Hydrogen peroxide affects abscisic acid binding to ABAP1 in barley aleurones

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Abstract
Dramatic increases in H2O2 levels have been observed following abscisic acid (ABA) treatment of plant tissues. Following ABA treatment in aleurone cells, H2O2 reached transient levels of approximately 115 µmol/L H2O2. To determine whether ABA perception was modified by such changes, the effect of H2O2 on a recently characterized ABA-binding protein (ABAP1), cloned from barley aleurone layers, was examined. ABA binding to the protein was weakened by H2O2 in a concentration-dependent manner. A concentration of 75 µmol/L H2O2 gave a 50% decline in ABA binding in a reaction following first-order kinetics, indicative of binding-site susceptibility to its microenvironment. We monitored the unfolding of ABAP1 using steady-state and time-resolved tryptophan fluorescence, while following the capacity of ABAP1 to bind ABA. ABA binding decreased by 50% following ABAP1 denaturation with 1 mol/L guanidine hydrochloride or 2 mol/L urea, while the maximum emission spectra (λemi) red shifted from 338 to 347 nm at 3.5 mol/L guanidine hydrochloride and 5 mol/L urea. However, only a slight blue shift of λemi was observed following either ABAP1 incubation with H2O2 or binding to (+)-ABA (physiologically active ABA). The equilibrium ABA dissociation rate accelerated in the presence of 250 µmol/L H2O2, with the half-time dissociation reduced to 8 min. A comparison of inactivation kinetics and conformational changes shows that inactivation of ABAP1 occurs before any noticeable conformational change. This suggests that the ABA binding site is highly responsive to its microenvironment and is situated in a region that is more flexible than the protein molecule as a whole. The results demonstrate that H2O2, generated by ABA treatment of aleurone layers, is sufficient to affect the ABA-binding capacity of ABAP1, suggesting that this may be another level of control of ABA signal transduction.