA). In the promoter regions of such ABA-regulated genes, conserved cis-elements, designated ABA-responsive elements (ABREs), control gene expression via bZIP-type AREB/ABF transcription factors. Although all three members of the AREB/ABF subfamily, AREB1, AREB2, and ABF3, are upregulated by ABA and water stress, it remains unclear whether these are functional homologs. Here, we report that all three AREB/ABF transcription factors require ABA for full activation, can form hetero- or homodimers to function in nuclei, and can interact with SRK2D/SnRK2.2, an SnRK2 protein kinase that was identified as a regulator of AREB1. Along with the tissue-specific expression patterns of these genes and the subcellular localization of their encoded proteins, these findings clearly indicate that AREB1, AREB2, and ABF3 have largely overlapping functions. To elucidate the role of these AREB/ABF transcription factors, we generated an areb1 areb2 abf3 triple mutant. Large-scale transcriptome analysis, which showed that stress-responsive gene expression is remarkably impaired in the triple mutant, revealed novel AREB/ABF downstream genes in response to water stress, including many LEA class and group-Ab PP2C genes and transcription factors. The areb1 areb2 abf3 triple mutant is more resistant to ABA than are the other single and double mutants with respect to primary root growth, and it displays reduced drought tolerance. Thus, these results indicate that AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent gene expression for ABA signaling under conditions of water stress.

A majority of the proteins of the chloroplast are encoded by the nuclear genome, and are post-translationally targeted to the chloroplast. From databases of tagged insertion lines at international seed stock centers and our own stock, we selected 3246 Ds/Spm (dissociator/suppressor-mutator) transposon- or T-DNA-tagged Arabidopsis lines for genes encoding 1369 chloroplast proteins (about 66% of the 2090 predicted chloroplast proteins) in which insertions disrupt the protein-coding regions. We systematically observed 3-week-old seedlings grown on agar plates, identified mutants with abnormal phenotypes and collected homozygous lines with wild-type phenotypes. We also identified insertion lines for which no homozygous plants were obtained. To date, we have identified 111 lines with reproducible seedling phenotypes, 122 lines for which we could not obtain homozygotes and 1290 homozygous lines without a visible phenotype. The Chloroplast Function Database presents the molecular and phenotypic information obtained from this resource. The database provides tools for searching for mutant lines using Arabidopsis Genome Initiative (AGI) locus numbers, tagged line numbers and phenotypes, and provides rapid access to detailed information on the tagged line resources. Moreover, our collection of insertion homozygotes provides a powerful tool to accelerate the functional analysis of nuclear-encoded chloroplast proteins in Arabidopsis. The Chloroplast Function Database is freely available at http://rarge.psc.riken.jp/chloroplast/. The homozygous lines generated in this project are also available from the various Arabidopsis stock centers. We have donated the insertion homozygotes to their originating seed stock centers.

Several hundred nucleus-encoded factors are required for regulating gene expression in plant organelles. Among them, the most numerous are the members of the pentatricopeptide repeat (PPR) protein family. We found that PPR protein OTP82 is essential for RNA editing of the ndhB-9 and ndhG-1 sites within transcripts encoding subunits of chloroplast NAD(P)H dehydrogenase. Despite the defects in RNA editing, otp82 did not show any phenotypes in NDH activity, stability or interaction with photosystem I, suggesting that the RNA editing events mediated by OTP82 are functionally silent even though they induce amino acid alterations. In agreement with this result, both sites are partially edited even in the wild type, implying the possibility that a single gene produces heterogeneous proteins that are functionally equivalent. Although only five nucleotides separate the ndhB-8 and ndhB-9 sites, the ndhB-8 site is normally edited in otp82 mutants, suggesting that both sites are recognized by different PPR proteins. OTP82 falls into the DYW subclass containing conserved C-terminal E and DYW motifs. As in CRR22 and CRR28, the DYW motif present in OTP82 is not essential for RNA editing in vivo.

Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.

Drought is the major environmental threat to agricultural production and distribution worldwide. Adaptation by plants to dehydration stress is a complex biological process that involves global changes in gene expression and metabolite composition. Here, using one type of functional genomics analysis, metabolomics, we characterized the metabolic phenotypes of Arabidopsis wild-type and a knockout mutant of the NCED3 gene (nc3-2) under dehydration stress. NCED3 plays a role in the dehydration-inducible biosynthesis of abscisic acid (ABA), a phytohormone that is important in the dehydration-stress response in higher plants. Metabolite profiling performed using two types of mass spectrometry (MS) systems, gas chromatography/time-of-flight MS (GC/TOF-MS) and capillary electrophoresis MS (CE-MS), revealed that accumulation of amino acids depended on ABA production, but the level of the oligosaccharide raffinose was regulated by ABA independently under dehydration stress. Metabolic network analysis showed that global metabolite-metabolite correlations occurred in dehydration-increased amino acids in wild-type, and strong correlations with raffinose were reconstructed in nc3-2. An integrated metabolome and transcriptome analysis revealed ABA-dependent transcriptional regulation of the biosynthesis of the branched-chain amino acids, saccharopine, proline and polyamine. This metabolomics analysis revealed new molecular mechanisms of dynamic metabolic networks in response to dehydration stress.

