植物的光敏色素与光信号转导

介绍了近年来植物光敏色素与光信号转导的研究进展。     

A Kinetic Theory of First Order Cyclical Processes—Phytochrome Controlled Red Light Induced Cereal Leaf Unfolding Compared with Theory

The phytochrome controlled unfolding of cereal leaves was studied as a function of irradiation time and light intensity (narrowband red light) over a wide energy range (5 decades). With different intensities, a family of similarly shaped response curves appear with distinct time-dependent maxima and minima. A theoretical kinetic model based upon a cyclical phytochrome photoconversion scheme has been calculated by us. The theoretical calculations and the experimental findings are in excellent agreement. The same model explains the early photoresponses (first maxima) as an effect of one active phytochrome form, P₂, and the delayed photoresponses as an effect of a second active form P_n. The active transitory form, P₂ (although it may not be the primary product), is formed upon light absorption from P₁. The P₂ decays by a first order dark reaction through several inactive intermediates to P_n (active). The effect of the intermediates is mainly to delay the production of the second active product. It is possible to identify the two active products, P₂ and P_n, as P_fr and P*_fr, respectively. The presented cyclical phytochrome reaction scheme is a special case of a general first order kinetic cycle which includes all possible feed back loops. The latter scheme also has been calculated and programmed since it has a more general application.     

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

Phytochromes are photoreceptors using a bilin tetrapyrrole as chromophore, which switch in canonical phytochromes between red (P_r) and far red (P_fr) light-absorbing states. Cph2 from Synechocystis sp., a noncanonical phytochrome, harbors besides a cyanobacteriochrome domain a second photosensory module, a P_r/P_fr-interconverting GAF-GAF bidomain (SynCph2(1-2)). As in the canonical phytochromes, a unique motif of the second GAF domain, the tongue region, seals the bilin-binding site in the GAF1 domain from solvent access. Time-resolved spectroscopy of the SynCph2(1-2) module shows four intermediates during P_r → P_fr phototransformation and three intermediates during P_fr → P_r back-conversion. A mutation in the tongue''s conserved PRXSF motif, S385A, affects the formation of late intermediate R3 and of a P_fr-like state but not the back-conversion to P_r via a lumi-F-like state. In contrast, a mutation in the likewise conserved WXE motif, W389A, changes the photocycle at intermediate R2 and causes an alternative red light-adapted state. Here, back-conversion to P_r proceeds via intermediates differing from SynCph2(1-2). Replacement of this tryptophan that is ∼15 Å distant from the chromophore by another aromatic amino acid, W389F, restores native P_r → P_fr phototransformation. These results indicate large scale conformational changes within the tongue region of GAF2 during the final processes of phototransformation. We propose that in early intermediates only the chromophore and its nearest surroundings are altered, whereas late changes during R2 formation depend on the distant WXE motifs of the tongue region. Ser-385 within the PRXSF motif affects only late intermediate R3, when refolding of the tongue and docking to the GAF1 domain are almost completed.     

Light maintains High Levels of Phytochrome Intermediates

THE plant photomorphogenetic pigment phytochrome exists in two forms, Pr and Pfr, interconvertible by light, which have peaks of absorption in the red and far-red regions of the spectrum respectively¹. Intermediates between Pr and Pfr have been demonstrated during photoconversion by Linschitz and his coworkers^2,3 using flash photolysis techniques. Low temperatures studies have also proved useful in identifying intermediates^4–8. Briggs and Fork^9,10 detected intermediates in vitro and in vivo in conditions of pigment cycling by mixed red and far-red light, but were restricted to studying the minor peaks of phytochrome absorption in the blue region of the spectrum because of the available instrumentation. In this type of measurement the problem is that actinic light has to be prevented from falling onto the photomultiplier. Briggs and Fork inserted a red cutoff filter, but this precluded measurement at the peaks of absorption of Pr and Pfr in the red and far-red regions of the spectrum. The design and construction of a sensitive quasi-continuous measuring spectrophotometer have now overcome this problem and made possible an investigation in vivo of phytochrome intermediates at any wavelength under conditions of pigment cycling, for example, in high intensity white incandescent light. The instrument can detect intermediates with a half life in excess of 0.2 ms. The longer lived intermediates between Pr and Pfr observed in the in vitro flash studies^2,3 should be readily detectable if they accumulate in conditions of cycling.     

