Aux/IAA proteins are phosphorylated by phytochrome in vitro

Auxin/indole-3-acetic acid (Aux/IAA) genes encode short-lived transcription factors that are induced as a primary response to the plant growth hormone IAA or auxin. Gain-of-function mutations in Arabidopsis genes, SHY2/IAA3, AXR3/IAA17, and AXR2/IAA7 cause pleiotropic phenotypes consistent with enha nced auxin responses, possibly by increasing Aux/IAA protein stability. Sem idominant mutations shy2-1D, shy2-2, axr3-1, and axr2-1 induce ectopic ligh t responses in dark-grown seedlings. Because genetic studies suggest that t he shy2-1D and shy2-2 mutations bypass phytochrome requirement for certain aspects of photomorphogenesis, we tested whether SHY2/IAA3 and related Aux/ IAA proteins interact directly with phytochrome and whether they are substr ates for its protein kinase activity. Here we show that recombinant Aux/IAA proteins from Arabidopsis and pea (Pisum sativum) interact in vitro with r ecombinant phytochrome A from oat (Avena sativa). We further show that reco mbinant SHY2/IAA3, AXR3/IAA17, IAA1, IAA9, and Ps-IAA4 are phosphorylated b y recombinant oat phytochrome A in vitro. Deletion analysis of Ps-IAA4 indi cates that phytochrome A phosphorylation occurs on the N-terminal half of t he protein. Metabolic labeling and immunoprecipitation studies with affinit y-purified antibodies to IAA3 demonstrate increased in vivo steady-state le vels of mutant IAA3 in shy2-2 plants and phosphorylation of the SHY2-2 prot ein in vivo. Phytochrome-dependent phosphorylation of Aux/IAA proteins is p roposed to provide one molecular mechanism for integrating auxin and light signaling in plant development.