Enzymes encoded by bacterial MurE genes catalyze the ATP-dependent formation of uridine diphosphate-N-acetylmuramic acid-tripeptide in bacterial peptidoglycan biosynthesis. The Arabidopsis thaliana genome contains one gene with homology to the bacterial MurE:AtMurE. Under normal conditions AtMurE is expressed in leaves and flowers, but not in roots or stems. Sequence-based predictions and analyses of GFP fusions of the N terminus of AtMurE, as well as the full-length protein, suggest that AtMurE localizes to plastids. We identified three T-DNA-tagged and one Ds-tagged mutant alleles of AtMurE in A. thaliana. All four alleles show a white phenotype, and A. thaliana antisense AtMurE lines showed a pale-green phenotype. These results suggest that AtMurE is involved in chloroplast biogenesis. Cells of the mutants were inhibited in thylakoid membrane development. RT-PCR analysis of the mutant lines suggested that the expression of genes that depend on a multisubunit plastid-encoded RNA polymerase was decreased. To analyze the functional relationships between the MurE genes of cyanobacteria, the moss Physcomitrella patens and higher plants, a complementation assay was carried out with a P. patens (Pp) MurE knock-out line, which exhibits a small number of macrochloroplasts per cell. Although the Anabaena MurE, fused with the N-terminal region of PpMurE, complemented the macrochloroplast phenotype in P. patens, transformation with AtMurE did not complement this phenotype. These results suggest that AtMurE is functionally divergent from the bacterial and moss MurE proteins.

The involvement of kinesin motor proteins in both cell-tip growth and cell-shape determination has been well characterized in various organisms. However, the functions of kinesins during cell morphogenesis in higher plants remain largely unknown. In the current study, we demonstrate that an armadillo repeat-containing kinesin-related protein, ARMADILLO REPEAT KINESIN1 (ARK1), is involved in root-hair morphogenesis. Microtubule polymers are more abundant in ark1 null allele root hairs, but analysis shows that these extra microtubules are concentrated in the endoplasm, and not in the cortical array, suggesting that ARK1 regulates tip growth by limiting the assembly and distribution of endoplasmic microtubules. The ARK1 gene has two homologues in the Arabidopsis genome, ARK2 and ARK3, and our results show that ARK2 is involved in root-cell morphogenesis. We further reveal that a NIMA-related protein kinase, NEK6, binds to the ARK family proteins and has pleiotropic effects on epidermal-cell morphogenesis, suggesting that NEK6 is involved in cell morphogenesis in Arabidopsis via microtubule functions associated with these armadillo repeat-containing kinesins. We discuss the function of NIMA-related protein kinases and armadillo repeat-containing kinesins in the cell morphogenesis of eukaryotes.

The phytohormone abscisic acid (ABA) regulates physiologically important stress and developmental responses in plants. To reveal the mechanism of response to ABA, we isolated several novel ABA-hypersensitive Arabidopsis thaliana mutants, named ahg (ABA-hypersensitive germination). ahg1-1 mutants showed hypersensitivity to ABA, NaCl, KCl, mannitol, glucose and sucrose during germination and post-germination growth, but did not display any significant phenotypes in adult plants. ahg1-1 seeds accumulated slightly more ABA before stratification and showed increased seed dormancy. Map-based cloning of AHG1 revealed that ahg1-1 has a nonsense mutation in a gene encoding a novel protein phosphatase 2C (PP2C). We previously showed that the ahg3-1 mutant has a point mutation in the AtPP2CA gene, which encodes another PP2C that has a major role in the ABA response in seeds (Yoshida et al., 2006b). The levels of AHG1 mRNA were higher in dry seeds and increased during late seed maturation--an expression pattern similar to that of ABI5. Transcriptome analysis revealed that, in ABA-treated germinating seeds, many seed-specific genes and ABA-inducible genes were highly expressed in ahg1-1 and ahg3-1 mutants compared with the wild-type. Detailed analysis suggested differences between the functions of AHG1 and AHG3. Dozens of genes were expressed more strongly in the ahg1-1 mutant than in ahg3-1. Promoter-GUS analyses demonstrated both overlapping and distinct expression patterns in seed. In addition, the ahg1-1 ahg3-1 double mutant was more hypersensitive than either monogenic mutant. These results suggest that AHG1 has specific functions in seed development and germination, shared partly with AHG3.

