CstPIF4 integrates temperature and circadian signals and interacts with CstMYB16 to represses anthocyanins in Crocus.

Crocus sativus has emerged as an important crop because it is the only commercial source of saffron which contains unique apocarotenoids. Saffron is composed of dried stigmas of Crocus flower and constitutes most priced spice of the world. Crocus floral organs are dominated by different classes of metabolites. While stigmas are characterized by the presence of apocarotenoids, tepals are rich in flavonoids and anthocyanins. Therefore, intricate regulatory network might play role in allowing different compounds to dominate in different organs. Work so far done on Crocus is focussed on apocarotenoid metabolism and its regulation. There are no reports describing regulation of flavonoids and anthocyanins in Crocus tepals. In this context we identified an R2R3 transcription factor, CstMYB16 which resembles subgroup 4 repressors of Arabidopsis. CstMYB16 is nuclear localized and acts as a repressor. Over-expression of CstMYB16 in Crocus down-regulated anthocyanin biosynthesis. C2/EAR motif was responsible for repressor activity of CstMYB16. CstMYB16 binds to promoter of anthocyanin biosynthetic pathway gene (LDOX) and reduces its expression. CstMYB16 also physically interacts with CstPIF4 which in turn is regulated by temperature and circadian clock. Thus CstPIF4 integrates these signals and forms a repressor complex with CstMYB16 which is involved in negative regulation of anthocyanin biosynthesis in Crocus. Independent of CstPIF4, CstMYB16 also represses CstPAP1 expression which is a component of MBW complex and positively controls anthocyanin biosynthesis. This is the first report on identifying and describing regulators of anthocyanin biosynthesis in Crocus.