植物的光敏色素

光敏色素作为植物体内的一种光受体,在植物的光形态建成过程中意义重大,本文对光敏色素的分子特性,生理功能,作用方式及基因表达调控等方面的研究作系统的总结。     

光敏色素对植物抗逆反应的调控研究进展

光敏色素是红光和远红光受体,不但在植物光形态建成中扮演着重要的角色,还参与调控植物抗逆信号通路。阐述了光敏色素及其互作的转录因子通过诱导植物激素信号途径调控植物对病原菌、害虫等生物胁迫的反应及作用机制,以及光敏色素调控植物对临近植物的竞争胁迫、干旱、低温、高温等非生物胁迫反应的作用机制研究进展,并讨论与展望了光敏色素研究领域所面临的挑战与发展方向。     

光敏色素分子及其信号转导途径

作为植物体内的一种光受体,光敏色素在植物的光形态建成过程中意义重大。植物光敏色素及由它介导的信号传导途径是目前细胞生物学、发育生物学和分子生物学研究的热点之一。本文介绍了光敏色素的分子特性、生理功能和信号转导途径等方面的研究进展。     

植物HY5蛋白结构与功能的研究进展

HY5（LONG HYPOCOTYL 5）为光形态建成的正调控因子,是光调控植物发育的分子开关。在光诱导的基因表达中,HY5调控着植物基因组中上千基因的表达,它既可以单独调控相关基因的表达,也可以与其他调控因子一起共同调控相关基因的表达。HY5蛋白除了在光形态建成中起作用外,还在植物激素的信号传递过程中起着极其重要的作用,它整合了光信号传递和植物激素的信号传递。本综述简要介绍HY5蛋白的结构、生理功能及其分子机制等方面有关的进展。     

光调控植物种子萌发分子机制研究进展

萌发是种子植物进入农业生态系统的重要发育阶段。对于需光类种子,光是调控其萌发最重要的环境信号因子之一,红光促进而远红光抑制种子萌发。光敏色素是调控种子萌发的主要光受体。活化的光敏色素诱导萌发主效抑制因子PIF1发生蛋白降解,调节赤霉素和脱落酸代谢和信号途径相关基因的表达,从而促进种子的萌发。同时,一系列的表观遗传因子通过改变染色质结构,动态调节萌发相关基因的表达从而影响种子的萌发进程。该论文重点论述了光调控种子萌发的转录及表观遗传机制研究进展,并对其在农业生产中的应用进行了展望。     

光控发育和光敏色素的研究进展

植物发育是其遗传基因在环境因子作用下,在一定的时间和空间组合下的顺序和协调的表达。光是环境因子中对发育调控作用最广泛、最明显的一个因子。绝大部分光调节反应是不可逆的形态建成反应。所谓光形态建成(photomor-phogenesis)就是光控发育的意思。它是由多个光受体参与调节的复杂过程,这些光受体是光敏色素(phytochrome)、隐花色素(cryptochrome)、紫外光-β受体。近一个世纪来光控发育研究从整体到细胞、最近到大分子水平不断地发展,近20年来积累了大量的资料,形成了一个分支学科。在植物的光生物学中,它是和光合作     

植物光敏素的反馈作用

植物正常的发育过程是一个光形态建成过程、一个需光调控的过程。从种子萌发、幼苗生长、叶片展开和叶绿体发育一直到开花、衰老和对光环境的各种适应都离不开光参与调节。在大多数情况下,光敏素就是这种调控发育的光的受体。它的作用的分子基础迄今还未研究清楚,但是人们已经开始了解光敏素可以调节高等植物的基因表达。最     

光敏色素A调控光响应基因表达及其翻译后修饰研究进展

光是调节植物生长发育最重要的环境信号因子之一。植物通过光受体感受自然环境中光的强度、方向以及光周期等信号的变化,从而调控其生长发育过程。光敏色素A (phytochrome A, PHYA)是植物中唯一的远红光受体蛋白,具有在黑暗下在细胞质中合成,而在照光后快速入核和降解的特性,并通过多种途径精确调节了植物光响应基因的转录网络。同时,蛋白质翻译后修饰在调节PHYA稳定性和活性的过程中发挥了重要的作用。该文论述了PHYA调节光响应基因表达以及PHYA翻译后修饰方向的研究进展,并展望了PHYA在农作物分子设计育种中的应用前景。     