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.

DREB1/CBFs and DREB2s are transcription factors that specifically interact with a cis-acting element, DRE/CRT, which is involved in the expression of genes responsive to cold and drought stress in Arabidopsis thaliana. The function of DREB1/CBFs has been precisely analyzed and it has been found to activate the expression of many genes responsive to cold stress containing a DRE/CRT sequence in their promoters. However, the regulation and function of DREB2-type transcription factors remained to be elucidated. In this research, we report the cloning of a DREB2 homolog from maize, ZmDREB2A, whose transcripts were accumulated by cold, dehydration, salt and heat stresses in maize seedlings. Unlike Arabidopsis DREB2A, ZmDREB2A produced two forms of transcripts, and quantitative real-time PCR analyses demonstrated that only the functional transcription form of ZmDREB2A was significantly induced by stresses. Moreover, the ZmDREB2A protein exhibited considerably high transactivation activity compared with DREB2A in Arabidopsis protoplasts, suggesting that protein modification is not necessary for ZmDREB2A to be active. Constitutive or stress-inducible expression of ZmDREB2A resulted in an improved drought stress tolerance in plants. Microarray analyses of transgenic plants overexpressing ZmDREB2A revealed that in addition to genes encoding late embryogenesis abundant (LEA) proteins, some genes related to heat shock and detoxification were also upregulated. Furthermore, overexpression of ZmDREB2A also enhanced thermotolerance in transgenic plants, implying that ZmDREB2A may play a dual functional role in mediating the expression of genes responsive to both water stress and heat stress.

The ZFHD recognition sequence (ZFHDRS) and NAC recognition sequence (NACRS) play an important role in the dehydration-inducible expression of the Arabidopsisthaliana EARLY RESPONSIVETO DEHYDRATION STRESS 1 (ERD1) gene. Using the yeast one-hybrid system, we isolated a cDNA encoding the ZFHD1 transcriptional activator that specifically binds to the 62 bp promoter region of ERD1, which contains the ZFHDRS. Both in vitro and in vivo analyses confirmed specific binding of the ZFHD1 to ZFHDRS, and the expression of ZFHD1 was induced by drought, high salinity and abscisic acid. The DNA-binding and activation domains of ZFHD1 were localized on the C-terminal homeodomain and N-terminal zinc finger domain, respectively. Microarray analysis of transgenic plants over-expressing ZFHD1 revealed that several stress-inducible genes were upregulated in the transgenic plants. Transgenic plants exhibited a smaller morphological phenotype and had a significant improvement of drought stress tolerance. Using the yeast two-hybrid system, we detected an interaction between ZFHD1 and NACRS-binding NAC proteins. Moreover, co-over-expression of the ZFHD1 and NAC genes restored the morphological phenotype of the transgenic plants to a near wild-type state and enhanced expression of ERD1 in both Arabidopsis T87 protoplasts and transgenic Arabidopsis plants.

Analysis of albino or pale-green (apg) mutants is important for identifying nuclear genes responsible for chloroplast development and pigment synthesis. We have identified 38 apg mutants by screening 11 000 Arabidopsis Ds-tagged lines. One mutant, apg6, contains a Ds insertion in a gene encoding APG6 (ClpB3), a homologue of the heat-shock protein Hsp101 (ClpB1). We isolated somatic revertants and identified two Ds-tagged and one T-DNA-tagged mutant alleles of apg6. All three alleles gave the same pale-green phenotype. These results suggest that APG6 is important for chloroplast development. The APG6 protein contains a transit peptide and is localized in chloroplasts. The plastids of apg6 pale-green cells were smaller than those of the wild type, and contained undeveloped thylakoid membranes. APG6 mRNA accumulated in response to heat shock in various organs, but not in response to other abiotic stresses. Under normal conditions, APG6 is constitutively expressed in the root tips, the organ boundary region, the reproductive tissues of mature plants where plastids exist as proplastids, and slightly in the stems and leaves. In addition, constitutive overexpression of APG6 in transgenic plants inhibited chloroplast development and resulted in a mild pale-green phenotype. The amounts of chloroplast proteins related to photosynthesis were markedly decreased in apg6 mutants. These results suggest that APG6 functions as a molecular chaperone involved in plastid differentiation mediating internal thylakoid membrane formation and conferring thermotolerance to chloroplasts during heat stress. The APG6 protein is not only involved in heat-stress response in chloroplasts, but is also essential for chloroplast development.

