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.

SummaryThe plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the generegulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.

GAMYB was first isolated as a positive transcriptional regulator of gibberellin (GA)-dependent ␣ -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 ␣ -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 ␣ -amylase in aleurone, OsGAMYB also is important for floral organ development and essential for pollen development.

SummaryTo show the involvement of microfilaments and microtubules in non-host resistance of barley, partially dissected coleoptiles which had been inoculated with a non-pathogen, Erysiphe pisi, were treated with several actin and tubulin inhibitors. If the coleoptiles were not treated with any of the inhibitors, the non-pathogen always failed to penetrate the coleoptile cells. However, when coleoptiles were treated with actin or tubulin polymerization or depolymerization inhibitors, the non-pathogen was able to penetrate successfully and to form haustoria in coleoptlie cells of a non-host plant, barley. Actin polymerization inhibitors, cytochalasins, were more effective in causing an increase in penetration efficiency of E. pisithan tubulin inhibitors. The effects of cytochalasins depended on the kind of cytochalasin; the strength of the actin depolymerizing activity correlated significantly with the efficiency of increasing the penetration of the non-pathogen. When both actin and tubulin inhibitors were added simultaneously, the polarization of defense-related responses, such as massive cytoplasmic aggregation, deposition of papillae and accumulation of autofluorescent compounds, at fungal penetration sites was suppressed. Actin inhibitors did not affect arrangement and stability of microtubules and vice versa, and a double treatment of coleoptile cells with both microfilament and microtubule inhibitors showed an additive effect in increasing the penetration efficiency of E. pisL Furthermore, cytochalasin A treatment allowed other non-pathogens, Colletotrichum lagenarium and Alternaria alternata, to penetrate successfully into the non-host barley cells. These results strongly suggest that microfilaments and microtubules might play important roles in the expression of non-host resistance of barley.

Abstract. The cytoskeleton in plant cells is a dynamic structure that can rapidly respond to extracellular stimuli. Alteration of the organization of microtubules and actin microfilaments was examined in mesophyll cells of flax, Linum usitatissimum L., during attempted infection by the flax rust fungus, MeIampsora lini (Ehrenb.) Lev. Flax leaves that had been inoculated with either a compatible (yielding a susceptible reaction) or an incompatible (yielding a resistant reaction) strain of M. lini were embedded in butyl-methylmethacrylate resin; sections of this material were immunofluorescently labelled with anti-tubulin or anti-actin and examined using confocal laser scanning microscopy. In uninfected leaves, microtubules in the mesophyll cells formed a transverse array in the cell cortex. Microfilaments radiated through the cytoplasm from the nucleus. In an incompatible interaction, microtubules and microfilaments were extensively reorganized in mesophyll cells that were in contact with fungal infection hyphae or haustorial mother cells before penetration of the cell by the infection peg. After the initiation of haustorium development, microtubules disappeared from the infected cells, and growth of the haustoria ceased. In an incompatible interaction, hypersensitive cell death occurred in more than 70% of infected cells but occurred in less than 20% of cells in compatible interactions. After the infected cell had undergone hypersensitive cell death, the cytoskeleton in neighbouring cells became focused on the walls shared with the necrotic cell. In compatible interactions, reorganization of the cytoskeleton was either not observed at all or was observed much less frequently up to 48 h after inoculation.