A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions

The stress hormone abscisic acid (ABA) induces expression of defense genes in many organs, modulates ion homeostasis and metabolism in guard cells and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H+ efflux (wat1-1D, over-expression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than wild type. This suggested that ABA could inhibit H+ efflux (H+-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibits the root plasma membrane H+-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H+ efflux from seedling roots). This inhibition involves the core ABA signaling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1) and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicate that ABA, acting through the PYR/PYL/RCAR receptors, induces membrane hyperpolarization (due to K+ efflux through GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H+-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots are different from those in guard cells. ABA does not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H+-ATPase in roots and SnRK2.2 phosphorylates in vitro the C-terminal regulatory domain of H+-ATPase while the guard cell kinase SnRK2.6/OST1 does not.
This work was funded by grants BFU2011-22526 (to RS) and BIO2011-23446 (to PLR) of the Spanish ‘Ministerio de Economía y Competitividad’, Madrid, Spain, and grant PROMETEO/2010/038 (to RS) of the ‘Generalitat Valenciana’, Valencia, Spain. MGG was funded by a JAE-DOC contract of the Spanish ‘Consejo Superior de Investigaciones Científicas’, Madrid, Spain.