Two calcium mobilizing pathways implicated within abscisic acid-induced stomatal closing in Arabidopsis thaliana

The present study investigated whether, depending on the abscisic acid (ABA) concentration, phospholipase C (PLC) would be implicated within a Ca2+ mobilizing pathway that would regulate stomatal aperture under standard watering conditions. Among Al sensitive mutants the als1-1 mutant of Arabidopsis thaliana (L.) Heynh. (Columbia-4 ecotype) was selected for a pharmacological approach of stomatal closing in leaf epidermal peels induced by 3, 20 or 30 µM ABA. Comparison with the wild type (WT) revealed that, exclusively in the als1-1 mutant, the stomatal response to 3 or 20 µM ABA was inhibited by about 40 %, whereas the stomatal response to 30 µM ABA and the wilting response to drought were unaffected. In WT, the Ca2+ buffer EGTA and the PLC inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), specifically inhibited by about 70 and 40 %, respectively, the response to 3 or 20 µM ABA, while the Ca2+ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibited by about 70 % the response to 3, 20 or 30 µM ABA. EGTA, BAPTA and U73122 did not inhibit the part of the response to 3 or 20 µM ABA that was unaffected by the als1-1 mutation. Together, these results showed that ABA closes the stomata through two different Ca2+ mobilizing pathways. Since PLC could be indirectly deactivated in the als1-1 mutant, these results might suggest that, under sufficient water supply, PLC-mediated Ca2+ mobilization is needed for the regulation of stomatal aperture by endogenous ABA resting at concentrations below a drought-specific threshold value.