Metabolite regulation of cryptochrome 2 activity and flowering time

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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).