Multiple bHLH Proteins form Heterodimers to Mediate CRY2-Dependent Regulation of Flowering-Time in Arabidopsis

Yawen Liu,Kunwu Li,Xu Li,Chentao Lin,Hongtao Liu,Peter H Quail

doi:10.1371/journal.pgen.1003861

Abstract

Arabidopsis thaliana cryptochrome 2 (CRY2) mediates light control of flowering time. CIB1 (CRY2-interacting bHLH 1) specifically interacts with CRY2 in response to blue light to activate the transcription of FT (Flowering Locus T). In vitro, CIB1 binds to the canonical E-box (CACGTG, also referred to as G-box) with much higher affinity than its interaction with non-canonical E-box (CANNTG) DNA sequences. However, in vivo, CIB1 binds to the chromatin region of the FT promoter, which only contains the non-canonical E-box sequences. Here, we show that CRY2 also interacts with at least CIB5, in response to blue light, but not in darkness or in response to other wavelengths of light. Our genetic analysis demonstrates that CIB1, CIB2, CIB4, and CIB5 act redundantly to activate the transcription of FT and that they are positive regulators of CRY2 mediated flowering. More importantly, CIB1 and other CIBs proteins form heterodimers, and some of the heterodimers have a higher binding affinity than the CIB homodimers to the non-canonical E-box in the in vitro DNA-binding assays. This result explains why in vitro CIB1 and other CIBs bind to the canonical E-box (G-box) with a higher affinity, whereas they are all associated with the non-canonical E-boxes at the FT promoter in vivo. Consistent with the hypothesis that different CIB proteins play similar roles in the CRY2-midiated blue light signaling, the expression of CIB proteins is regulated specifically by blue light. Our study demonstrates that CIBs function redundantly in regulating CRY2-dependent flowering, and that different CIBs form heterodimers to interact with the non-canonical E-box DNA in vivo.

Highlights

Cryptochromes are photolyase-like photoreceptors regulating photomorphogenesis in plants and the circadian clock in plants and animals [1,2,3,4]
We report here that in addition to CIB1, cryptochrome 2 (CRY2) interact with CIB1 related bHLH proteins, CIBs
We examined their ability to interact with CRY2. 4 of the 6 CIB1-related bHLH proteins examined (CIB2-At5g48560, CIB3-At3g07340, CIB4-At1g10120, CIB5-At1g26260) interacted with CRY2 in vitro in a pull-down assay (Figure S1B)

Summary

Introduction

Cryptochromes are photolyase-like photoreceptors regulating photomorphogenesis in plants and the circadian clock in plants and animals [1,2,3,4]. The Arabidopsis thaliana genome encodes at least two cryptochromes, cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2). Cryptochromes may mediate photoperiodic control of floral initiation by at least three different mechanisms: 1. Cryptochromes mediate light suppression of the COP1-dependent degradation of CONSTANS (CO) [7,8,9], which is a major transcription regulator of floral initiation. CO is a critical positive regulator of flowering in long day condition, CO promotes the flowering initiation by activating transcription of the florigen gene FT [10], which encodes a mobile transcriptional regulator that migrates from leaves to the apical meristem to activate transcription of floral meristem identity genes [11,12]. 2. Cryptochromes regulate the light entrainment of the circadian clock [13], and affect the expression of CO. Cryptochromes directly modulate the transcription of FT through interaction with CIB1, a basic-helix-loop-helix (bHLH) transcription factor, which was isolated in a blue light differentiated yeasttwo-hybrid screen [14]

Methods

Results

Discussion

Conclusion