A quadruple photoreceptor mutant still keeps track of time.
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Time measurement and light detection are inextricably linked. Cryptochromes, the blue-light photoreceptors shared between plants and animals, are critical for circadian rhythms in flies and mice [1-3]. WC-1, a putative blue-light photoreceptor, is also essential for the maintenance of circadian rhythms in Neurospora [4]. In contrast, we report here that in Arabidopsis thaliana the double mutant lacking the cryptochromes cry1 and cry2, and even a quadruple mutant lacking the red/ far-red photoreceptor phytochromes phyA and phyB as well as cry1 and cry2, retain robust circadian rhythmicity. Interestingly, the quadruple mutant was nearly blind for developmental responses but perceived a light cue for entraining the circadian clock. These results indicate that cryptochromes and phytochromes are not essential components of the central oscillator in Arabidopsis and suggest that plants could possess specific photosensory mechanisms for temporal orientation, in addition to cryptochromes and phytochromes, which are used for both spatial and temporal adaptation. (+info)
HFR1 encodes an atypical bHLH protein that acts in phytochrome A signal transduction.
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Phytochromes are informational photoreceptors through which plants adapt their growth and development to prevailing light conditions. These adaptations are effected primarily through phytochrome regulation of gene expression by mechanisms that remain unclear. We describe a new mutant, hfr1 (long hypocotyl in far-red), that exhibits a reduction in seedling responsiveness specifically to continuous far-red light (FRc), thereby suggesting a locus likely to be involved in phytochrome A (phyA) signal transduction. Using an insertionally tagged allele, we cloned the HFR1 gene and subsequently confirmed its identity with additional alleles derived from a directed genetic screen. HFR1 encodes a nuclear protein with strong similarity to the bHLH family of DNA-binding proteins but with an atypical basic region. In contrast to PIF3, a related bHLH protein previously shown to bind phyB, HFR1 did not bind either phyA or B. However, HFR1 did bind PIF3, suggesting heterodimerization, and both the HFR1/PIF3 complex and PIF3 homodimer bound preferentially to the Pfr form of both phytochromes. Thus, HFR1 may function to modulate phyA signaling via heterodimerization with PIF3. HFR1 mRNA is 30-fold more abundant in FRc than in continuous red light, suggesting a potential mechanistic basis for the specificity of HFR1 to phyA signaling. (+info)
Resonance Raman spectroscopic study of the tryptic 39-kDa fragment of phytochrome.
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The 39-kDa fragment of oat phytochrome phyA, obtained by tryptic digestion at the amino acids 65 and 425, was studied by resonance Raman spectroscopy. The parent state P(r) reveals far-reaching similarities with that of the native phytochrome implying that the structures of the tetrapyrrole chromophore and its immediate protein environment are not affected by the proteolysis. However, the resonance Raman spectrum of the final product of the P(r) phototransformation, denoted as P(bl), is more closely related to that of the P(fr) precursor of the native phytochrome, i.e. meta-R(C), rather than to that of P(fr) itself. The resonance Raman spectra indicate a high conformational flexibility of the chromophore in P(bl) so that, unlike in P(fr), the tetrapyrrole rings C and D adopt a largely coplanar conformation. The protein interactions with ring D of the chromophore, which in the native phytochrome stabilize the specific chromophore structure of P(fr), cannot be established in the 39-kDa fragment due to the lack of the major C-terminal part of the protein. These findings, furthermore, support the view that the meta-R(C)-->P(fr) transition is associated with a coupling of chromophore and protein structural changes that represent crucial events for the photoactivation of phytochrome. (+info)
Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix-loop-helix factor PIF3 in a reaction requiring the PAS domain of PIF3.
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The signaling pathways by which the phytochrome (phy) family of photoreceptors transmits sensory information to light-regulated genes remain to be fully defined. Evidence for a relatively direct pathway has been provided by the binding of one member of the family, phyB, to a promoter-element-bound, basic helix-loop-helix protein, PIF3, specifically upon light-induced conversion of the photoreceptor molecule to its biologically active conformer (Pfr). Here, we show that phyA also binds selectively and reversibly to PIF3 upon photoconversion to Pfr, but that the apparent affinity of PIF3 for phyA is 10-fold lower than for phyB. This result is consistent with previous in vivo data from PIF3-deficient Arabidopsis, indicating that PIF3 has a major role in phyB signaling, but a more minor role in phyA signaling. We also show that phyB binds stoichiometrically to PIF3 at an equimolar ratio, suggesting that the resultant complex is the unit active in transcriptional regulation at target promoters. Deletion mapping suggests that a 37-aa segment present at the N terminus of phyB, but absent from phyA, contributes strongly to the high binding affinity of phyB for PIF3. Conversely, deletion mapping and point mutation analysis of PIF3 for determinants involved in recognition of phyB indicates that the PAS domain of PIF3 is a major contributor to this interaction, but that a second determinant in the C-terminal domain is also necessary. (+info)
REP1, a basic helix-loop-helix protein, is required for a branch pathway of phytochrome A signaling in arabidopsis.
