Differential Activation of the Rice Sucrose Nonfermenting1–Related Protein Kinase2 Family by Hyperosmotic Stress and Abscisic Acid

To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1–related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain

Abscisic Acid–Induced Transcription Is Mediated by Phosphorylation of an Abscisic Acid Response Element Binding Factor, TRAB1

The rice basic domain/Leu zipper factor TRAB1 binds to abscisic acid (ABA) response elements and mediates ABA signals to activate transcription. We show that TRAB1 is phosphorylated rapidly in an in vivo labeling experiment and by phosphatase-sensitive mobility shifts on SDS–polyacrylamide gels. We had shown previously that a chimeric promoter containing GAL4 binding sites became ABA inducible when a GAL4 binding domain–TRAB1 fusion protein was present. This expression system allowed us to assay the ABA response function of TRAB1. Using this system, we show that Ser-102 of TRAB1 is critical for this function. Because no ABA-induced mobility shift was observed when Ser-102 was replaced by Ala, we suggest that this Ser residue is phosphorylated in response to ABA. Cell fractionation experiments, as well as fluorescence microscopy observations of transiently expressed green fluorescent protein–TRAB1 fusion protein, indicated that TRAB1 was localized in the nucleus independently of ABA. Our results suggest that the terminal or nearly terminal event of the primary ABA signal transduction pathway is the phosphorylation in the nucleus of preexisting TRAB1

アブラナ科野菜の穂発芽原因遺伝子の解明

application/pdf日本では、アブラナ科野菜の採種時期は梅雨と重なるため、高い頻度で穂発芽が発生し、種苗業界では深刻な問題となっている。本研究では、アブラナ科の穂発芽の原因遺伝子を同定するために、遺伝学的および分子生物学的解析を行った。ゲノム解析、遺伝子発現解析およびプロモーター発現解析によりアブラナ科野菜では、胚成長停止制御因子IAA30のプロモーターに普遍的な変異が生じていることを見出した。この変異が、アブラナ科野菜で穂発芽が生じる品種が多発する主要な原因であることが示唆された。さらに加えて、アブラナ科野菜の穂発芽は母性効果により生じ、母性効果で穂発芽をもたらす複数のQTLの存在を見出した。In Japan, because seeds of Brassicaceae are harvested during the rainy season, high frequency of preharvest sprouting is a serious problem in the seed industry. In this study, we performed genetic and molecular analyses for preharvest sprouting to elucidate the responsible genes in Brassicaceae. Genomic, gene expression, and promoter expression analyses revealed the conserved mutations in the promoters of the embryo growth arrest regulator IAA30 in Brassicaceae. These mutations were suggested to be responsible for the high incidence of preharvest sprouting in Brassicaceae. Furthermore, we found that preharvest sprouting in Brassicaceae is caused by maternal effects regulated via multiple QTLs.2019年度～2021年度科学研究費補助金(基盤研究(C))研究成果報告書19K0596

Ectopic gene expression and organogenesis in Arabidopsis mutants missing BRU1 required for genome maintenance

Chromatin reconstitution after DNA replication and repair is essential for the inheritance of epigenetic information, but mechanisms underlying such a process are still poorly understood. Previously, we proposed that Arabidopsis BRU1 functions to ensure the chromatin reconstitution. Loss-of-function mutants of BRU1 are hypersensitive to genotoxic stresses and cause release of transcriptional gene silencing of heterochromatic genes. In this study, we show that BRU1 also plays roles in gene regulation in euchromatic regions. bru1 mutations caused sporadic ectopic expression of genes, including those that encode master regulators of developmental programs such as stem cell maintenance and embryogenesis. bru1 mutants exhibited adventitious organogenesis, probably due to the misexpression of such developmental regulators. The key regulatory genes misregulated in bru1 alleles were often targets of PcG SET-domain proteins, although the overlap between the bru1-misregulated and PcG SET-domain-regulated genes was limited at a genome-wide level. Surprisingly, a considerable fraction of the genes activated in bru1 were located in several subchromosomal regions ranging from 174 to 944 kb in size. Our results suggest that BRU1 has a function related to the stability of subchromosomal gene regulation in the euchromatic regions, in addition to the maintenance of chromatin states coupled with heritable epigenetic marks

Double-Locking Mechanism of Self-Compatibility in Arabidopsis thaliana: The Synergistic Effect of Transcriptional Depression and Disruption of Coding Region in the Male Specificity Gene

Self-compatibility in Arabidopsis thaliana represents the relatively recent disruption of ancestral obligate cross pollination, recognized as one of the prevalent evolutionary pathways in flowering plants, as noted by Darwin. Our previous study found that inversion of the male specificity gene (SP11/SCR) disrupted self-incompatibility, which was restored by overexpressing the SCR with the reversed inversion. However, SCR in A. thaliana has other mutations aside from the pivotal inversion, in both promoter and coding regions, with probable effects on transcriptional regulation. To examine the functional consequences of these mutations, we conducted reciprocal introductions of native promoters and downstream sequences from orthologous loci of self-compatible A. thaliana and self-incompatible A. halleri. Use of this inter-species pair enabled us to expand the scope of the analysis to transcriptional regulation and deletion in the intron, in addition to inversion in the native genomic background. Initial analysis revealed that A. thaliana has a significantly lower basal expression level of SCR transcripts in the critical reproductive stage compared to that of A. halleri, suggesting that the promoter was attenuated in inducing transcription in A. thaliana. However, in reciprocal transgenic experiments, this A. thaliana promoter was able to restore partial function if coupled with the functional A. halleri coding sequence, despite extensive alterations due to the self-compatible mode of reproduction in A. thaliana. This represents a synergistic effect of the promoter and the inversion resulting in fixation of self-compatibility, primarily enforced by disruption of SCR. Our findings elucidate the functional and evolutionary context of the historical transition in A. thaliana thus contributing to the understanding of the molecular events leading to development of self-compatibility