title: Phytochrome signaling in green Arabidopsis seedlings: impact assessment of a mutually negative phyB-PIF feedback loop.
abstract: The reversibly red (R)/far-red (FR)-light-responsive phytochrome (phy) photosensory system initiates both the deetiolation process in dark-germinated seedlings upon first exposure to light, and the shade-avoidance process in fully deetiolated seedlings upon exposure to vegetational shade. The intracellular signaling pathway from the light-activated photoreceptor conformer (Pfr) to the transcriptional network that drives these responses involves direct, physical interaction of Pfr with a small subfamily of bHLH transcription factors, termed Phy-Interacting Factors (PIFs), which induces rapid PIF proteolytic degradation. In addition, there is evidence of further complexity in light-grown seedlings, whereby phyB-PIF interaction reciprocally induces phyB degradation, in a mutually-negative, feedback-loop configuration. Here, to assess the relative contributions of these antagonistic activities to the net phenotypic readout in light-grown seedlings, we have examined the magnitude of the light- and simulated-shade-induced responses of a pentuple phyBpif1pif3pif4pif5 (phyBpifq) mutant and various multiple pif-mutant combinations. The data (1) reaffirm that phyB is the predominant, if not exclusive, photoreceptor imposing the inhibition of hypocotyl elongation in deetiolating seedlings in response to prolonged continuous R irradiation and (2) show that the PIF quartet (PIF1, PIF3, PIF4, and PIF5) retain and exert a dual capacity to modulate hypocotyl elongation under these conditions, by concomitantly promoting cell elongation through intrinsic transcriptional-regulatory activity, and reducing phyB-inhibitory capacity through feedback-loop-induced phyB degradation. In shade-exposed seedlings, immunoblot analysis shows that the shade-imposed reduction in Pfr levels induces increases in the abundance of PIF3, and mutant analysis indicates that PIF3 acts, in conjunction with PIF4 and PIF5, to promote the known shade-induced acceleration of hypocotyl elongation. Conversely, although the quadruple pifq mutant displays clearly reduced hypocotyl elongation compared to wild-type in response to prolonged shade, immunoblot analysis detects no elevation in phyB levels in the mutant seedlings compared to the wild-type during the majority of the shade-induced growth period, and phyB levels are not robustly correlated with the growth phenotype across the pif-mutant combinations compared. These results suggest that PIF feedback modulation of phyB abundance does not play a dominant role in modulating the magnitude of the PIF-promoted, shade-responsive phenotype under these conditions. In seedlings grown under diurnal light-dark cycles, the data show that FR-pulse-induced removal of Pfr at the beginning of the dark period (End-of-Day-FR (EOD-FR) treatment) results in longer hypocotyls relative to no EOD-FR treatment and that this effect is attenuated in the pif-mutant combinations tested. This result similarly indicates that the PIF quartet members are capable of intrinsically promoting hypocotyl cell elongation in light-grown plants, independently of the effects of PIF feedback modulation of photoactivated-phyB abundance.
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Pablo
authors.initials: P
authors.lastname: Leivar
authors.forename: Elena
authors.initials: E
authors.lastname: Monte
authors.forename: Megan M
authors.initials: MM
authors.lastname: Cohn
authors.forename: Peter H
authors.initials: PH
authors.lastname: Quail
mesh_terms: D017360:Arabidopsis / Q000254:growth & development* / Q000378:metabolism* / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D025461:Feedback, Physiological / Q000528:radiation effects; D008027:Light; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D051939:Phytochrome B / Q000378:metabolism*; D036226:Seedlings / Q000254:growth & development* / Q000528:radiation effects; D015398:Signal Transduction / Q000528:radiation effects
chemical_list: D029681:Arabidopsis Proteins; D051792:Basic Helix-Loop-Helix Proteins; D051939:Phytochrome B; C497952:PHYB protein, Arabidopsis