Mutant lines covering all Arabidopsis genes allow us to pursue systematic functional genomics. A comprehensive phenotype description, called a phenome, is highly sought after in the profiling of -omics data. We selected 4000 transposon-insertional lines with transposon insertions in their gene-coding regions, and systematically observed the visible phenotype of each line. For the first 3 weeks after germination, plants were grown on agar plates and the juvenile phenotypes were recorded. Then the plants were transferred to soil and their phenotypes were recorded at each growth stage. About 140 lines showed clear and reproducible visible phenotypes, including novel phenotypic mutants as well as previously reported ones. All descriptions of the mutants showing visible phenotypes were classified into eight primary categories (seedling, leaves, flowering and growth, stems, branching, flowers, siliques and seed yield) and 43 secondary categories of morphological phenotypes. Phenotypic images have been entered into a searchable database (http://rarge.gsc.riken.jp/phenome/). One example investigated through the use of plural alleles was a mutant of a novel gene related to glycerolipid biosynthesis, with a unique visible phenotype of sepal opening. Our results suggest that we can find more novel visible phenotypes and their corresponding genes, and that phenotypic mutants of gene knockouts are not exhausted yet. This study provides basic data on large-scale phenotyping of gene knockout lines in plants, and will contribute to the completion of an international effort to develop a phenome database of all the functional genes in Arabidopsis.

Accumulating evidence suggests that mRNA degradation systems are crucial for various biological processes in eukaryotes. Here we provide evidence that an mRNA degradation system is associated with some plant hormones and stress responses in plants. We analysed a novel Arabidopsis abscisic acid (ABA)-hypersensitive mutant, ahg2-1, that showed ABA hypersensitivity not only in germination, but also at later developmental stages, and that displayed pleiotropic phenotypes. We found that ahg2-1 accumulated more endogenous ABA in seeds and mannitol-treated plants than did the wild type. Microarray experiments showed that the expressions of ABA-, salicylic acid- and stress-inducible genes were increased in normally grown ahg2-1 plants, suggesting that the ahg2-1 mutation somehow affects various stress responses as well as ABA responses. Map-based cloning of AHG2 revealed that this gene encodes a poly(A)-specific ribonuclease (AtPARN) that is presumed to function in mRNA degradation. Detailed analysis of the ahg2-1 mutation suggests that the mutation reduces AtPARN production. Interestingly, expression of AtPARN was induced by treatment with ABA, high salinity and osmotic stress. These results suggest that both upregulation and downregulation of gene expression by the mRNA-destabilizing activity of AtPARN are crucial for proper ABA, salicylic acid and stress responses.

In this study we characterized the sensitive to low humidity 1 (slh1) mutant of Arabidopsis ecotype No-0 which exhibits normal growth on agar plate medium but which on transfer to soil shows growth arrest and development of necrotic lesions. cDNA microarray hybridization and RNA gel blot analysis revealed that genes associated with activation of disease resistance were upregulated in the slh1 mutants in response to conditions of low humidity. Furthermore, the slh1 mutants accumulate callose, autofluorescent compounds and salicylic acid (SA). We demonstrate that SA is required for the slh1 phenotype but not PAD4 or NPR1. SLH1 was isolated by map-based cloning and it encodes a resistance (R)-like protein consisting of a domain with Toll and interleukin-1 receptor homology (TIR), a nucleotide-binding domain (NB), leucine-rich repeats (LRR) and a carboxy-terminal WRKY domain. SLH1 is identical to the R gene RRS1-R of the Arabidopsis ecotype Nd-1, a gene which confers resistance to the bacterial pathogen Ralstonia solanacearum GMI1000 and also functions as an R gene to this pathogen in No-0. We identified a 3 bp insertion mutation in slh1 that results in the addition of a single amino acid in the WRKY domain; thereby impairing its DNA-binding activity. Our data suggest that SLH1 disease resistance signaling may be negatively regulated by its WRKY domain in the R protein and that the constitutive defense activation conferred by the slh1 mutation is inhibited by conditions of high humidity.