Red Light-Independent Instability of Oat Phytochrome mRNA in Vivo

Phytochrome A (phyA) mRNA abundance decreased rapidly in total RNA samples isolated from 4-day-old etiolated oat seedlings following a red light pulse. Putative in vivo phyA mRNA degradation products were detectable both before and after red light treatment. Cordycepin-treated coleoptiles were unable to accumulate the chlorophyll a/b-binding protein mRNA in response to red light, indicating that cordycepin effectively inhibited mRNA synthesis. In cordycepin-treated coleoptiles, phyA mRNA rapidly decreased in abundance, consistent with the hypothesis that phyA mRNA is inherently unstable, rather than being destabilized after red light treatment of etiolated oat seedlings.     

Ultrafast Red Light Activation of Synechocystis Phytochrome Cph1 Triggers Major Structural Change to Form the Pfr Signalling-Competent State

Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel ‘head-to-head’ arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response.     

Differential Exposure of Tryptophan Residues in the Red and Far-Red Light Absorbing Forms of Phytochrome, as Revealed by Chemical Modification

The effects of the chemical modification of tryptophan residuesin native pea (Pisum sativum L.) phytochrome by 2-hydroxy-5-nitrobenzylbromide (HNB-Br) were examined. Such treatment had no effecton the spectral properties or on the pattern of tryptic digestionof phytochrome, which indicated that no major conformationalchange in phytochrome had occurred. Amino acid analysis of theHNB-Br-treated phytochrome indicated that the number of modifiedTrp residues after the treatment was dependent on the light-absorbingform. The values were three for P_R and five for P_FR (out ofa total of ten) per monomer. The results indicate that two additionalTrp residues are exposed on the molecular surface of P_FR whenthe photoconversion of P_R to P_FR occurs. The amino acid analysisof a 58-kDa tryptic fragment of phytochrome (a mixture of peptides,residues 63–583 and 66–587) showed that one Trpresidue in the fragment from P_R and two in that from P_FR (outof six) were modified by HNB-Br. In the 56-kDa fragment (a mixtureof peptides, residues 598–1121 and 603–1124), therewere two modified Trp residues in P_R and three in P_FR (out offour). The Trp residue in a 36-kDa fragment (residues 66–383),which includes the tetrapyrrolic chromophore, was not modifiedin the either case. These results indicate that new exposedsites that are generated by the photoconversion of P_R to P_FRare in the region between Trp–456 and Trp–567 andin that between Trp–644 and Trp–787. (Received February 25, 1993; Accepted August 16, 1993)     

A Temporarily Red Light-Insensitive Mutant of Tomato Lacks a Light-Stable,B-Like Phytochrome

We have selected four recessive mutants in tomato (Lycopersicon esculentum Mill.) that, under continuous red light (R), have long hypocotyls and small cotyledons compared to wild type (WT), a phenotype typical of phytochrome B (phyB) mutants of other species. These mutants, which are allelic, are only insensitive to R during the first 2 days upon transition from darkness to R, and therefore we propose the gene symbol tri (temporarily red light insensitive). White light-grown mutant plants have a more elongated growth habit than that of the WT. An immunochemically and spectrophotometrically detectable phyB-like polypeptide detectable in the WT is absent or below detection limits in the tri1 mutant. In contrast to the absence of an elongation growth response to far-red light (FR) given at the end of the daily photoperiod (EODFR) in all phyB-deficient mutants so far characterized, the tri1 mutant responds to EODFR treatment. The tri1 mutant also shows a strong response to supplementary daytime far-red light. We propose that the phyB-like phytochrome deficient in the tri mutants plays a major role during de-etiolation and that other light-stable phytochromes can regulate the EODFR and shade-avoidance responses in tomato.     