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Phytochromes are primary photoreceptors mediating diverse responses ranging from induction of germination to floral induction in higher plants. We have isolated novel recessive rep1 (reduced phytochrome signaling 1) mutants, which exhibit a long-hypocotyl phenotype only under far-red light but not under red light. Physiological characterization showed that rep1 mutations greatly reduced a subset of phytochrome A-regulated responses, including the inhibition of hypocotyl elongation, cotyledon expansion, modulation of gravitropic growth of hypocotyl, and induction of the CAB (encoding chlorophyll a/b binding protein) gene, without affecting the accumulation of anthocyanin, far-red-preconditioned blocking of greening, induction of germination, and induction of CHS (encoding chalcone synthase) and FNR (encoding ferredoxin-NADP(+) oxidoreductase) genes. These results suggest that REP1 is a positive signaling component, functioning in a branch of the phytochrome A signaling pathway. Molecular cloning and characterization of the REP1 gene revealed that it encodes a light-inducible, putative transcription factor containing the basic helix-loop-helix motif. (+info)
Both subunits of the dimeric plant photoreceptor phytochrome require chromophore for stability of the far-red light-absorbing form.
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The dimeric plant photoreceptor phytochrome is converted from its inactive red light-absorbing form (Pr) into the active far-red light-absorbing form (Pfr) upon light absorption. Dynamics of Pfr generation and of thermal Pfr-to-Pr conversion are of fundamental importance for inducing adequate responses to light signals. Here, we analyzed the role of subunit interactions on spectroscopic properties of dimeric phytochrome A. Using a coexpression system and affinity chromatography, we prepared mixed phytochrome dimers that can incorporate the essential chromophore only in one subunit. We demonstrate that such mixed dimers have unaltered difference spectra. In contrast, dark reversion differed greatly between Pfr-Pfr homodimers and Pfr-Pr heterodimers, the former being about 100-fold more stable. Temperature dependence of reaction rates revealed an additional stabilization of about 4 kcal/mol in homodimers. Consequences of these findings are discussed in relation to the biological function of, and functional diversification between, phytochrome family members. (+info)
Aux/IAA proteins are phosphorylated by phytochrome in vitro.
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Auxin/indole-3-acetic acid (Aux/IAA) genes encode short-lived transcription factors that are induced as a primary response to the plant growth hormone IAA or auxin. Gain-of-function mutations in Arabidopsis genes, SHY2/IAA3, AXR3/IAA17, and AXR2/IAA7 cause pleiotropic phenotypes consistent with enhanced auxin responses, possibly by increasing Aux/IAA protein stability. Semidominant mutations shy2-1D, shy2-2, axr3-1, and axr2-1 induce ectopic light responses in dark-grown seedlings. Because genetic studies suggest that the shy2-1D and shy2-2 mutations bypass phytochrome requirement for certain aspects of photomorphogenesis, we tested whether SHY2/IAA3 and related Aux/IAA proteins interact directly with phytochrome and whether they are substrates for its protein kinase activity. Here we show that recombinant Aux/IAA proteins from Arabidopsis and pea (Pisum sativum) interact in vitro with recombinant phytochrome A from oat (Avena sativa). We further show that recombinant SHY2/IAA3, AXR3/IAA17, IAA1, IAA9, and Ps-IAA4 are phosphorylated by recombinant oat phytochrome A in vitro. Deletion analysis of Ps-IAA4 indicates that phytochrome A phosphorylation occurs on the N-terminal half of the protein. Metabolic labeling and immunoprecipitation studies with affinity-purified antibodies to IAA3 demonstrate increased in vivo steady-state levels of mutant IAA3 in shy2-2 plants and phosphorylation of the SHY2-2 protein in vivo. Phytochrome-dependent phosphorylation of Aux/IAA proteins is proposed to provide one molecular mechanism for integrating auxin and light signaling in plant development. (+info)
Choice of tracks, microtubules and/or actin filaments for chloroplast photo-movement is differentially controlled by phytochrome and a blue light receptor.
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Light induced chloroplast movement has been studied as a model system for photoreception and actin microfilament (MF)-based intracellular motilities in plants. Chloroplast photo-accumulation and -avoidance movement is mediated by phytochrome as well as blue light (BL) receptor in the moss Physcomitrella patens. Here we report the discovery of an involvement of a microtubule (MT)-based system in addition to an MF-based system in photorelocation of chloroplasts in this moss. In the dark, MTs provided tracks for rapid movement of chloroplasts in a longitudinal direction and MFs contributed the tracks for slow movement in any direction. We found that phytochrome responses utilized only the MT-based system, while BL responses had an alternative way of moving, either along MTs or MFs. MT-based systems were mediated by both photoreceptors, but chloroplasts showed movements with different velocity and pattern between them. No apparent difference in the behavior of chloroplast movement between the accumulation and avoidance movement was detected in phytochrome responses or BL responses, except for the direction of the movement. The results presented here demonstrate that chloroplasts use both MTs and MFs for motility and that phytochrome and a BL receptor control directional photo-movement of chloroplasts through the differential regulation of these motile systems. (+info)