Molecular analyses of Phytochrome-PIF-LHY interaction in poplar photoperiodic response

As sessile organism, plants require adaptive mechanisms to fit its development with their local environment. The adaptative versatility is especially meaningful for woody perennials trees from temperate and boreal regions, long-life organisms that have to deal with the harsh winter conditions year after year. With the aim to survive to this season, perennials have evolved the vegetative growth-dormancy phenological transition. During winter dormancy plants enter in a growth quiescent state resistant to freezing and drought stress. In poplar, the shortening of daylength leads to shoot apical growth cessation that is the gateway of dormancy establishment. During last decade, molecular components mediating this photoperiodic control of growth cessation have started to emerge, however understanding the mechanism involved require deeper analysis. Previous studies performed in the laboratory, using poplar as the experimental system, proved that the photoperiod integrator and growth inductor FLOWERING LOCUS T 2 (FT2), is quantitatively repressed in response to the extension of the night. An investigation of the photoperiodic repression route was initiated revealing that the clock gene LATE ELONGATED HYPOCOTYL 2 (LHY2) is involved. Moreover, it was identified the phytochrome signalling pathway as the responsible for perceiving and transferring the photoperiodic external information to LHY2. These results have led to hypothesize that a novel pathway senses the progressive extension of the night and eventually represses FT2 expression, causing poplar shoot apical growth cessation. The purpose of this work is to provide new experimental evidences that support this model, focusing on understanding the photoperiodic control of LHY2 transcription. This study evidences that night break assays, where poplars were subjected to a red-light pulse in the middle of the night, release the repression of FT2 and subsequently the bud set establishment triggered under short days. Moreover, in these conditions LHY2 expression, as well as the other growth repressors acting up/downstream of LHY2 are downregulated, supporting the model hypothesized. Furthermore, it is shown that red-light illumination inhibits LHY2 activation at the end of the night, pointing that phytochrome signalling pathway could control its transcription. Remarkably, it is demonstrated that the ELONGATED HYPOCOTYL 5 chromosome 10 (HY5_10) gene, whose homologue in Arabidopsis is known to act downstream of the phytochromes and physically interact with PHYTOCHROME-INTERACTING FACTOR 1 (PIF1), binds to cis-regulatory elements of the LHY2 promoter, previously identified as essentials for its correct photoperiodic transcriptional activation. Thus, a new model of LHY2 transcriptional regulation based on PIF1/HY5_10 ratio is proposed