Metabolite regulation of cryptochrome 2 activity and flowering time

UV-A/蓝光受体隐花色素2(Cry2)在光周期花诱导中起核心作用。Cry2信号通路中的重要组成部分是bHLH转录因子(CIB1、CIB2、CIB4、CIB5)，它们在蓝光下与Cry2相关。这些异源二聚体然后与开花基因T(FT)启动子区域的非规范E-box结合，从而诱导这个重要的开花基因的表达。此外，Cry2与SPA蛋白相互作用，从而抑制E3泛素连接酶构成的光形态发生酶1(COP1)的活性。COP1在FT激活剂Constans(CO)的降解中起主要作用。我们已经证明，在LIT状态下，Cry2的黄素发色团是以中性半喹酮形式存在的，这种氧化还原状态的形成被代谢产物如ATP和NADPH所促进。CRY2的结构定点突变揭示了代谢物控制所需的残基。这些信息将被用来阐明代谢产物在光周期开花中的作用。为此，我们计划原位表达Cry2变异体，并分析它们的开花时间、FT表达以及与下游伙伴(CIB、SPA、COP1)的相互作用。这些研究将得到重组蛋白体外研究的补充。与野生型Cry1不同的是，在短日照条件下，该突变等位基因能诱导CO和FT的表达，并能提前开花。结果表明，Cry1L407F蛋白在黑暗中的胰酶消化模式与野生型Cry1蛋白在蓝光下的消化模式相似，提示该蛋白具有不依赖于光的光态结构。我们将确定是否使用与Cry2相同的信号成分来诱导FT。为此，我们将分析该突变株在CIB1/CIB2/cib5突变背景下的开花时间和FT表达，以及该突变株与已知的Cry2合作伙伴之间的相互作用。我们打算与Christian Jung(甜菜开花控制)和Markus Schmids(通过海藻糖-6-磷酸信号调节开花时间)小组合作。

The UV-A/blue light receptor cryptochrome 2 (cry2) has a central role in photoperiodic flower induction. Important components in cry2 signaling are bHLH transcription factors (CIB1, CIB2, CIB4, CIB5), which associate with cry2 under blue light. These heterodimers then bind to non-canonical E-boxes in the promoter region of FLOWERING LOCUS T (FT) and thereby induce expression of this important flowering gene. Moreover, cry2 interacts with SPA proteins and thereby inhibits the activity of the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1). COP1 plays a major role in degradation of the FT activator CONSTANS (CO). We have shown that the flavin chromophore of cry2 in the lit state is in the neutral semiquinone form, and formation of this redox state is enhanced by metabolites such as ATP and NADPH. Structure-based site-directed mutagenesis of CRY2 revealed which residues are required for metabolite control. This information will be used for elucidating the role of metabolites in photoperiodic flowering. For this purpose, we plan to express the cry2 variants in situ and to analyze their flowering time, FT expression, and interaction with downstream partners (CIB, SPA, COP1). These studies will be complemented by in vitro studies of recombinant proteins. In contrast to wild-type cry1, the cry1L407F mutant allele induces the expression of CO and FT and flowers early under short-day conditions. We showed that the tryptic digestion pattern of the cry1L407F protein in the dark is similar to that of wild-type cry1 in blue light, which suggests a light-independent lit-state structure of cry1L407F. We will determine whether cry1L407F uses the same signaling components as cry2 for the induction of FT. For this purpose, we will analyze flowering time and FT expression of the cry1L407F mutant in the cib1/cib2/cib5 mutant background as well as the interaction of cry1L407F with known partners of cry2. We intend to collaborate with the groups of Christian Jung (flowering control in Beta vulgaris) and Markus Schmid (regulation of flowering time by trehalose-6-phosphate signaling).