Tiling arrays of high-density oligonucleotide probes spanning the entire genome are powerful tools for the discovery of new genes. However, it is difficult to determine the structure of the spliced product of a structurally unknown gene from noisy array signals only. Here we introduce a statistical method that estimates the precise splicing points and the exon/intron structure of a structurally unknown gene by maximizing the odds or the ratio of posterior probabilities of the structure under the observation of array signal intensities and nucleic acid sequences. Our method more accurately predicted the gene structures than the simple threshold-based method, and more correctly estimated the expression values of structurally unknown genes than the window-based method. It was observed that the Markov model contributed to the precision of splice points, and that the statistical significance of expression (P-value) represented the reliability of the estimated gene structure and expression value well. We have implemented the method as a program ARTADE (ARabidopsis Tiling Array-based Detection of Exons) and applied it to the Arabidopsis thaliana whole-genome array data analysis. The database of the predicted results and the ARTADE program are available at http://omicspace.riken.jp/ARTADE/.

Phosphatidic acid (PA) functions as a lipid signaling molecule in plants. Physiological analysis showed that PA triggers early signal transduction events that lead to responses to abscisic acid (ABA) during seed germination. We measured PA production during seed germination and found increased PA levels during early germination. To investigate the role of PA during seed germination, we focused on the PA catabolic enzyme lipid phosphate phosphatase (LPP). LPP catalyzes the conversion of PA to diacylglycerol (DAG). There are 4 LPP genes in the Arabidopsis genome. Among them, AtLPP2 and AtLPP3 are expressed during seed germination. Two AtLPP2 T-DNA insertional mutants (lpp2-1 and lpp2-2) showed hypersensitivity to ABA and significant PA accumulation during germination. Furthermore, double-mutant analysis showed that ABA-insensitive 4 (ABI4) is epistatic to AtLPP2 but ABA-insensitive 3 (ABI3) is not. These results suggest that PA is involved in ABA signaling and that AtLPP2 functions as a negative regulator upstream of ABI4, which encodes an AP2-type transcription factor, in ABA signaling during germination.

Xyloglucan endotransglucosylases/hydrolases (XTHs) are a class of enzymes capable of catalyzing the molecular grafting between xyloglucans and/or the endotype hydrolysis of a xyloglucan molecule. They are encoded by 33 genes in Arabidopsis. Whereas recent studies have revealed temporally and spatially specific expression profiles for individual members of this family in plants, their biological roles are still to be clarified. To identify the role of each member of this gene family, we examined phenotypes of mutants in which each of the Arabidopsis XTH genes was disrupted. This was undertaken using a reverse genetic approach, and disclosed two loss-of-function mutants for the AtXTH27 gene, xth27-1 and xth27-2. These exhibited short-shaped tracheary elements in tertiary veins, and reduced the number of tertiary veins in the first leaf. In mature rosette leaves of the mutant, yellow lesion-mimic spots were also observed. Upon genetic complementation by introducing the wild-type XTH27 gene into xth27-1 mutant plants, the number of tertiary veins was restored, and the lesions disappeared completely. Extensive expression of the pXTH27::GUS fusion gene was observed in immature tracheary elements in the rosette leaves. The highest level of AtXTH27 mRNA expression in the rosette leaves was observed during leaf expansion, when the tracheary elements were elongating. These findings indicate that AtXTH27 plays an essential role during the generation of tracheary elements in the rosette leaves of Arabidopsis.

In plants, the last step in the synthesis of p-aminobenzoate (PABA) moiety of folate remains to be elucidated. In Escherichia coli, this step is catalyzed by the PabC protein, a beta-lyase that converts 4-amino-4-deoxychorismate (ADC)--the reaction product of the PabA and PabB enzymes--to PABA and pyruvate. So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E. coli PabA and PabB and is located in plastids. ADC synthase has no lyase activity, implying that plants have a separate ADC lyase. No such lyase is known in any eukaryote. Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast-targeting peptides. These cDNAs were shown to encode functional enzymes by complementation of an E. coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA. Plant ADC lyase is active as dimer and is not feedback inhibited by physiologic concentrations of PABA, its glucose ester, or folates. The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments. These data indicate that ADC lyase, like ADC synthase, is present in plastids. As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated.

The transcriptional factor DREB/CBF (dehydration-responsive element/C-repeat-binding) specifically interacts with the dehydration-responsive element (DRE)/C-repeat (CRT) cis-acting element (A/GCCGAC) and controls the expression of many stress-inducible genes in Arabidopsis. Transgenic plants overexpressing DREB1A showed activated expression of many stress-inducible genes and improved tolerance to not only drought, salinity, and freezing but also growth retardation. We searched for downstream genes in transgenic plants overexpressing DREB1A using the full-length cDNA microarray and Affymetrix GeneChip array. We confirmed candidate genes selected by array analyses using RNA gel blot and identified 38 genes as the DREB1A downstream genes, including 20 unreported new downstream genes. Many of the products of these genes were proteins known to function against stress and were probably responsible for the stress tolerance of the transgenic plants. The downstream genes also included genes for protein factors involved in further regulation of signal transduction and gene expression in response to stress. The identified genes were classified into direct downstream genes of DREB1A and the others based on their expression patterns in response to cold stress. We also searched for conserved sequences in the promoter regions of the direct downstream genes and found A/GCCGACNT in their promoter regions from -51 to -450 as a consensus DRE. The recombinant DREB1A protein bound to A/GCCGACNT more efficiently than to A/GCCGACNA/G/C.

