Metabolite regulation of cryptochrome 2 activity and flowering time

UV-A/蓝光受体隐花色素2（cry 2）在光周期花诱导中具有核心作用。cry 2信号传导中的重要组分是bHLH转录因子（CIB 1、CIB 2、CIB 4、CIB 5），其在蓝光下与cry 2结合。这些异源二聚体然后结合到FLOWERLOCUS T（FT）的启动子区域中的非典型E盒，从而诱导该重要开花基因的表达。此外，cry 2与SPA蛋白相互作用，从而抑制E3泛素连接酶组成性光形态发生1（COP 1）的活性。COP 1在FT活化剂CONSTANS（CO）的降解中起主要作用。我们已经表明，cry 2在点燃状态下的黄素发色团是中性半醌形式，并且这种氧化还原状态的形成被代谢物如ATP和NADPH增强。基于结构的定点突变的EST 2揭示了哪些残基是代谢物控制所需的。这些信息将用于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cry 2变体，并分析其开花时间，FT表达以及与下游合作伙伴（CIB，SPA，COP 1）的相互作用。这些研究将通过重组蛋白的体外研究进行补充。与野生型cry 1相比，cry 1 L407 F突变等位基因诱导CO和FT的表达，并在短日照条件下提前开花。我们发现，在黑暗中的cry 1 L407 F蛋白的胰蛋白酶消化模式是类似的野生型cry 1在蓝光下，这表明一个光独立的光状态结构的cry 1 L407 F。我们将确定cry 1 L407 F是否使用与cry 2相同的信号成分来诱导FT。为此，我们将分析开花时间和FT表达的cry 1 L407 F突变体的cib 1/cib 2/cib 5突变体的背景，以及cry 1 L407 F与已知的合作伙伴的cry 2的相互作用。我们打算与Christian Jung（甜菜开花控制）和Markus Schmid（通过海藻糖-6-磷酸信号调节开花时间）的小组合作。

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).