These results suggest that endogenous auxin in the hypocotyl sect

These results suggest that endogenous auxin in the hypocotyl sections becomes rapidly depleted after removal of the cotyledons. When 10 mM IAA was applied to the auxin depleted hypocotyl sections, elongation began after a short lag phase of around 10 min . Elongation reached a maximum rate of 8.8 mm min21 approximately 25 min after the addition of IAA; this rate was maintained for at least 60 min . The time course of the IAAinduced hypocotyl elongation was identical to that seen in a variety of previously studied plants . Vanadate, an inhibitor of P type ATPase, including the plasma membrane H ATPase , suppressed the IAA induced elongation , suggesting that H ATPase activity is required for auxin induced elongation. Auxin Induces Phosphorylation of the H ATPase in Hypocotyl Sections The fungal toxin FC is known to enhance H ATPase activity through phosphorylation of the penultimate Thr as well as to induce elongation . Therefore, we examined the FC induced hypocotyl elongation and H ATPase phosphorylation to confirm that our assay system was usable for analysis of the phosphorylation status of the H ATPase in response to auxin.
The amount Quizartinib selleckchem of H ATPase and the phosphorylation status of its penultimate Thr were detected by immunoblot analysis using anti H ATPase and anti pThr 947, respectively. These antibodies were raised against the catalytic domain of Arabidopsis H ATPase2 and the phosphorylated penultimate Thr 947 of AHA2 . As shown in Supplemental Figure S2, FC induced hypocotyl elongation and phosphorylation of H ATPase were detected, indicating that this assay system is suitable for analyzing H ATPase phosphorylation in Arabidopsis hypocotyls. Next, we examined the phosphorylation status of the penultimate Thr of the H ATPase in hypocotyl sections in response to auxin. Exogenous IAA induced the phosphorylation of the H ATPase within 10 min. The phosphorylation level peaked 20 min after the addition of IAA and was maintained at this level for at least 60 min . Phosphorylation of the H ATPase preceded an increase in the hypocotyl elongation rate by about 5 min .
Furthermore, IAA induced the binding of a 14 3 3 protein to the H ATPase and enhanced ATP hydrolysis by the plasma membrane H ATPase in hypocotyl sections . In this study, we detected only 20% stimulation of ATP hydrolysis by auxin. It is most likely that the phosphorylated H ATPase is Sunitinib subsequently dephosphorylated during the ATP hydrolysis assay, because the reaction mixture for this assay contains Mg2 . Our previous work indicates that the phosphorylated H ATPase is dephosphorylated in the presence of Mg2 in vitro . We further examined the dose responses of H ATPase phosphorylation and hypocotyl elongation to exogenous IAA.