Red Light-enhanced Phytochrome Pelletability: Re-examination and Further Characterization

Red light-enhanced pelletability of phytochrome was observed in extracts of all 11 plants tested: Avena sativa L., Secale cereale L., Zea mays L., Cucurbita pepo L., Sinapis alba L., Pisum sativum L., Helianthus anuus L., Raphanus sativus L., Glycine max (L.) Merr., Phaseolus vulgaris L., and Lupinus albus L. This enhanced pelletability was observed in all 11 plants following in situ irradiation (in vivo binding) but only in Sinapis and Cucurbita after irradiation of crude extracts (in vitro binding). In vivo binding was not strongly dependent upon pH and, with few exceptions, was not markedly sensitive to high salt concentration, whereas in vitro binding was completely reversed by both high pH and high salt concentration. However, both binding phenomena were observed only with a divalent cation in the extract buffer. In vivo binding was further characterized using Avena which showed an increase in pelletability from less than 10% in dark control extracts to more than 60% in extracts of red light-irradiated shoots. The half-life for binding was 40 seconds at 0.5 C and was strongly temperature-dependent, binding being complete within 5 to 10 sec at 22 C. If pelletable phytochrome in the far red-absorbing form was photoconverted back to the red-absorbing form in situ, phytochrome was released from the pelletable condition with a half-life of 25 minutes at 25 C and 100 minutes at both 13 C and 3 C. No cooperativity in red light-enhanced pelletability with respect to phytochrome-far red-absorbing form was observed.     

Phytochrome A Mediates the Promotion of Seed Germination by Very Low Fluences of Light and Canopy Shade Light in Arabidopsis

Seeds of the wild type (WT) and of the phyA and phyB mutants of Arabidopsis thaliana were exposed to single red light (R)/far-red light (FR) pulses predicted to establish a series of calculated phytochrome photoequilibria (Pfr/P). WT and phyB seeds showed biphasic responses to Pfr/P. The first phase, i.e. the very-low-fluence response (VLFR), occurred below Pfr/P = 10-1%. The second phase, i.e. the low-fluence response, occurred above Pfr/P = 3%. The VLFR was similarly induced by either a FR pulse saturating photoconversion or a subsaturating R pulse predicted to establish the same Pfr/P. The VLFR was absent in phyA seeds, which showed a strong low-fluence response. In the field, even brief exposures to the very low fluences of canopy shade light (R/FR ratio < 0.05) promoted germination above dark controls in WT and phyB seeds but not in the phyA mutant. Seeds of the phyA mutant germinated normally under canopies providing higher R/FR ratios or under deep canopy shade light supplemented with R from light-emitting diodes. We propose that phytochrome A mediates VLFR of A. thaliana seeds.     

Light Regulation of Hypocotyl Elongation and Greening in Transgenic Tobacco Seedlings That Over-Express Rice Phytochrome A

Previous analysis of a transgenic tobacco line (BN1) that over-expressedrice phytochrome A (PhyA) indicated that the introduced PhyAwas spectrally and biologically active [Kay et al. (1989) PlantCell 1: 775, Nagatani et al. (1991) Proc. Natl. Acad. Sci. USA88: 5207]. In the present study, we have further investigatedresponses of the BN1 plants to light. Fluence rate dependenceanalysis of the inhibition of hypocotyl elongation indicatedthat the response is biphasic. The amplitude of the low fluencerate component increased by 2 to 3 fold in the BN1 plants comparedto the wild type. In contrast, the presence of rice PhyA didnot alter the level of chlorophyll in the BN1 seedlings grownunder the same light conditions. Ultrastructure studies showedthat chloroplasts in the BN1 plants were not significantly differentfrom those in the wild type plants, except that chloroplastsin the guard cells of the BN1 plants appeared to be more developedthan those of the wild type plants. The fluence response analysisof the potentiation of chlorophyll accumulation indicated nosignificant difference between the BN1 and the wild type plants.Thus, the introduced rice PhyA greatly influenced hypocotylelongation but did not significantly affect the greening process. ⁴Present address: NSFC Center for Biological Timing, Universityof Virginia Charlottesville, VA 22901, U.S.A. ⁵Present address: Advanced Research Laboratory, Hitachi Ltd.Hatoyama, Saitama, 350-03 Japan     

Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. In Arabidopsis thaliana, there are genes encoding at least five phytochromes, and it is of interest to learn if the different phytochromes have overlapping or distinct functions. To address this question for two of the phytochromes in Arabidopsis, we have compared light responses of the wild type with those of a phyA null mutant, a phyB null mutant, and a phyA phyB double mutant. We have found that both phyA and phyB mutants have a deficiency in germination, the phyA mutant in far-red light and the phyB mutant in the dark. Furthermore, the germination defect caused by the phyA mutation in far- red light could be suppressed by a phyB mutation, suggesting that phytochrome B (PHYB) can have an inhibitory as well as a stimulatory effect on germination. In red light, the phyA phyB double mutant, but neither single mutant, had poorly developed cotyledons, as well as reduced red-light induction of CAB gene expression and potentiation of chlorophyll induction. The phyA mutant was deficient in sensing a flowering response inductive photoperiod, suggesting that PHYA participates in sensing daylength. In contrast, the phyB mutant flowered earlier than the wild type (and the phyA mutant) under all photoperiods tested, but responded to an inductive photoperiod. Thus, PHYA and PHYB appear to have complementary functions in controlling germination, seedling development, and flowering. We discuss the implications of these results for possible mechanisms of PHYA and PHYB signal transduction.     

Pea Mutants with Reduced Sensitivity to Far-Red Light Define an Important Role for Phytochrome A in Day-Length Detection

In garden pea (Pisum sativum L.), a long-day plant, long photoperiods promote flowering by reducing the synthesis or transport of a graft-transmissible inhibitor of flowering. Previous physiological studies have indicated that this promotive effect is predominantly achieved through a response that requires long exposures to light and for which far-red (FR) light is the most effective. These characteristics implicate the action of phytochrome A (phyA). To investigate this matter further, we screened ethylmethane sulfonate-mutagenized pea seedlings for FR-unresponsive, potentially phyA-deficient mutants. Two allelic, recessive mutants were isolated and were designated fun1 for FR unresponsive. The fun1-1 mutant is specifically deficient in the PHYA apoprotein and has a seedling phenotype indistinguishable from wild type when grown under white light. However, fun1-1 plants grown to maturity under long photoperiods show a highly pleiotropic phenotype, with short internodes, thickened stems, delayed flowering and senescence, longer peduncles, and higher seed yield. This phenotype results in large part from an inability of fun1-1 to detect day extensions. These results establish a crucial role for phyA in the control of flowering in pea, and show that phyA mediates responses to both red and FR light. Furthermore, grafting and epistasis studies with fun1 and dne, a mutant deficient in the floral inhibitor, show that the roles of phyA in seedling deetiolation and in day-length detection are genetically separable and that the phyA-mediated promotion of flowering results from a reduction in the synthesis or transport of the floral inhibitor.     

Different Phototransduction Kinetics of Phytochrome A and Phytochrome B in Arabidopsis thaliana

The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of β-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h⁻¹ (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h⁻¹ caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.     

高等植物光敏色素的分子结构、生理功能和进化特征

光敏色素是植物感受外界环境变化的最重要光受体之一,对红光和远红外光非常敏感。本文综述了光敏色素的分子结构、它所包含的结构域和相应功能以及植物各主要类群中光敏色素基因家族的成员组成与进化关系;重点在分子水平上介绍了光敏色素的生理功能与作用机制。最后,基于最新的研究进展提出了将来的研究方向。