Phytochrome Signaling in Green Arabidopsis Seedlings： Impact Assessment of a Mutually Negative phyB-PIF Feedback Loop

The reversibly red （R）/far-red （FR）-Iight-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 inter- action of Pfr with a small subfamily of bHLH transcription factors, termed Phy-lnteracting 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 phyBpiflpif3pif4pif5 （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 pro- longed 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 degrada- tion. 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.

Newly Described Components and Regulatory Mechanisms of Circadian Clock Function in Arabidopsis thaliana

The circadian clock temporally coordinates plant growth and metabolism in close synchronization with the diurnal and seasonal environmental changes. Research over the last decade has identified a number of clock components and a variety of regulatory mechanisms responsible for the rhythmic oscillations in metabolic and physiological activities. At the core of the clock, transcriptional/translational feedback loops modulate the expression of a significant proportion of the genome. In this article, we briefly describe some of the very recent advances that have improved our understanding of clock organization and function in Arabidopsis thaliana. The new studies illustrate the role of clock protein complex for- mation on circadian gating of plant growth and identify alternative splicing as a new regulatory mechanism for clock function. Examination of key clock properties such as temperature compensation has also opened new avenues for func- tional research within the plant clockwork. The emerging connections between the circadian clock and metabolism, hor- mone signaling and response to biotic and abiotic stress also add new layers of complexity to the clock network and underscore the significance of the circadian clock regulating the daily life of plants.