Novel components identified using activation tagging reveal new aspects of brassinosteroid signaling.

Abstract

Brassinosteroids (BRs) are a unique class of plant polyhydroxylated steroids that play important roles throughout plant life cycle. Unlike their animal counterparts that are recognized by the cognate nuclear receptors, BRs are perceived by a transmembrane receptor kinase, BRI1. BR perception at the cell surface initiates a phosphorylation-mediated signaling cascade, involving a GSK3-like kinase, BIN2, and its substrates, BES1 and BZR1, to regulate expression of a variety of BR-responsive genes. To identify additional components of BR signaling, we used a gain-of-function approach, activation tagging, to identify genes whose overexpression can suppress a weak <italic> bri1</italic> mutant. Two dominant <italic>bri1</italic> suppressors, <italic> abs1-D</italic> and <italic>abs2-D</italic> for activation-tagged <italic> bri1</italic> suppressor 1 and 2, respectively, were isolated from this screen. <italic>ABS1</italic> encodes a small transcription factor with an atypical basic helix-loop-helix (bHLH) domain. Genetic and biochemical studies suggest that overaccumulated ABS1 stimulates BR signaling by sequestering a negative regulator of the BR signaling pathway. A yeast two-hybrid screen using ABS1 as bait identified AIF1 (ABS1- Interacting Factor 1), another atypical bHLH protein, as a potential candidate for such a negative regulator. Overexpression of <italic>AIF1</italic> gave rise to a <italic>bri1</italic>-like morphology, whereas RNAi-mediated gene silencing of <italic>AIF1</italic> partially suppressed the <italic>bri1</italic> mutation. My results suggest that heterodimerization between two atypical bHLH proteins might be an important mechanism to regulate expression of BR-responsive genes. <italic>ABS2</italic> encodes a plasma membrane localized protein containing a plant specific domain of unknown function (DUF) 740. Its overexpression constitutively activates the BR signaling pathway, leading to a phenotype similar to that of mutant lacking BIN2 and its two closest homologs or transgenic plant accumulating BIN2 substrates. Genetic and biochemical studies suggest that ABS2 activates BR signaling by inhibiting BIN2 activity. ABS2 interacts with BIN2 and ABS2 overexpression led to accumulation of non-phosphorylated BES1, whereas overexprssion of mutated ABS2 proteins incapable of BIN2 interaction failed to produce a visible morphological phenotype. Taken together, my studies not only reveal a novel regulatory mechanism to control the nuclear activity of the BR signaling pathway but also identify a potential signaling component that links the BR perception at the cell surface to BR signaling in the cytosol.