1. Inhibition of root plasma membrane H + -ATPase by drought is mediated by the abscisic acid signalling pathway M. Dolores Planes 1, Hassan M. Younis 2 and Ramón Serrano 1 1 Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-C.S.I.C., Camino de Vera s/n, 46022 Valencia, Spain 2 Department of Pesticide Chemistry, Faculty of Agriculture, El-Shatby, 21545 Alexandria, Egypt BACKGROUND AND SUMMARY The plasma membrane H + -ATPase of stomata gueard cells is inhibited by abscisisc acid (ABA) during drought stress and this is important for stomata closure (Merlot et al, 2007, EMBO J 26: 3216). In the present work we have investigated the regulation of the enzyme by drought and ABA in wheat and Arabidopsis roots. Root H + -ATPase is inhibited by drought. Addition of ABA was also inhibitory and no inhibition by drought was observed in an ABA- insensitive Arabidopsis mutant. Inhibition of H + -ATPase correlates with neither decreased phosphorylation of the penultimate threonine nor decreased amount of the enzyme. An inhibitory phosphorylation mediated by ABA-activated protein kinases (SnRK2) is proposed. ATP ADP + Pi H+H+ K, P, N Anions Wheat plants before and after 4 h drying out of hydroponics Control Desiccation Arabidopsis plants before and after 4 h drying out of hydroponics Wheat and Arabidopsis plants were grown hydroponically and, when indicated, roots were stressed by drying in the air for 4 h. Roots were homogenized and the plasma membrane purified by sucrose gradient centrifugation. ATPase activity resistant to molybdate, azide and nitrate but sensitive to vanadate was determined. Average and standard error of 4-6 experiments are given. nmoles Pi x min -1 x µg protein -1 -------------------------------------------------------------- Wheat control0.23 ± 0.03 Wheat desiccated0.18 ± 0.04 (78 %) Arabidopsis control0.26 ± 0.02 Arabidopsis desiccated0.19 ± 0.01 (73 %) DROUGHT STRESS INHIBITS ROOT PLASMA MEMBRANE H + -ATPase IN WHEAT AND IN ARABIDOPSIS DROUGHT INHIBITION OF H + -ATPase IS MEDIATED BY ABSCISIC ACID (ABA) nmoles Pi x min -1 x µg protein -1 ---------------------------------------- Arabidopsis wild type control0.26 ± 0.02 Drought stress0.19 ± 0.01 ABA 10 µM0.11 ± 0.01 Over-expression HAB1*0.20 ± 0.02 Over-expression HAB1, drought stress0.20 ± 0.01 *ABA insensitive The hypothesis to explain these results is that ABA, via PYR/PYL receptors, PP2Cs and SnRK2s, triggers phosphorylation of the plasma membrane H + -ATPase in an inhibitory site different from the activating phosphorylation of the penultimate threonine. Inhibition of root plasma membrane H + - ATPase by ABA may participate in the inhibition of root and general plant functions under stress conditions. ABA INHIBITION OF H + -ATPase IS ACCOMPANIED BY NEITHER DECREASED AMOUNT OF ENZYME NOR DECREASED PHOSPHORYLATION OF PENULTIMATE THREONINE Detection of the amount of H + -ATPase with antibody against the central conserved domain of AHA3 and detection of the phosphorylation state of the penultimate threonine of the H+- ATPase by reaction with antibody against the AHA2 phosphopeptide CDTAGHHYTpV. markers Protein staining Anti P- peptide Anti central domain - + - + - + ABA H + -ATPase