FLOWERING PROMOTING FACTOR 1 (FPF1) family genes control shade-induced development in Arabidopsis thaliana

Abstract

The perception of environmental light conditions and the generation of appropriate developmental responses are key to plant fitness. Plants must be capable of detecting information in the light environment, such as the time of day, the progression of seasons, and the proximity of competing neighbours, and translate those signals into coordinated alterations in development. Among the most well-characterized developmental programmes is photoperiodic flowering - the capacity to measure day length and time vegetative and reproductive transitions accordingly. The molecular mechanisms underlying this process have been extensively studied in Arabidopsis thaliana (Arabidopsis), where CONSTANS (CO) and the mobile flowering signal FLOWERING LOCUS T (FT) have been established as primary mediators of day-length-dependent floral induction (Suárez-López et al., 2001; Valverde et al., 2004; Shim et al., 2017). However, the light environment through which this mechanistic understanding was developed, with standard fluorescent and LED growth chambers producing red to far-red ratios (R:FR) exceeding 2.0, is not replicated by those found in nature, where sunlight carries an R:FR ratio of approximately 1.0 (Song et al., 2018b). Work from our laboratory first identified this discrepancy. Arabidopsis, under outdoor natural long-day conditions, or under artificial light adjusted to match the natural R:FR ratio showed a bimodal pattern of circadian FT expression, exhibiting a morning peak that was entirely absent under standard laboratory conditions (Song et al., 2018b). This finding established that far-red light - present at biologically significant levels in natural sunlight but systematically suppressed in laboratory settings - exerts a significant influence on FT induction (Song et al., 2018b) and, by extension, on the developmental transitions it controls. The molecular mechanisms mediating this FR-dependent regulation had not been characterized, representing a conspicuous gap in our understanding of how plants respond to the light environment. The four chapters of this dissertation are united by this reframing. Chapter 1 provides a comprehensive account of the multilayered regulatory mechanisms governing CO and FT in Arabidopsis, synthesising the molecular processes responsible for day-length-dependent floral induction across transcriptional, post-translational, and chromatin-remodelling levels. In doing so, we make clear how sparsely characterised FR-dependent interactions are relative to those mediated by blue light and red light. Despite FR's influence on FT induction under natural conditions, the molecular mechanisms by which phyA stabilises CO under FR-enriched conditions (Valverde et al., 2004; Song et al., 2018b), and the interactions governing the morning peak of FT, remained largely undescribed at the time of writing. Chapter 2 addresses that gap directly. Morning FT induction, previously uncharacterized, is shown to represent a phytochrome A-mediated high irradiance response - a class of far-red-dependent regulation long described physiologically in long-day plants but not previously incorporated into the molecular framework of photoperiodic flowering in Arabidopsis. Morning FT levels correlate positively with the duration of FR exposure, and full induction requires FR enrichment spanning both the afternoon and the following morning. Our findings also implicate PIF7, known primarily as a contributor to shade avoidance (Zhang, R et al., 2019), as a component of natural long-day flowering - connecting the shade avoidance machinery to the photoperiodic pathway in a manner not previously described. Chapter 3 examines the downstream consequences of FR enrichment in florigen-producing cells. Absent under standard laboratory conditions, FLP1 expression is confined to phloem companion cells, the primary site of FT expression (Takada & Goto, 2003), and is induced only under FR-enriched long-day conditions, requiring both the appropriate photoperiod and R:FR ratio, similar to the morning FT peak. The small size of FLP1 (14 kDa), its specific localisation to phloem companion cells, and the observation that FLP1-overexpressing plants exhibit accelerated flowering and stem elongation while loss-of-function mutants exhibit the opposite, together position FLP1 as a component of the developmental program activated by far-red light in florigen-producing cells. FLP1 promotes flowering independently of FT, acting in parallel through the induction of the floral homeotic gene SEP3 (Teper-Bamnolker & Samach, 2005). The first three chapters of this dissertation approach FR light primarily in the context of natural long-day conditions, where an R:FR ratio of approximately 1.0 was sufficient to reveal novel regulatory mechanisms masked under standard laboratory conditions. Chapter 4 extends this investigation into shaded conditions (R:FR <0.5). The induction of FLP1 exclusively under FR-enriched conditions, combined with the observation that FLP1-overexpressing plants exhibit a phenotype consistent with shade avoidance, prompted an investigation of FLP1 and, more broadly, FPF1 gene family function under shade-simulating conditions. Under FR-enriched shade, the FPF1 gene family is shown to contribute substantially to the shade avoidance response. FLP1 overexpression broadly phenocopies the developmental trajectory and morphology of phyB mutants, while loss-of-function fpf1flp1flp2 triple mutants exhibit a severely attenuated shade avoidance response. We found that FLP1 and FPF1 are upregulated under shade conditions in a phytochrome B (phyB)-dependent manner downstream of PIFs, positioning them as components of the shade avoidance pathway. These findings implicate the FPF1 gene family as essential mediators of the transition from shade perception to adaptive developmental change. The studies presented in this dissertation collectively characterise a previously underappreciated contribution of far-red light to plant development. Under natural light conditions, FR drives a morning peak of FT expression through a phyA-mediated high irradiance response, induces a suite of genes in florigen-producing cells - among them FLP1, which promotes flowering independently of FT - and activates the FPF1 gene family as essential mediators of the phyB shade avoidance response. That these regulatory relationships remained undescribed is a direct consequence of their dependence on FR irradiance absent from standard laboratory conditions, and their characterisation clarifies the phyB-PIF shade avoidance pathway by identifying the FPF1 gene family as previously unrecognised mediators of the developmental and morphological transitions it controls.