Both auxin responses changed dramatically between concentrations of 1 nM and 1 mM ; the responses were similarly concentration dependent and highly correlated . The dose response curve of IAA induced hypocotyl elongation in Arabidopsis hypocotyls resembled those of other plant organs described previously . Taken together, these results indicate that auxin mediates the activation of the H ATPase in hypocotyl sections via phosphorylation of its penultimate Thr, with subsequent binding of a 14 3 3 protein to the phosphorylated H ATPase. Auxin Does Not Induce H ATPase Expression As shown in Figure 1, the amount of H ATPase protein did not change for at least 60 min after IAA application. We analyzed the transcriptional level of H ATPase in the IAA treated hypocotyls by quantitative reverse transcription PCR assays . Relative transcriptional levels of AHA1 and AHA2, which are the major H ATPase isogenes expressed in etiolated seedlings , were not changed by IAA treatment. In contrast, levels of the known auxin inducible genes KAT1 and IAA1 increased more than 10 fold in response to IAA.
Hence, an increase in the expression level of H ATPase is not required for early phase auxin induced hypocotyl elongation. Auxin Induces H ATPase Phosphorylation in tir1 1 afb2 3 and axr1 3 Mutant Plants IAA induced hypocotyl elongation was essentially unchanged, relative to the wild type, in both an auxinreceptor TIR1 AFB mutant, tir afb, and in themutant axr1 12, the regulatory IOX2 selleck chemicals component of the SCFTIR1 AFB complex , suggesting that auxin induces hypocotyl elongation in Arabidopsis without SCFTIR1 AFB signals. We then examined whether auxin induces H ATPase phosphorylation in the tir1 1 afb2 3 double mutant and the axr1 3 mutant . IAA induced phosphorylation of the H ATPase in these mutants, and the phosphorylation level was increased to the same extent as in wild type plants , suggesting that auxin increased the phosphorylation level of the penultimate Thr of the H ATPase without the involvement of TIR1 AFBs, although the IAA induced expression of KAT1 and IAA1, and elongation in these mutants, were less than those in wild type plants .
Auxin Induced Phosphorylation of the H ATPase Is Not Affected by PEO IAA and MG132 We next examined the effect of the auxin antagonist a indole 3 acetic acid , which specifically binds to the auxin receptor TIR1 AFBs and blocks TIR1 AFB functions . Pretreatment with 100 mM PEO IAA had no effect on the IAA induced phosphorylation Valproate of the H ATPase , although it suppressed the IAAinduced expression of KAT1 and IAA1 . In corroboration, pretreatment with 50 mM MG132, a proteasome inhibitor that inhibits ubiquitin ligase complex SCFTIR1 AFB dependent responses , also did not change the H ATPase phosphorylation level but rather suppressed the IAA induced expression of KAT1 and IAA1 .