+
H -ATPase
Inhibition of root plasma membrane
by the abscisic acid signalling pathway
by drought is mediated
M. Dolores Planes1, Hassan M. Younis2 and Ramón Serrano1
1Instituto
de Biología Molecular y Celular de Plantas, Universidad Politécnica
de Valencia-C.S.I.C., Camino de Vera s/n, 46022 Valencia, Spain
2Department 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 ABAinsensitive 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.
DROUGHT STRESS INHIBITS ROOT PLASMA MEMBRANE
H+-ATPase IN WHEAT AND IN ARABIDOPSIS
Anions
K, P, N
H+
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 plants before and after
4 h drying out of hydroponics
Control
Desiccation
Arabidopsis plants before and after
4 h drying out of hydroponics
DROUGHT INHIBITION OF H+-ATPase IS
MEDIATED BY ABSCISIC ACID (ABA)
nmoles Pi x min-1 x µg protein-1
---------------------------------------Arabidopsis wild type control
0.26 ± 0.02
Drought stress
0.19 ± 0.01
ABA 10 µM
0.11 ± 0.01
Over-expression HAB1*
0.20 ± 0.02
Over-expression HAB1, drought stress
0.20 ± 0.01
Wheat control
Wheat desiccated
Arabidopsis control
Arabidopsis desiccated
0.23 ± 0.03
0.18 ± 0.04 (78 %)
0.26 ± 0.02
0.19 ± 0.01 (73 %)
ABA INHIBITION OF H+-ATPase IS ACCOMPANIED
BY NEITHER DECREASED AMOUNT OF ENZYME
NOR DECREASED PHOSPHORYLATION OF
PENULTIMATE THREONINE
MW
170
130
-
+
-
+ -
+
ABA
H+-ATPase
95
72
55
*ABA insensitive
43
34
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.
26
markers
Protein Anti P- Anti central
staining peptide domain
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.