植物的光受体及其调控机制的研究

近年来,通过对植物的分子遗传学研究,在植物光受体及其在光形态建成中对植物生长发育的调控机制方面取得了显著进展。从光受体及基因家族的概况,包括光敏色素、隐花色素、向光素的基本结构、分子特征、基因和信号转导等,介绍了光受体在光控发育调节机制方面的研究进展情况。     

光色素信号通路中磷酸化修饰研究进展

光是植物的唯一能量来源, 植物在进化过程中产生不同的光敏色素来感知光信号。光信号通路中元件通常被特异翻译后修饰调节。光敏色素是一种自磷酸化的丝氨酸/苏氨酸蛋白激酶, 可以被一些蛋白磷酸酶去磷酸化。通过对光敏色素A (phyA)和光敏色素B (phyB)的自磷酸化位点研究, 发现自磷酸化对光敏色素的功能及其介导的信号通路起着非常重要的作用。光激活的光敏色素诱导光敏色素作用因子(PIF)磷酸化, 这对于PIF的正常降解及光形态建成的起始是必需的。该文主要介绍了光敏色素信号通路磷酸化修饰的最新进展, 以期为深入研究光敏色素信号转导机制提供参考。     

Functional analysis of a 450-amino acid N-terminal fragment of phytochrome B in Arabidopsis

Phytochrome, a major photoreceptor in plants, consists of two domains: the N-terminal photosensory domain and the C-terminal domain. Recently, the 651-amino acid photosensory domain of phytochrome B (phyB) has been shown to act as a functional photoreceptor in the nucleus. The phytochrome (PHY) domain, which is located at the C-terminal end of the photosensory domain, is required for the spectral integrity of phytochrome; however, little is known about the signal transduction activity of this domain. Here, we have established transgenic Arabidopsis thaliana lines expressing an N-terminal 450-amino acid fragment of phyB (N450) lacking the PHY domain on a phyB-deficient background. Analysis of these plants revealed that N450 can act as an active photoreceptor when attached to a short nuclear localization signal and beta-glucuronidase. In vitro spectral analysis of reconstituted chromopeptides further indicated that the stability of the N450 Pfr form, an active form of phytochrome, is markedly reduced in comparison with the Pfr form of full-length phyB. Consistent with this, plants expressing N450 failed to respond to intermittent light applied at long intervals, indicating that N450 Pfr is short-lived in vivo. Taken together, our findings show that the PHY domain is dispensable for phyB signal transduction but is required for stabilizing the Pfr form of phyB.     

NDPK2 as a signal transducer in the phytochrome-mediated light signaling

Nucleoside-diphosphate kinase (NDPK) 2 in Arabidopsis has been identified as a phytochrome-interacting protein by using the C-terminal domain of phytochrome A (PhyA) as the bait in yeast two-hybrid screening. The far-red light-absorbing form of phytochrome (Pfr) A stimulates NDPK2 gamma-phosphate exchange activity in vitro. To better understand the multiple functions of NDPK and its role in phytochrome-mediated signaling, we characterized the interaction between phytochrome and NDPK2. Domain studies revealed that PER-ARNT-SIM domain A in the C-terminal domain of phytochrome is the binding site for NDPK2. Additionally, phytochrome recognizes both the NDPK2 C-terminal fragment and the NDPK2 hexameric structure to fulfill its binding. To illustrate the mechanism of how the Pfr form of phytochrome stimulates NDPK2, His-197-surrounding residue mutants were made and tested. Results suggested that the H-bonding with His-197 inside the nucleotide-binding pocket is critical for NDPK2 functioning. The pH dependence profiles of NDPK2 indicated that mutants with different activities from the wild type have different pK(a) values of His-197 and that NDPK2 hyperactive mutants possess lower pK(a) values. Because a lower pK(a) value of His-197 accelerates NDPK2 autophosphorylation and the phospho-transfer between the phosphorylated NDPK2 and its kinase substrate, we concluded that the Pfr form of phytochrome stimulates NDPK2 by lowering the pK(a) value of His-197.     