An activation-tagged allele of activated disease resistance 1 (ADR1) has previously been shown to convey broad spectrum disease resistance. ADR1 was found to encode a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) protein, which possessed domains of homology with serine/threonine protein kinases. Here, we show that either constitutive or conditional enhanced expression of ADR1 conferred significant drought tolerance. This was not a general feature of defence-related mutants because cir (constitutive induced resistance)1, cir2 and cpr (constitutive expressor of PR genes)1, which constitutively express systemic acquired resistance (SAR), failed to exhibit this phenotype. Cross-tolerance was not a characteristic of adr1 plants, rather they showed increased sensitivity to thermal and salinity stress. Hence, adr1-activated signalling may antagonise some stress responses. Northern analysis of abiotic marker genes revealed that dehydration-responsive element (DRE)B2A but not DREB1A, RD (response to dehydration)29A or RD22 was expressed in adr1 plant lines. Furthermore, DREB2A expression was salicylic acid (SA) dependent but NPR (non-expressor of PR genes)1 independent. In adr1/ADR1 nahG (naphthalene hydroxylase G), adr1/ADR1 eds (enhanced disease susceptibility)1 and adr1/ADR1 abi1 double mutants, drought tolerance was significantly reduced. Microarray analyses of plants containing a conditional adr1 allele demonstrated that a significant number of the upregulated genes had been previously implicated in responses to dehydration. Therefore, biotic and abiotic signalling pathways may share multiple nodes and their outputs may have significant functional overlap.

To study the functions of the nuclear genes involved in chloroplast development, we systematically analyzed albino and pale-green Arabidopsis thaliana mutants by using a two-component transposon system based on the Ac/Ds element of maize as a mutagen. One of the pale-green mutants, albino or pale green mutant 1 (designated as apg1), did not survive beyond the seedling stage, when germinated on soil. The chloroplasts of the apg1 plants contained decreased numbers of lamellae with reduced levels of chlorophyll. A gene encoding a 37 kDa polypeptide precursor of the chloroplast inner envelope membrane was disrupted by insertion of the Ds transposon in apg1. The 37 kDa protein had partial sequence similarity to the S-adenosylmethionine-dependent methyltransferase. The apg1 plants lacked plastoquinone (PQ), suggesting that the APG1 protein is involved in the methylation step of PQ biosynthesis, which is localized at the envelope membrane. Our results demonstrate the importance of the 37 kDa protein of the chloroplast inner envelope membrane for chloroplast development in Arabidopsis.

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.

In the photorespiratory process, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to 2-oxoglutarate and glycine. Although GGAT has been assumed to play important roles for the transamination in photorespiratory carbon cycles, the gene encoding GGAT has not been identified. Here, we report that an alanine:2-oxoglutarate aminotransferase (AOAT)-like protein functions as GGAT in peroxisomes. Arabidopsis has four genes encoding AOAT-like proteins and two of them (namely AOAT1 and AOAT2) contain peroxisomal targeting signal 1 (PTS1). The expression analysis of mRNA encoding AOATs and EST information suggested that AOAT1 was the major protein in green leaves. When AOAT1 fused to green fluorescent protein (GFP) was expressed in BY-2 cells, it was found to be localized to peroxisomes depending on PTS1. By screening of Arabidopsis T-DNA insertion lines, an AOAT1 knockout line (aoat1-1) was isolated. The activity of GGAT and alanine:glyoxylate aminotransferase (AGAT) in the above-ground tissues of aoat1-1 was reduced drastically and, AOAT and glutamate:pyruvate aminotransferase (GPAT) activity also decreased. Peroxisomal GGAT was detected in the wild type but not in aoat1-1. The growth rate was repressed in aoat1-1 grown under high irradiation or without sugar, though differences were slight in aoat1-1 grown under low irradiation, high-CO2 (0.3%) or high-sugar (3% sucrose) conditions. These phenotypes resembled those of photorespiration-deficient mutants. Glutamate levels increased and serine levels decreased in aoat1-1 grown in normal air conditions. Based on these results, it was concluded that AOAT1 is targeted to peroxisomes, functions as a photorespiratory GGAT, plays a markedly important role for plant growth and the metabolism of amino acids.

