guard cells responds to ABA with rapid increase in cytosolic
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asIn guard cells of open stomata under daylight, long actin filaments are arranged at the cortex, radiating out from the stomatal pore. Abscisic acid (ABA), a signal for stomatal closure, induces rapid depolymerization of cortical actin filaments and the slower formation of a new type of actin that is randomly oriented throughout the cell. This change in actin organization has been suggested to be important in signaling pathways involved in stomatal closing movement, since actin antagonists interfere with normal stomatal closing responses to ABA. Here we present evidence that the actin changes induced by ABA in guard cells of dayflower (Commelina communis) are mediated by cytosolic calcium levels and by protein phosphatase and protein kinase activities. Treatment of guard cells with CaCl2 induced changes in actin organization similar to those induced by ABA. Removal of extracellular calcium with EGTA inhibited ABA-induced actin changes. These results suggest that Ca2+ acts as a signal mediator in actin reorganization during guard cell response to ABA. A protein kinase inhibitor, staurosporine, inhibited actin reorganization in guard cells treated with ABA or CaCl2, and also increased the population of cells with long radial cortical actin filaments in untreated control cells. A protein phosphatase inhibitor, calyculin A, induced fragmentation of actin filaments in ABA- or CaCl2-treated cells and in control cells, and inhibited the formation of randomly oriented long actin filaments induced by ABA or CaCl2. These results suggest that protein kinase(s) and phosphatase(s) participate in actin remodeling in guard cells during ABA-induced stomatal closure.
In guard cells of open stomata under daylight, long actin filaments are arranged at the cortex, radiating out from the stomatal pore (Kim et al., 1995; Eun and Lee, 1997). When the guard cells detect abscisic acid (ABA), a signal for stomatal closure, these cortical actin filaments disintegrate (Eun and Lee, 1997). This change in actin organization appears to be important in stomatal closing movement, since actin antagonists alter the normal stomatal responses to ABA (Hwang et al., 2000). Although many lines of evidence suggest that actin participates in signaling pathways involved in stomatal movement, upstream regulators and downstream targets of actin have not been well characterized.
One of the earliest responses of guard cells to stomatal closing signals is an increase in the intracellular calcium ion concentration ([Ca2+]i). Experimental elevation of [Ca2+]i induces stomatal closure and mimics several effects of ABA on ion channels in guard cells (Assmann, 1993; McAinsh et al., 1995). In addition, Ca2+ regulates cellular actin dynamics in animal cells via Ca2+-dependent actin-binding proteins including gelsolin, filamin, fimbrin, and α-actinin (Puius et al., 1998). Therefore, cytosolic free Ca2+ is a second messenger for stomatal closing signals and is a candidate mediator of actin changes during stomatal closure.