Tsukaho Hattori

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.

Loss-of-Function Mutations of the Rice GAMYB Gene Impair α-Amylase Expression in Aleurone and Flower Development

GAMYB was first isolated as a positive transcriptional regulator of gibberellin (GA)-dependent α-amylase expression in barley aleurone cells, and its molecular and biochemical properties have been well characterized. However, the role of GAMYB elsewhere in the plant is not well understood. To investigate the molecular function of GAMYB outside of the aleurone cells, we isolated loss-of-function mutants from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17. Through PCR screening using primers for rice GAMYB (OsGAMYB) and Tos17, we isolated three independent mutant alleles that contained Tos17 inserted in the exon region. No α-amylase expression in the endosperm was induced in these mutants in response to GA treatment, indicating that the Tos17 insertion had knocked out OsGAMYB function. We found no significant defects in the growth and development of the mutants at the vegetative stage. After the phase transition to the reproductive stage, however, shortened internodes and defects in floral organ development, especially a defect in pollen development, were observed. On the other hand, no difference was detected in flowering time. High-level OsGAMYB expression was detected in the aleurone cells, inflorescence shoot apical region, stamen primordia, and tapetum cells of the anther, but only low-level expression occurred in organs at the vegetative stage or in the elongating stem. These results demonstrate that, in addition to its role in the induction of α-amylase in aleurone, OsGAMYB also is important for floral organ development and essential for pollen development.

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.

Molecular cloning of Sdr4, a regulator involved in seed dormancy and domestication of rice

Seed dormancy provides a strategy for flowering plants to survive adverse natural conditions. It is also an important agronomic trait affecting grain yield, quality, and processing performance. We cloned a rice quantitative trait locus, Sdr4, which contributes substantially to differences in seed dormancy between japonica (Nipponbare) and indica (Kasalath) cultivars. Sdr4 expression is positively regulated by OsVP1, a global regulator of seed maturation, and in turn positively regulates potential regulators of seed dormancy and represses the expression of postgerminative genes, suggesting that Sdr4 acts as an intermediate regulator of dormancy in the seed maturation program. Japonica cultivars have only the Nipponbare allele (Sdr4-n), which endows reduced dormancy, whereas both the Kasalath allele (Srd4-k) and Sdr4-n are widely distributed in the indica group, indicating prevalent introgression. Srd4-k also is found in the wild ancestor Oryza rufipogon, whereas Sdr4-n appears to have been produced through at least two mutation events from the closest O. rufipogon allele among the accessions examined. These results are discussed with respect to possible selection of the allele during the domestication process.

Arabidopsis NF-YB subunits LEC1 and LEC1-LIKE activate transcription by interacting with seed-specific ABRE-binding factors

LEAFY COTYLEDON 1 (LEC1) plays vital roles in the regulation of seed maturation in Arabidopsis. LEC1 encodes a homolog of yeast HAP3 or mammalian NF-YB/CBF-A subunit of trimeric CCAAT binding factor (CBF). Among the nine paralogs of NF-YB in Arabidopsis, LEC1-LIKE (L1L) is most closely related to LEC1, and can complement the lec1 mutation when expressed under the control of the LEC1 promoter. Although the nature of the B3-type seed maturation regulators as transcription factors have been investigated, knowledge of the molecular action of LEC1 is limited. When co-expressed with NF-YC2 in the presence of ABA, we found that LEC1 or L1L, but not other NF-YBs, activated the promoter of CRUCIFERIN C (CRC), which encodes a seed storage protein. However, additional expression of an NF-YA subunit interfered with the activation. The LEC1/L1L-[NF-YC2] activation depended on ABA-response elements present in the promoter, which led to the finding that LEC1/L1L-[NF-YC2] can strongly activate the CRC promoter in the absence of ABA when co-expressed with a seed-specific ABA-response element (ABRE)-binding factor, bZIP67. Functional coupling of LEC1/L1L-[AtNF-YC2] and bZIP67 was also observed in the regulation of sucrose synthase 2 (SUS2). Immunoprecipitation experiments revealed that L1L and bZIP67 formed a protein complex in vivo. These results demonstrate a novel plant-specific mechanism for NF-Y subunit function that enables LEC1 and L1L to regulate a defined developmental network.