The transcription factors DREBs/CBFs specifically interact with the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element (core motif: G/ACCGAC) and control the expression of many stress-inducible genes in Arabidopsis. In rice, we isolated five cDNAs for DREB homologs: OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, and OsDREB2A. Expression of OsDREB1A and OsDREB1B was induced by cold, whereas expression of OsDREB2A was induced by dehydration and high-salt stresses. The OsDREB1A and OsDREB2A proteins specifically bound to DRE and activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts. Over-expression of OsDREB1A in transgenic Arabidopsis induced over-expression of target stress-inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought, high-salt, and freezing stresses. This indicated that OsDREB1A has functional similarity to DREB1A. However, in microarray and RNA blot analyses, some stress-inducible target genes of the DREB1A proteins that have only ACCGAC as DRE were not over-expressed in the OsDREB1A transgenic Arabidopsis. The OsDREB1A protein bound to GCCGAC more preferentially than to ACCGAC whereas the DREB1A proteins bound to both GCCGAC and ACCGAC efficiently. The structures of DREB1-type ERF/AP2 domains in monocots are closely related to each other as compared with that in the dicots. OsDREB1A is potentially useful for producing transgenic monocots that are tolerant to drought, high-salt, and/or cold stresses.

Full-length cDNAs are essential for functional analysis of plant genes in the post-sequencing era of the Arabidopsis genome. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. We have prepared a full-length cDNA microarray containing approximately 7000 independent, full-length cDNA groups to analyse the expression profiles of genes under drought, cold (low temperature) and high-salinity stress conditions over time. The transcripts of 53, 277 and 194 genes increased after cold, drought and high-salinity treatments, respectively, more than fivefold compared with the control genes. We also identified many highly drought-, cold- or high-salinity- stress-inducible genes. However, we observed strong relationships in the expression of these stress-responsive genes based on Venn diagram analysis, and found 22 stress-inducible genes that responded to all three stresses. Several gene groups showing different expression profiles were identified by analysis of their expression patterns during stress-responsive gene induction. The cold-inducible genes were classified into at least two gene groups from their expression profiles. DREB1A was included in a group whose expression peaked at 2 h after cold treatment. Among the drought, cold or high-salinity stress-inducible genes identified, we found 40 transcription factor genes (corresponding to approximately 11% of all stress-inducible genes identified), suggesting that various transcriptional regulatory mechanisms function in the drought, cold or high-salinity stress signal transduction pathways.

Mitogen-activated protein kinase (MAP kinase, MAPK) cascades play pivotal roles in signal transduction of extracellular stimuli, such as environmental stresses and growth regulators, in various organisms. Arabidopsis thaliana MAP kinases constitute a gene family, but stimulatory signals for each MAP kinase have not been elucidated. Here we show that environmental stresses such as low temperature, low humidity, hyper-osmolarity, touch and wounding induce rapid and transient activation of the Arabidopsis MAP kinases ATMPK4 and ATMPK6. Activation of ATMPK4 and ATMPK6 was associated with tyrosine phosphorylation but not with the amounts of mRNA or protein. Kinetics during activation differ between these two MAP kinases. These results suggest that ATMPK4 and ATMPK6 are involved in distinct signal transduction pathways responding to these environmental stresses.

Summary We identified a Dissociation (Ds) transposon-inserted Arabidopsis mutant of a gene (AtRPS13A) homologous to cytoplasmic ribosomal protein (RP) S13. We named our mutant pointed first leaf (pfl) 2 because of its similar phenotype to the pfl1 mutant of the RPS18 gene. This mutant caused multiple phenotypic changes, including aberrant leaf and trichome morphology, retarded root growth, and late flowering. Microscopic analysis showed that the first leaf blade of pfl2 contained a significantly reduced number of palisade cells, which suggests that the mutant phenotype was caused by reduced cell division. However, no phenotypic changes were observed during reproductive growth. In Arabidopsis, the RPS13 protein was encoded by a small expressed gene family including AtRPS13A. A pfl1 pfl2 double mutant showed no additive effect. These results suggest that RPS13 functions in quantitative and pleiotropic ways during growth and development, and that mutations at different kinds of RP gene loci are accumulatable without serious growth defects because they belong to small gene families.

