Dataset Information

Altered dark- and photoconversion of phytochrome B mediate extreme light sensitivity and loss of photoreversibility of the phyB-401 mutant.

ABSTRACT: The phyB-401 mutant is 10(3) fold more sensitive to red light than its wild-type analogue and shows loss of photoreversibility of hypocotyl growth inhibition. The phyB-401 photoreceptor displays normal spectral properties and shows almost no dark reversion when expressed in yeast cells. To gain insight into the molecular mechanism underlying this complex phenotype, we generated transgenic lines expressing the mutant and wild-type phyB in phyB-9 background. Analysis of these transgenic lines demonstrated that the mutant photoreceptor displays a reduced rate of dark-reversion but normal P(fr) to P(r) photoconversion in vivo and shows an altered pattern of association/dissociation with nuclear bodies compared to wild-type phyB. In addition we show (i) an enhanced responsiveness to far-red light for hypocotyl growth inhibition and CAB2 expression and (ii) that far-red light mediated photoreversibility of red light induced responses, including inhibition of hypocotyl growth, formation of nuclear bodies and induction of CAB2 expression is reduced in these transgenic lines. We hypothesize that the incomplete photoreversibility of signalling is due to the fact that far-red light induced photoconversion of the chromophore is at least partially uncoupled from the P(fr) to P(r) conformation change of the protein. It follows that the phyB-401 photoreceptor retains a P(fr)-like structure (P(r) (*)) for a few hours after the far-red light treatment. The greatly reduced rate of dark reversion and the formation of a biologically active P(r) (*) conformer satisfactorily explain the complex phenotype of the phyB-401 mutant and suggest that amino acid residues surrounding the position 564 G play an important role in fine-tuning phyB signalling.

SUBMITTER: Adam E

PROVIDER: S-EPMC3207837 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Publications

Altered dark- and photoconversion of phytochrome B mediate extreme light sensitivity and loss of photoreversibility of the phyB-401 mutant.

Ádám Éva É Hussong Andrea A Bindics János J Wüst Florian F Viczián András A Essing Marcus M Medzihradszky Mátyás M Kircher Stefan S Schäfer Eberhard E Nagy Ferenc F

PloS one 20111103 11

The phyB-401 mutant is 10(3) fold more sensitive to red light than its wild-type analogue and shows loss of photoreversibility of hypocotyl growth inhibition. The phyB-401 photoreceptor displays normal spectral properties and shows almost no dark reversion when expressed in yeast cells. To gain insight into the molecular mechanism underlying this complex phenotype, we generated transgenic lines expressing the mutant and wild-type phyB in phyB-9 background. Analysis of these transgenic lines demo ...[more]

PMID: 22073299

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Project description: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.

Project description:The photoprocesses of native (phyA of oat), and of C-terminally truncated recombinant phytochromes, assembled instead of the native phytochromobilin with phycocyanobilin (PCB-65 kDa-phy) and iso-phycocyanobilin (iso-PCB-65 kDa-phy) chromophores, have been studied by femtosecond transient absorption spectroscopy in both their red absorbing phytochrome (P(r)) and far-red absorbing phytochrome (P(fr)) forms. Native P(r) phytochrome shows an excitation wavelength dependence of the kinetics with three main picosecond components. The formation kinetics of the first ground-state intermediate I(700), absorbing at approximately 690 nm, is mainly described by 28 ps or 40 ps components in native and PCB phytochrome, respectively, whereas additional approximately 15 and 50 ps components describe conformational dynamics and equilibria among different local minima on the excited-state hypersurface. No significant amount of I(700) formation can be observed on our timescale for iso-PCB phytochrome. We suggest that iso-PCB-65 kDa-phy either interacts with the protein differently leading to a more twisted and/or less protonated configuration, or undergoes P(r) to P(fr) isomerization primarily via a different configurational pathway, largely circumventing I(700) as an intermediate. The isomerization process is accompanied by strong coherent oscillations due to wavepacket motion on the excited-state surface for both phytochrome forms. The femto- to (sub-)nanosecond kinetics of the P(fr) forms is again quite similar for the native and the PCB phytochromes. After an ultrafast excited-state relaxation within approximately 150 fs, the chromophores return to the first ground-state intermediate in 400-800 fs followed by two additional ground-state intermediates which are formed with 2-3 ps and approximately 400 ps lifetimes. We call the first ground-state intermediate in native phytochrome I(fr 750), due to its pronounced absorption at that wavelength. The other intermediates are termed I(fr 675) and pseudo-P(r). The absorption spectrum of the latter already closely resembles the absorption of the P(r) chromophore. PCB-65 kDa-phy shows a very similar kinetics, although many of the detailed spectral features in the transients seen in native phy are blurred, presumably due to wider inhomogeneous distribution of the chromophore conformation. Iso-PCB-65 kDa-phy shows similar features to the PCB-65 kDa-phy, with some additional blue-shift of the transient spectra of approximately 10 nm. The sub-200 fs component is, however, absent, and the picosecond lifetimes are somewhat longer than in 124 kDa phytochrome or in PCB-65 kDa-phy. We interpret the data within the framework of two- and three-dimensional potential energy surface diagrams for the photoisomerization processes and the ground-state intermediates involved in the two photoconversions.

Dataset Information

Altered dark- and photoconversion of phytochrome B mediate extreme light sensitivity and loss of photoreversibility of the phyB-401 mutant.

Publications

Altered dark- and photoconversion of phytochrome B mediate extreme light sensitivity and loss of photoreversibility of the phyB-401 mutant.

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