Sequence and functional analyses of the rice gene homologous to the maize Vp1

The homologous gene of the maize Vp1 gene was isolated from rice (Oryza sativa). Sequence analysis revealed that the rice Vp1 gene (Osvp1) encodes a protein of 728 amino acids and is interupted by 5 introns at positions identical to those of the maize gene. Osvp1 transcript was detected in developing embryo as early as 10 days after flowering and decreased toward maturity. Osvp1 transcript was also detected in dry as well as imbided mature embryos. The ability of Osvp1 gene product to activate a target gene was shown by transient expression experiments in rice suspension-cultured cell protoplasts using a reporter gene construct carrying the bacterial β-glucuronidase (GUS) gene fused to the promoter of OsEm gene, the rice homologue of the wheat Em gene.

High-level expression of tuberous root storage protein genes of sweet potato in stems of plantlets grown in vitro on sucrose medium

Sporamin, the tuberous root storage protein of the sweet potato, accounts for about 60 to 80% of the total soluble protein of this organ. The amount of sporamin present in other organs is very low, or even not detectable, in the normal field-grown plants. However, the stem of sweet potato plantlets grown axenically on agar medium containing sucrose was found to accumulate large amounts of sporamin. Two-dimensional gel electrophoretic profiles of sporamin precursors synthesized in vitro by poly(A)+ RNA are indistinguishable between tuberous roots of the field-grown plants and stems of the axenically cultured plants, suggesting that an essentially identical set of the members of sporamin multigene family are expressed in these two organs under different growth conditions. Transgenic tobacco plants having a CAT (chloramphenicol acetyltransferase) fusion gene with the 5′ upstream region of a sporamin A gene, gSPO-A1, show preferential expression of CAT activity in stems when the plants are maintained in axenic culture on sucrose medium as is the case for sporamin in sweet potato. Deletion analysis revealed that the DNA sequence of gSPO-A1 between −94 and −305, relative to the transcription start site, is important for its expression in tobacco. This region contains two of the previously postulated putative regulatory elements conserved between sporamin A and B genes.

Molecular cloning and nucleotide sequence of cDNA for sporamin, the major soluble protein of sweet potato tuberous roots

SummarySporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)+ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.

RSS1 regulates the cell cycle and maintains meristematic activity under stress conditions in rice

Plant growth and development are sustained by continuous cell division in the meristems, which is perturbed by various environmental stresses. For the maintenance of meristematic functions, it is essential that cell division be coordinated with cell differentiation. However, it is unknown how the proliferative activities of the meristems and the coordination between cell division and differentiation are maintained under stressful conditions. Here we show that a rice protein, RSS1, whose stability is controlled by cell cycle phases, contributes to the vigour of meristematic cells and viability under salinity conditions. These effects of RSS1 are exerted by regulating the G1–S transition, possibly through an interaction of RSS1 with protein phosphatase 1, and are mediated by the phytohormone, cytokinin. RSS1 is conserved widely in plant lineages, except eudicots, suggesting that RSS1-dependent mechanisms might have been adopted in specific lineages during the evolutionary radiation of angiosperms.

Pleiotropic Effects of the Wheat Dehydrin DHN-5 on Stress Responses in Arabidopsis

We have previously reported that transgenic Arabidopsis plants overexpressing the wheat dehydrin DHN-5 show enhanced tolerance to osmotic stresses. In order to understand the mechanisms through which DHN-5 exerts this effect, we performed transcriptome profiling using the Affymetrix ATH1 microarray. Our data show an altered expression of 77 genes involved mainly in transcriptional regulation, cellular metabolism, stress tolerance and signaling. Among the up-regulated genes, we identified those which are known to be stress-related genes. Several late embryogenesis abundant (LEA) genes, ABA/stress-related genes (such as RD29B) and those involved in pathogen responses (PR genes) are among the most up-regulated genes. In addition, the MDHAR gene involved in the ascorbate biosynthetic pathway was also up-regulated. This up-regulation was correlated with higher ascorbate content in two dehydrin transgenic lines. In agreement with this result and as ascorbate is known to be an antioxidant, we found that both transgenic lines show enhanced tolerance to oxidative stress caused by H₂O₂. On the other hand, multiple types of transcription factors constitute the largest group of the down-regulated genes. Moreover, three members of the jasmonate-ZIM domain (JAZ) proteins which are negative regulators of jasmonate signaling were severely down-regulated. Interestingly, the dehydrin-overexpressing lines exhibit less sensitivity to jasmonate than wild-type plants and changes in regulation of jasmonate-responsive genes, in a manner similar to that in the jasmonate-insensitive jai3-1 mutant. Altogether, our data unravel the potential pleiotropic effects of DHN-5 on both abiotic and biotic stress responses in Arabidopsis.