Taishi Umezawa

Genome sequence of the palaeopolyploid soybean

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create a chromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70% more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78% of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies. The two duplication events were followed by gene diversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.

Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis

Abscisic acid (ABA) signaling is important for stress responses and developmental processes in plants. A subgroup of protein phosphatase 2C (group A PP2C) or SNF1-related protein kinase 2 (subclass III SnRK2) have been known as major negative or positive regulators of ABA signaling, respectively. Here, we demonstrate the physical and functional linkage between these two major signaling factors. Group A PP2Cs interacted physically with SnRK2s in various combinations, and efficiently inactivated ABA-activated SnRK2s via dephosphorylation of multiple Ser/Thr residues in the activation loop. This step was suppressed by the RCAR/PYR ABA receptors in response to ABA. However the abi1–1 mutated PP2C did not respond to the receptors and constitutively inactivated SnRK2. Our results demonstrate that group A PP2Cs act as ‘gatekeepers’ of subclass III SnRK2s, unraveling an important regulatory mechanism of ABA signaling.

Molecular basis of the core regulatory network in ABA responses: sensing, signaling and transport.

ABA is a major phytohormone that regulates a broad range of plant traits and is especially important for adaptation to environmental conditions. Our understanding of the molecular basis of ABA responses in plants improved dramatically in 2009 and 2010, banner years for ABA research. There are three major components; PYR/PYL/ RCAR (an ABA receptor), type 2C protein phosphatase (PP2C; a negative regulator) and SNF1-related protein kinase 2 (SnRK2; a positive regulator), and they offer a double negative regulatory system, [PYR/PYL/RCAR—| PP2C—| SnRK2]. In the absence of ABA, PP2C inactivates SnRK2 by direct dephosphorylation. In response to environmental or developmental cues, ABA promotes the interaction of PYR/PYL/RCAR and PP2C, resulting in PP2C inhibition and SnRK2 activation. This signaling complex can work in both the nucleus and cytosol, as it has been shown that SnRK2 phosphorylates basic-domain leucine zipper (bZIP) transcription factors or membrane proteins. Several structural analyses of PYR/PYL/RCAR have provided the mechanistic basis for this ‘core signaling’ model, by elucidating the mechanism of ABA binding of receptors, or the ‘gate–latch–lock’ mechanism of interaction with PP2C in inhibiting activity. On the other hand, intercellular ABA transport had remained a major issue, as had intracellular ABA signaling. Recently, two plasma membrane-type ABC transporters were identified and shed light on the influx/efflux system of ABA, resolving how ABA is transported from cell to cell in plants. Our knowledge of ABA responses in plants has been greatly expanded from intracellular signaling to intercellular transport of ABA.

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.

Three Arabidopsis SnRK2 Protein Kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, Involved in ABA Signaling are Essential for the Control of Seed Development and Dormancy

ABA is an important phytohormone regulating various plant processes, including stress tolerance, seed development and germination. SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3 are redundant ABA-activated SNF1-related protein kinases 2 (SnRK2s) in Arabidopsis thaliana. We examined the role of these protein kinases in seed development and germination. These SnRK2 proteins were mainly expressed in the nucleus during seed development and germination. The triple mutant (srk2d srk2e srk2i) was sensitive to desiccation and showed severe growth defects during seed development. It exhibited a loss of dormancy and elevated seed ABA content relative to wild-type plants. The severity of these phenotypes was far stronger than that of any single or double SRK2D, SRK2E and SRK2I mutants, including the srk2d srk2i mutant. The triple mutant had greatly reduced phosphorylation activity in in-gel kinase experiments using basic leucine zipper (bZIP) transcription factors including ABI5. Microarray experiments revealed that 48 and 30% of the down-regulated genes in abi5 and abi3 seeds were suppressed in the triple mutant seeds, respectively. Moreover, disruption of the three protein kinases induced global changes in the up-regulation of ABA-repressive gene expression, as well as the down-regulation of ABA-inducible gene expression. These alterations in gene expression result in a loss of dormancy and severe growth defects during seed development. Collectively, these results indicate that SRK2D, SRK2E and SRK2I protein kinases involved in ABA signaling are essential for the control of seed development and dormancy through the extensive control of gene expression.

