Cooperative Function of PLDδ and PLDα1 in Abscisic Acid-Induced Stomatal Closure in Arabidopsis    

Abstract

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.

Abscisic Acid Synthesis and Response

“…Structural differences among the PLDs result in different requirements for Ca 2+ or phospholipids as cofactors, as well as different substrate preferences (reviewed in Guo and Wang, 2012). Many of the PLDs are induced by a variety of stresses (Wang et al, 2000) , but only PLDα1 (AT3G15730) and PLDd (At4g35790) expression and activity are increased by ABA (Uraji et al, 2012). Consistent with roles for these family members in ABA signaling, mutations in these genes impair ABA effects on stomatal closure and germination inhibition.…”

Calcium-Dependent Protein Kinase CPK6 Positively Functions in Induction by Yeast Elicitor of Stomatal Closure and Inhibition by Yeast Elicitor of Light-Induced Stomatal Opening in Arabidopsis

“…Elevation of [Ca 2+ ] cyt activates slow anion (S-type) channels and down-regulates inward-rectifying potassium (K in ) channels in guard cells (Schroeder and Hagiwara, 1989;Grabov and Blatt, 1999). The activation of S-type channels is a hallmark of stomatal closure, and the suppression of K in channels is favorable to stomatal closure but not to stomatal opening (Pei et al, 1997;Kwak et al, 2001;Xue et al, 2011;Uraji et al, 2012).…”

“…Inhibition of K in channels is preferred in stomatal closure and impairs light-induced stomatal opening (Schroeder et al, 1987;Kwak et al, 2001;Khokon et al, 2011;Uraji et al, 2012). A 22-amino acid sequence of the conserved N-terminal part of flagellin, flg22, inhibits K in channels in Arabidopsis guard cells (Zhang et al, 2008).…”

“…The luminescence was measured for 6 s. The experiments were repeated six times. H 2 O 2 accumulation in guard cells was evaluated using 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCF-DA; Uraji et al, 2012). Epidermal tissues were isolated after leaves of 4-to 6-week-old plants shredded with a commercial blender.…”

“…Current measurements of K in , I Ca , and S-type channels in Arabidopsis guard cells were performed as described previously (Mori et al, 2006;Uraji et al, 2012). Arabidopsis GCPs were prepared from rosette leaves of 4-to 6-week-old plants with the digestion solution containing 1.0% (w/v) Cellulase R10, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) bovine serum albumin, 0.1% (w/v) kanamycin, 10 mM ascorbic acid, 0.1 mM KCl, 0.1 mM CaCl 2 , and 500 mM D-mannitol (pH 5.5) with KOH.…”