Many organisms, including higher plants, accumulate free proline (Pro) in response to osmotic stress. Although various studies have focused on the ability of Pro as a compatible osmolyte involved in osmotolerance, its specific role throughout plant growth is still unclear. It has been reported that Pro is synthesized from Glu catalyzed by a key enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), in plants. To elucidate essential roles of Pro, we generated antisense transgenic Arabidopsis plants with a P5CS cDNA. Several transgenics accumulated Pro at a significantly lower level than wild-type plants, providing direct evidence for a key role of P5CS in Pro production in Arabidopsis. These antisense transgenics showed morphological alterations in leaves and a defect in elongation of inflorescences. Furthermore, transgenic leaves were hypersensitive to osmotic stress. Microscopic analysis of transgenic leaves, in which the mutated phenotype clearly occurred, showed morphological abnormalities of epidermal and parenchymatous cells and retardation of differentiation of vascular systems. These phenotypes were suppressed by exogenous L-Pro but not by D-Pro or other Pro analogues. In addition, Pro deficiency did not broadly affect all proteins but specifically affected structural proteins of cell walls in the antisense transgenic plants. These results indicate that Pro is not just an osmoregulator in stressed plants but has a unique function involved in osmotolerance as well as in morphogenesis as a major constituent of cell wall structural proteins in plants.

Summary A cDNA (1122 bp) was isolated from a cDNA library prepared from Arabidopsis thaliana L. that had been subjected to drought stress for 1 h. The sequencing of a genomic clone corresponding to the cDNA and S1 mapping analysis revealed that the cDNA lacked the first 6 bp from its translational start (ATG). The resulting open reading frame encodes a polypeptide of 321 amino acids, and the calculated molecular weight of this polypeptide is 36 423 Da. The deduced amino acid sequence shows a high degree of similarity to C terminal halves of those of soluble epoxide hydrolases (sEHs) of human, mouse and rat, 35.5%, 34.1% and 33.1%, respectively. The cDNA was expressed in Escherichia coli cells, and the expressed protein migrates at 40 kDa when analyzed by SDS—PAGE. The recombinant protein at 40 kDa is much smaller than the mammalian sEH (58 kDa) but has characteristics of activity and inhibition similar to the mammalian sEHs when assayed with the substrate trans-stilbene oxide and the inhibitors 4-fluorochalcone oxide (4FCO), (2R,3R)-3-(4-nitrophenyl) glycidol (RRNPG), and (2S,3S)-3-(4-nitrophenyl)glycidol (SSNPG), which indicates that the cDNA did encode a soluble epoxide hydrolase of A. thaliana (AtsEH). Drought stress, but not heat or cold stress, slightly increased the accumulation of the mRNAs for AtsEH. The level of AtsEH transcripts increased strongly after treatment with a plant hormone, auxin (2,4-dichlorophenoxyacetic acid, 2,4-D; naphthaleneacetic acid, NAA; and indole-3-acetic acid, IAA) in young, pre-bolting plants. Treatment with cytokinin (6-benzylaminopurine, BA), abscisic acid (ABA) or gibberellin (GA3) had no detectable effect on AtsEH transcript levels. The transcripts for AtsEH gene were detected in the aerial vegetative organs of bolting plants (i.e. stems and leaves), but not in roots, flowers and seeds. The possible function of AtsEH is discussed. A similar sEH cDNA has recently been characterized in potato (Stapleton et al., 1994).

Summary Two cDNA clones, cATMPK1 and CATMPK2, encoding MAP kinases (mitogen-activated protein kinases) have been cloned from Arabidopsis thaliana and their nucleotide sequences have been determined. Putative proteins encoded by ATMPK1 and ATMPK2 genes, designated ATMPK1 and ATMPK2, contain 370 and 376 amino acid residues, respectively, and are 88.7% identical at the amino acid sequence level. ATMPK1 and ATMPK2 exhibit significant similarity to rat ERK2 (49%) and Xenopus MAP kinase (50%). The amino acid residues corresponding to the sites of phosphorylation (Thr-Glu-Tyr) that are involved in the activation of MAP kinases are conserved in ATMPK1 and ATMPK2. Northern blot analysis indicates that the ATMPK1 and ATMPK2 mRNAs are significantly present in all the organs except seeds. Genomic Southern blot analysis suggests that there are a few additional genes that are related to ATMPK1 and ATMPK2 in the Arabidopsis genome. Purified Xenopus MAP kinase kinase (MAPK kinase) phosphorylates ATMPK1 and ATMPK2 proteins that have been expressed in Escherichia coli, activating these enzymes. A rapid and transient activation of 46-kDa protein kinase activity that phosphorylated myelin basic protein (MBP) was detected when auxinstarved tobacco BY-2 cells were treated with synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). Protein kinase activities which phosphorylated the recombinant ATMPK2 protein also increased rapidly after auxin treatment in the auxin-starved BY-2 cells. These results suggest that auxin may function as an activator of plant MAP kinase homologues, as do various mitogens in animal systems.