Three SnRK2 Protein Kinases are the Main Positive Regulators of Abscisic Acid Signaling in Response to Water Stress in Arabidopsis

Responses to water stress are thought to be mediated by transcriptional regulation of gene expression via reversible protein phosphorylation events. Previously, we reported that bZIP (basic-domain leucine zipper)-type AREB/ABF (ABA-responsive element-binding protein/factor) transcription factors are involved in ABA signaling under water stress conditions in Arabidopsis. The AREB1 protein is phosphorylated in vitro by ABA-activated SNF1-related protein kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). Consistent with this, we now show that SRK2D/E/I and AREB1 co-localize and interact in nuclei in planta. Our results show that unlike srk2d, srk2e and srk2i single and double mutants, srk2d srk2e srk2i (srk2d/e/i) triple mutants exhibit greatly reduced tolerance to drought stress and highly enhanced insensitivity to ABA. Under water stress conditions, ABA- and water stress-dependent gene expression, including that of transcription factors, is globally and drastically impaired, and jasmonic acid (JA)-responsive and flowering genes are up-regulated in srk2d/e/i triple mutants, but not in other single and double mutants. The down-regulated genes in srk2d/e/i and areb/abf triple mutants largely overlap in ABA-dependent expression, supporting the view that SRK2D/E/I regulate AREB/ABFs in ABA signaling in response to water stress. Almost all dehydration-responsive LEA (late embryogenesis abundant) protein genes and group-A PP2C (protein phosphatase 2C) genes are strongly down-regulated in the srk2d/e/i triple mutants. Further, our data show that these group-A PP2Cs, such as HAI1 and ABI1, interact with SRK2D. Together, our results indicate that SRK2D/E/I function as main positive regulators, and suggest that ABA signaling is controlled by the dual modulation of SRK2D/E/I and group-A PP2Cs.

The Regulatory Domain of SRK2E/OST1/SnRK2.6 Interacts with ABI1 and Integrates Abscisic Acid (ABA) and Osmotic Stress Signals Controlling Stomatal Closure in Arabidopsis

ABI1 and ABI2 encode PP2C-type protein phosphatases and are thought to negatively regulate many aspects of abscisic acid (ABA) signaling, including stomatal closure in Arabidopsis. In contrast, SRK2E/OST1/SnRK2.6 encodes an Arabidopsis SnRK2 protein kinase and acts as a positive regulator in the ABA-induced stomatal closure. SRK2E/OST1 is activated by osmotic stress as well as by ABA, but the independence of the two activation processes has not yet been determined. Additionally, interaction between SRK2E/OST1 and PP2C-type phosphatases (ABI1 and ABI2) is not understood. In the present study, we demonstrated that the abi1-1 mutation, but not the abi2-1 mutation, strongly inhibited ABA-dependent SRK2E/OST1 activation. In contrast, osmotic stress activated SRK2E/OST1 even in abi1-1 and aba2-1 plants. The C-terminal regulatory domain of SRK2E/OST1 was required for its activation by both ABA and osmotic stress in Arabidopsis. The C-terminal domain was functionally divided into Domains I and II. Domain II was required only for the ABA-dependent activation of SRK2E/OST1, whereas Domain I was responsible for the ABA-independent activation. Full-length SRK2E/OST1 completely complemented the wilty phenotype of the srk2e mutant, but SRK2E/OST1 lacking Domain II did not. Domain II interacted with the ABI1 protein in a yeast two-hybrid assay. Our results suggested that the direct interaction between SRK2E/OST1 and ABI1 through Domain II plays a critical role in the control of stomatal closure.

