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.

Cell transformations accompany alterations in cell morphology and microfilament patterns. Calvasculin encodes mRNA termed pEL‐98, 18A2, 42A, p9Ka, or mtsl, found to be elevated in several metastatic cell lines. We report the elevation of calvasculin expression in SR‐3Y1 cells, which show disappearance of ordered microfilaments, compared to that in 3Y1 cells and that the similar distribution of calvasculin to that of actin filaments. Interestingly, calvasculin co‐sediments with F‐actin and bundles actin filaments in a Ca2+‐dependent manner. This activity, along with the elevation of calvasculin following transformation, suggests that the disorganization of filaments in SR‐3Y1 cell is due to the cross‐linking activity of calvasculin.

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It is generally known that unilamellar vesicles prepared from zwitterionic phosphatidylcholines (PCs) do not aggregate in the presence of multivalent cations, as opposed to the case of electrostatically stabilized vesicles of acidic phospholipids. However, we have found that Be2+ can induce the aggregation of PC vesicles. The aggregation exhibited characteristic features concerning the Be2+ concentration and temperature, as follows:  (i) there exists an optimum concentration range of Be2+ to induce the aggregation, and (ii) the aggregation becomes most pronounced at the temperature corresponding to the bilayer phase transition of the vesicle membranes; it is suppressed completely at higher temperatures. In addition, the Be2+-induced aggregation of PC vesicles was found to be reversible with respect to the Be2+ concentration and temperature. The reversibility of the aggregation suggests that vesicles are trapped in a secondary minimum of intervesicular potential in the aggregating entities. This in turn leads to the speculation that the repulsive hydration force characteristic of PC vesicles is partially destroyed by the action of Be2+.