Hyperosmotic stress induces the rapid phosphorylation of a soybean phosphatidylinositol transfer protein homolog through activation of the protein kinases SPK1 and SPK2.

Although phosphatidylinositol transfer proteins (PITPs) are known to serve critical functions in regulating a varied array of signal transduction processes in animals and yeast, the discovery of a similar class of proteins in plants occurred only recently. Here, we report the participation of Ssh1p, a soybean PITP-like protein, in the early events of osmosensory signal transduction in plants, a function not attributed previously to animal or yeast PITPs. Exposure of plant tissues to hyperosmotic stress led to the rapid phosphorylation of Ssh1p, a modification that decreased its ability to associate with membranes. An osmotic stress-activated Ssh1p kinase activity was detected in several plant species by presenting recombinant Ssh1p as a substrate in in-gel kinase assays. Elements of a similar osmosensory signaling pathway also were conserved in yeast, an observation that facilitated the identification of soybean protein kinases SPK1 and SPK2 as stress-activated Ssh1p kinases. This study reveals the activation of SPK1 and/or SPK2 and the subsequent phosphorylation of Ssh1p as two early successive events in a hyperosmotic stress-induced signaling cascade in plants. Furthermore, Ssh1p is shown to enhance the activities of a plant phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, an observation that suggests that the ultimate function of Ssh1p in cellular signaling is to alter the plant's capacity to synthesize phosphoinositides during periods of hyperosmotic stress.

Relationship between changes in the guard cell abscisic-acid content and other stress-related physiological parameters in intact plants.

The relationships of guard cell ABA content to eight stress-related physiological parameters were determined on intact Vicia faba L. plants that were grown hydroponically with split-root systems. Continuous stress was imposed by the addition of PEG to part of the root system. The water potentials of roots sampled after the addition of PEG were 0.25 MPa lower than the water potentials of other roots of the same plant, which were similar to the roots of untreated plants. The leaflet water potentials of plants sampled within 2 h of stress imposition were similar to those of control plants. However, leaf conductance was lower in plants sampled after only 20 min of stress imposition, and the root- and leaflet apoplastic ABA concentrations of these plants were higher than those of untreated plants. As the essence of this report, there was a linear relationship between guard cell ABA content and leaf conductance. Leaflet apoplastic ABA concentrations <150 nM were also linearly related to leaf conductance, but higher leaflet apoplastic ABA concentration did not cause equally large further declines in leaf conductance. It is suggested that evaporation from guard cell walls caused ABA to accumulate in the guard cell apoplast and this pool was saturated at high leaflet apoplastic ABA concentrations.

Abg1: a novel gene up-regulated by abscisic acid in guard cells of Vicia faba L.

A novel gene (abg1) was isolated by differential display RT-PCR from guard cells of Vicia faba L. Abg1 transcript accumulated in guard cells that were incubated with 5 microM S(+)-ABA for 1 h. The full-length abg1 cDNA was 753 bp, which included a 513 bp coding region. The deduced 17.8 kDa protein shared sequence similarity with several desiccation-related proteins reported in plants.

In vivo phosphorylation of phosphoenolpyruvate carboxylase in guard cells of Vicia faba L. is enhanced by fusicoccin and suppressed by abscisic acid.

Plants regulate water loss and CO2 gain by modulating the aperture sizes of stomata that penetrate the epidermis. Aperture size itself is increased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma. Guard cell phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Regulation of this cytosolic enzyme by effectors is well documented, but additional regulation by posttranslational modification is predicted by the alteration of PEPC kinetics during stomatal opening (FEBS Lett. 352, 45-48). In this study, we have investigated whether this alteration is associated with the phosphorylation status of this enzyme. Using sonicated epidermal peels ("isolated" guard cells) preloaded with 32PO4, we induced stomatal opening and guard cell malate accumulation by incubation with 5 microM fusicoccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 microM abscisic acid (ABA). The phosphorylation status of PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. PEPC was phosphorylated when stomata were stimulated to open, and phosphorylation was lessened by incubation with ABA. Thus, we conclude that regulation of guard cell PEPC in vivo is multifaceted; the effects of regulatory metabolites and the activation status of the enzyme are integrated to control malate synthesis. These results, together with the coincident alteration in the kinetics of the enzyme (FEBS Lett. 352, 45-48), constitute the first unequivocal demonstration of regulatory posttranslational modification of a guard cell protein that is specifically implicated in stomatal movements.