Antagonistic Interaction between Systemic Acquired Resistance and the Abscisic Acid–Mediated Abiotic Stress Response in Arabidopsis

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR development in dicotyledonous plants, such as tobacco (Nicotiana tabacum) and Arabidopsis thaliana, is mediated by salicylic acid (SA). Here, using two types of SAR-inducing chemicals, 1,2-benzisothiazol-3(2H)-one1,1-dioxide and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, which act upstream and downstream of SA in the SAR signaling pathway, respectively, we show that treatment with abscisic acid (ABA) suppresses the induction of SAR in Arabidopsis. In an analysis using several mutants in combination with these chemicals, treatment with ABA suppressed SAR induction by inhibiting the pathway both upstream and downstream of SA, independently of the jasmonic acid/ethylene-mediated signaling pathway. Suppression of SAR induction by the NaCl-activated environmental stress response proved to be ABA dependent. Conversely, the activation of SAR suppressed the expression of ABA biosynthesis–related and ABA-responsive genes, in which the NPR1 protein or signaling downstream of NPR1 appears to contribute. Therefore, our data have revealed that antagonistic crosstalk occurs at multiple steps between the SA-mediated signaling of SAR induction and the ABA-mediated signaling of environmental stress responses.

Arabidopsis DREB2A-Interacting Proteins Function as RING E3 Ligases and Negatively Regulate Plant Drought Stress–Responsive Gene Expression

The DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) transcription factor controls water deficit–inducible gene expression and requires posttranslational modification for its activation. The activation mechanism is not well understood; however, the stability of this protein in the nucleus was recently found to be important for its activation. Here, we report the isolation of Arabidopsis thaliana DREB2A-INTERACTING PROTEIN1 (DRIP1) and DRIP2, C3HC4 RING domain–containing proteins that interact with the DREB2A protein in the nucleus. An in vitro ubiquitination assay showed that they function as E3 ubiquitin ligases and are capable of mediating DREB2A ubiquitination. Overexpression of DRIP1 in Arabidopsis delayed the expression of DREB2A-regulated drought-responsive genes. Drought-inducible gene expression was slightly enhanced in the single T-DNA mutants of drip1-1 and drip2-1. By contrast, significantly enhanced gene expression was revealed in the drip1 drip2 double mutant under dehydration stress. Collectively, these data imply that DRIP1 and DRIP2 function negatively in the response of plants to drought stress. Moreover, overexpression of full-length DREB2A protein was more stable in drip1-1 than in the wild-type background. These results suggest that DRIP1 and DRIP2 act as novel negative regulators in drought-responsive gene expression by targeting DREB2A to 26S proteasome proteolysis.

Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase

The Arabidopsis thaliana K+ channel KAT1 has been suggested to have a key role in mediating the aperture of stomata pores on the surface of plant leaves. Although the activity of KAT1 is thought to be regulated by phosphorylation, the endogenous pathway and the primary target site for this modification remained unknown. In the present study, we have demonstrated that the C-terminal region of KAT1 acts as a phosphorylation target for the Arabidopsis calcium-independent ABA (abscisic acid)-activated protein kinase SnRK2.6 (Snf1-related protein kinase 2.6). This was confirmed by LC-MS/MS (liquid chromatography tandem MS) analysis, which showed that Thr306 and Thr308 of KAT1 were modified by phosphorylation. The role of these specific residues was examined by single point mutations and measurement of KAT1 channel activities in Xenopus oocyte and yeast systems. Modification of Thr308 had minimal effect on KAT1 activity. On the other hand, modification of Thr306 reduced the K+ transport uptake activity of KAT1 in both systems, indicating that Thr306 is responsible for the functional regulation of KAT1. These results suggest that negative regulation of KAT1 activity, required for stomatal closure, probably occurs by phosphorylation of KAT1 Thr306 by the stress-activated endogenous SnRK2.6 protein kinase.