Temporal and spatial frameworks supporting plant responses to vegetation proximity

After perception of vegetation proximity by the phytochrome photoreceptors, shade-avoider plants initiate a set of responses known as the Shade Avoidance Syndrome (SAS). Shade perception by the phytochrome B (phyB) photoreceptor unleashes the PHYTOCHROME INTERACTING FACTORs (PIFs) and initiates SAS responses. In Arabidopsis thaliana seedlings, shade perception involves rapid and massive changes in gene expression, increases auxin production and promotes hypocotyl elongation. Other components, such as phyA and ELONGATED HYPOCOTYL 5 (HY5), also participate in the shade regulation of the hypocotyl elongation response by repressing it. However, it remains unclear why and how so many regulators with either positive or negative activities modulate the same response. Our physiological, genetic, cellular and transcriptomic analyses showed that (1) these components are organized in two main branches or modules (phyA/HY5 and PIFs/HFR1/SAV3) and (2) the connection between them is dynamic and changes with the time of shade exposure. We propose a model for the regulation of shade-induced hypocotyl elongation in which the temporal and spatial functional importance of the various SAS regulators analyzed in here helps to explain the co-existence of differentiated regulatory branches with overlapping activities. Despite the temporal differences observed between phyA, HY5 and PIFs/HFR1/SAV3, their activities overlap and eventually converge in controlling hypocotyl elongation. Hence, we aimed to further investigate possible convergence points between these two groups of regulators.To expand our understanding of the role and interaction of HY5 and PIF4, PIF5 and PIF7 (PIF457) activities, we carried out RNA sequencing (RNA-seq) of seedlings exposed to different times of shade exposure of four genotypes: wild-type (Col-0), the single mutant hy5, the triple pif457 and the quadruple hy5 pif457. Seedlings of wild-type (Col-0), hy5, pif457 and hy5 pif457 were grown from the day of germination under continuous white light (W). On day seven, seedlings were transferred to W enriched in far-red light (W+FR). Three biological replicas of seedlings were harvested at 0, 1 and 8 h of W+FR (about 35 mg per sample) and frozen in liquid nitrogen (FOUR genotypes x THREE time points x THREE biological replicas = 36 samples). RNA was extracted using commercial kits. Total RNA libraries were prepared for each biological replicate, genotypes and treatments (36 samples s and sequenced at the Centre Nacional d’Anàlisi Genòmica (CNAG-CRG) on an Illumina NovaSeq6000 in paired-end 50 bp read length.