Role of TCP transcription factors in seedling development, leaf morphogenesis and senescence in Arabidopsis thaliana

Abstract

The TCP gene family encodes non-canonical bHLH transcription factors that act as key regulatory molecules in diverse developmental processes in plants including organ morphogenesis, plant architecture, leaf maturation, and flowering transition. In this study, we assign a number of new functions to the CINCINNATA-like TCP (CIN-TCP) proteins throughout the life of Arabidopsis thaliana starting from light-mediated seedling morphogenesis, regulation of simple leaf architecture, and hormone homeostasis during leaf senescence. PART-I: Role of TCP transcription factors in light-mediated Arabidopsis seedling development Plant growth and morphogenesis rely heavily on the coordination between external and internal cues to cope with the ever-changing surroundings. In Arabidopsis seedlings grown under low light intensity, the embryonic stem (hypocotyl) elongates more in an attempt to reach the light source, a process called skotomorphogenesis. By contrast, seedlings grown under sufficient light grow shorter (photomorphogenesis). We had earlier shown that the CIN-TCPs promote cell elongation during photomorphogenesis. Here we show that this effect of CIN-TCPs is abolished in darkness, suggesting that CIN-TCP-mediated cell elongation is dependent on the light-signaling pathway. By analyzing hypocotyl elongation under various light qualities, we show that TCP4-mediated hypocotyl cell elongation is dependent on phytochrome B (PhyB) photoreceptor under diverse light conditions. Using various biochemical and genetic assays, we demonstrate that TCP4 activation leads to the stabilization of several phytochrome-interacting factor (PIF) proteins through protein-protein interaction. Enhanced PIF level leads to the destabilization of PhyB and indirectly represses the HFR1 protein to promote hypocotyl elongation. Thus, CIN-TCP functions as a major negative regulator of photomorphogenic seedling growth together with PIF. PART-II: CIN-TCPs actively suppress leaflet emergence to promote simple leaf form Though all angiosperm leaves are initiated as simple rod-like primordia at the flank of the shoot apical meristem (SAM), they show extensive shape diversity at maturity, based on which they are broadly divided into two forms; simple leaves with intact lamina and compound leaves with lamina dissected into leaflets. Although genetic intervention has converted compound leaves into simpler or more complex variants, it is not clear whether, or to what extent, simple leaves can initiate leaflets and form compound architecture upon endogenous gene manipulation. Here, we show that simultaneous down-regulation of CIN-TCP and class II KNOTTED1-LIKE (KNOX-II) proteins converts simple Arabidopsis lamina to super-compound form with reiterative and indeterminate leaflet emergence, accompanied with sustained reactivation of the meristem-specific genes including KNOX-I and CUPSHAPED COTYLEDON (CUC). CIN-TCPs activate KNOX-II and a dominant CIN-TCP member restores simple leaf form. These results offer a framework of simple leaf development wherein CIN-TCP-KNOX-II forms a strong differentiation module that suppresses the KNOX-I-CUC network and leaflet initiation in the primordia. PART-III: CIN-TCPs maintain jasmonic acid homeostasis during leaf senescence through an incoherent feed forward loop (IFFL) The class I and class II TCP transcription factors, divided based on their sequence diversity, display functional antagonism in regulating multiple cellular and physiological processes including jasmonic acid (JA) biosynthesis during leaf senescence, the final stage of leaf development. Five members of miR319-regulated class II TCPs (TCP2, 3, 4, 10 & 24), also called CIN-TCPs, redundantly promote the JA-biosynthetic enzyme-encoding gene LIPOXYGENASE2 (LOX2). For example, TCP4 binds to the LOX2 promoter and directly activates its transcription that induces leaf senescence. By contrast, the class I TCP members TCP9 and TCP20, together with TCP8 and TCP22, are recruited on the LOX2 promoter to repress its transcription. However, a molecular link between these two TCP groups in regulating LOX2 transcription has not been demonstrated. We here demonstrate a novel type I incoherent feed forward loop (IFFL) formed by the direct transcriptional link between class I (TCP9) and class II TCP proteins (TCP4/ TCP10) to balance the LOX2 expression dynamics. By combined mathematical modelling and genetic manipulation, we show that this IFFL filters out the stochastic noise in TCP and maintains a robust level of LOX2. Thus, the TCP4/TCP10-TCP9-LOX2 module regulates JA homeostasis and leaf senescence in Arabidopsis. In conclusion, we show that the CIN-TCP proteins, along with their class II members, regulate important developmental processes throughout the life of Arabidopsis starting from the seedling establishment, simple leaf shape, and hormone homeostasis. Studies on the role of CIN-TCP homologs in other species would test whether these functions are conserved in evolution.