ABSTRACT

In this study, we examined the involvement of endogenous abscisic acid (ABA) in methyl jasmonate (MeJA)-induced stomatal closure using an inhibitor of ABA biosynthesis, fluridon (FLU), and an ABA-deficient Arabidopsis (Arabidopsis thaliana) mutant, aba2-2. We found that pretreatment with FLU inhibited MeJA-induced stomatal closure but not ABA-induced stomatal closure in wild-type plants. The aba2-2 mutation impaired MeJA-induced stomatal closure but not ABA-induced stomatal closure. We also investigated the effects of FLU and the aba2-2 mutation on cytosolic free calcium concentration ( ] cyt elevation. We also tested the effects of the aba2-2 mutation and FLU on the expression of MeJA-inducible VEGETATIVE STORAGE PROTEIN1 (VSP1). In the aba2-2 mutant, MeJA did not induce VSP1 expression. In wild-type leaves, FLU inhibited MeJA-induced VSP1 expression. Pretreatment with ABA at 0.1 mM, which is not enough concentration to evoke ABA responses in the wild type, rescued the observed phenotypes of the aba2-2 mutant. Finally, we found that in wild-type leaves, MeJA stimulates the expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE3, which encodes a crucial enzyme in ABA biosynthesis. These results suggest that endogenous ABA could be involved in MeJA signal transduction and lead to stomatal closure in Arabidopsis guard cells.Stomatal pores are surrounded by pairs of guard cells in the leaf epidermis of higher plants. Guard cells respond to a variety of external and internal stimuli such as light, drought, external Ca 2+ , pathogen attack, and the phytohormones abscisic acid (ABA) and methyl jasmonate (MeJA) and regulate CO 2 uptake into leaves for photosynthesis, control of transpirational water loss, and innate immunity (Schroeder et al

Yeast elicitor (YEL) induces stomatal closure. We investigated reactive oxygen species (ROS) production, nitric oxide (NO) production and [Ca(2+)](cyt) oscillations to clarify YEL signaling in Arabidopsis guard cells. YEL induced ROS accumulation in guard cells. A peroxidase inhibitor [salicylhydroxamic acid (SHAM)] inhibited the stomatal closure and the ROS accumulation, but neither the atrbohD atrbohF mutation nor an NADPH oxidase inhibitor [diphenylene iodonium chloride (DPI)] had any effect. An NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)] inhibited the YEL-induced stomatal closure and SHAM abolished NO production. YEL-elicited [Ca(2+)](cyt) oscillations were inhibited by SHAM but not by the atrbohD atrbohF mutation. These results indicate that YEL induces stomatal closure accompanied by ROS production mediated by peroxidases and NO production.

Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDa1 and PLDd, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, plda1 and pldd single mutants, and a plda1 pldd double mutant. ABA-induced stomatal closure was suppressed in the plda1 pldd double mutant but not in the pld single mutants. The plda1 and pldd mutations reduced ABAinduced phosphatidic acid production in epidermal tissues. Expression of either PLDa1 or PLDd complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both plda1 and pldd single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDa1 and PLDd function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the plda1 pldd double mutant. Inward-rectifying K + channel currents of guard cells were inhibited by ABA in the wild type but not in the plda1 pldd double mutant. ABA inhibited stomatal opening in the wild type and the pldd mutant but not in the plda1 mutant. In wild-type rosette leaves, ABA significantly increased PLDd transcript levels but did not change PLDa1 transcript levels. Furthermore, the plda1 and pldd mutations mitigated ABA inhibition of seed germination. These results suggest that PLDa1 and PLDd cooperate in ABA signaling in guard cells but that their functions do not completely overlap.

Thioglucoside glucohydrolase (myrosinase), TGG1, is a strikingly abundant protein in Arabidopsis guard cells. We investigated responses of tgg1-3, tgg2-1 and tgg1-3 tgg2-1 mutants to abscisic acid (ABA) and methyl jasmonate (MeJA) to clarify whether two myrosinases, TGG1 and TGG2, function during stomatal closure. ABA, MeJA and H(2)O(2) induced stomatal closure in wild type, tgg1-3 and tgg2-1, but failed to induce stomatal closure in tgg1-3 tgg2-1. All mutants and wild type showed Ca(2+)-induced stomatal closure and ABA-induced reactive oxygen species (ROS)production. A model is discussed in which two myrosinases redundantly function downstream of ROS production and upstream of cytosolic Ca(2+) elevation in ABA and MeJA signaling in guard cells.