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Periodic Report Summary 1 - WINCLOCK (Underpinning the significance of circadian clock winter disruption in Poplar)

WinClock project investigates the crosstalk between the environmental signals and developmental programs in woody plants. Specifically, WinClock is focused on the study of how photoperiod and temperature are integrated by the biological clock defining the temporal control of the winter dormancy in woody plants.
In this project two complementary angles of this research line have been addressed, the first one is focused on decipher how the winter environmental cues controls poplar circadian clock, and the second one, aims to undercover the mechanism of action of the circadian transcription factor RAV1 in promoting dormant axillary bud outgrowth.

WinClock Objetive 1: Deciphering the molecular mechanism involved in winter disruption of the circadian clock in poplar.

The circadian clock sustains the plant growth by anticipating daily and seasonal changes, through a complex mechanism that integrates the environmental cues with endogenous developmental programs. Perennial plants stop growing during winter undergoing dormancy. Winter environmental signals disrupt circadian clock function, however, the molecular mechanism is unknown. In this objective we aims to find out how the winter environmental signals control the clock function at transcriptional level.
We have generated a poplar circadian reporter line fusing 5’ upstream regulatory region of the circadian clock LATE ELONGATED HYPOCOTYL (LHY) gene with the LUCIFERASE (LUC) reporter gene to obtain the reporter construct PtaLHY::LUC. Initially we carried out a complete characterization of the PtaLHY::LUC responses to photoperiod and cold temperature in parallel with qRT-PCR studies of endogenous LHY . The results indicated the strong influence of the photoperiod in LHY transcription. By using a combination of promoter loss of function analysis, reverse genetics approaches, and functional studies in transient assays, we identified potential transcriptional regulators of LHY in poplar. The analysis of these candidates will let to understand how the environment transcriptionally controls the biological clock. Future functional studies in poplar trees will reveal their role in winter dormancy.
A temporal control of growth-dormancy transitions is important from an environmental point of view, given the need to exploit a wider range of sustainable energy sources and the need of plants to adapt to the new conditions imposed by climate change. The function of the circadian clock in controlling seasonal development of trees has recently emerged, however its regulation is not understood. This work dissects how the poplar clock is controlled transcriptionally by the environmental cues, as well as it sheds light on clock regulation through the identification of novel regulators. Clock functioning and its role in plant development have a broader interest in plant biology.

WinClock Objetive 2: The spatiotemporal investigation of rav1 gene network and its transcriptional mechanism

Earlier work has shown that overexpression of the circadian chestnut RAV1 (CsRAV1) transcription factor in poplar exhibits dormant axillary bud outgrowth or syllepsis without any visible impairment of wood anatomy. The aim of this objective is to decipher the regulatory principles of dormancy eluding and the seasonal control of lateral branch development through the understanding of the gene network of the circadian gene RAV1 in poplar.
To accomplish this objective, we are setting up a transcription factor gene regulatory network methods of analysis in poplar. Firstly, we constructed a Dexamethasone inducible version of poplar RAV1 gene as a transcriptional fusion to the glucocorticoid receptor domain (GR). We generated and evaluated independent poplar lines showing a conditionally activation RAV1 expression. This tool will permit the identification of RAV1 primary response genes by temporal analysis of trancriptome after induction by Dexamethasone and Cycloheximide. Having this information we will identify RAV1 gene network and will get information about its mode of action. Future ChIP-seq analyses will be setting up to identify direct binding of RAV1 to its target genes.
Branching has a direct influence in plant shoot architecture and it is positively correlated with high biomass yields in poplar plantations. Although very little is known about its regulation, it has been recently shown that strigolatones play a role by repressing axillary bud outgrowth, however how this trait is seasonally regulated is not understood. The circadian RAV1 transcription factor may play a role ticking the time of branching, a mechanism that is completely unknown. Therefore, the impact of uncovering RAV1 gene network will be important to find a molecular link between seasons and control of branching.

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Life Sciences
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