Identification of a highly conserved domain on phytochrome from angiosperms to algae

A monoclonal antibody (Pea-25) directed to phytochrome from etiolated peas (Pisum sativum L., cv Alaska) binds to an antigenic domain that has been highly conserved throughout evolution. Antigenic cross-reactivity was evaluated by immunoblotting sodium dodecyl sulfate sample buffer extracts prepared from lyophilized tissue samples or freshly harvested algae. Pea-25 immunostained an approximately 120-kilodalton polypeptide from a variety of etiolated and green plant tissues, including both monocotyledons and dicotyledons. Moreover, Pea-25 immunostained a similarly sized polypeptide from the moss Physcomitrella, and from the algae Mougeotia, Mesotaenium, and Chlamydomonas. Because Pea-25 is directed to phytochrome, and because it stains a polypeptide about the size of oat phytochrome, it is likely that Pea-25 is detecting phytochrome in each case. The conserved domain that is recognized by Pea-25 is on the nonchromophore bearing, carboxyl half of phytochrome from etiolated oats. Identification of this highly conserved antigenic domain creates the potential to expand investigations of phytochrome at a cellular and molecular level to organisms, such as Chlamydomonas, that offer unique experimental advantages.     

Structure function studies on phytochrome. Identification of light-induced conformational changes in 124-kDa Avena phytochrome in vitro

Light-mediated conformational changes in highly purified 124-kDa phytochrome preparations from etiolated oat seedlings have been identified by steric exclusion high performance liquid chromatography and limited proteolytic studies. Steric exclusion high performance liquid chromatography studies of oat and rye phytochromes show photoreversible changes in retention times, with the red absorbing form of phytochrome (Pr form) eluting later than the far red absorbing form of phytochrome produced by saturating red light illumination of Pr (Pfr form) in a variety of different mobile phase buffers. Molecular mass calibration with globular protein standards in Tris-glycol buffers provides estimates of 318-349 and 363-366 kDa for the molecular sizes of the Pr and Pfr forms, respectively. These analyses support earlier studies that phytochrome is a nonglobular homodimer of 124-kDa subunits in vitro. Limited proteolytic dissection of phytochrome in nondenaturing buffers with seven different endoproteases provides evidence for two "operational" domains within the 124-kDa subunit with molecular mass values of 69-72 and 52-55 kDa. The larger 69-72-kDa domain contains the site for the chromophore attachment as shown by gel electrophoresis derived enzyme-linked immunosorbent assay utilizing site-directed rabbit antiserum to a synthetic undecapeptide which is homologous with the chromophore binding site on oat phytochrome. This chromophore domain exhibits a compact structure, resistant to further proteolysis except near its N terminus. By contrast, the 52-55-kDa nonchromophore domain contains multiple sites for further proteolytic cleavage as revealed by rapid cleavage to smaller polypeptide fragments. Detailed kinetic analyses of the limited proteolytic cleavage of phytochrome with four endoproteases, subtilisin BPN', thermolysin, trypsin, and clostripain, has mapped specific regions within the 124-kDa subunit that participate in light-induced conformational changes. These include a 4-10-kDa region near the N terminus of the chromophore binding domain and at least two regions within the nonchromophore domain. A comprehensive peptide map of the oat phytochrome subunit is presented, which incorporates the results of these proteolytic studies with the recent, yet unpublished sequence analyses of Avena phytochrome cDNA clones which show the N-terminal localization of the chromophore binding site (Hershey, H. P., Colbert, J. T., Lissemore, J. L., Barker, R. F., and Quail, P. H. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2332-2336).     

Molecular topography of phytochrome as deduced from the tritium-exchange method

The hydrogen-tritium-exchange measurements on phytochrome have been performed to detect the conformational differences between the red-absorbing (Pr) and the far-red absorbing (Pfr) forms of phytochrome. The large and small Pfr molecules revealed more exchangeable protons that did the corresponding Pr molecules by 96 and 70 protons, respectively. These results suggest that the Pr leads to Pfr phototransformation is accompanied by an additional exposure of the peptide chains in the Pfr molecule. Of 1682 theoretically exchangeable hydrogens in undegraded phytochrome, only 442 (26%) and 346 (21%) protons were found to be exchangeable (excluding instantaneously exchangeable protons that cannot be determined by the present method). Thus, the phytochrome protein appears to be compact and highly folded. The kinetic analyses of the tritium exchange-out curves indicate that two kinetically different groups are responsible for the conformational differences between the Pr and Pfr forms of phytochrome. These components are due to (1) the exposure of hydrogen-bonded peptide segments (alpha helix and/or beta-pleated sheet) in the chromophore vicinity of Pfr and (2) the exposure of hydrogen-bonded peptide segments on the chromophore peptide domain as well as on the chromophore-free tryptic domain of undegraded phytochrome.     

Arabidopsis phytochrome a is modularly structured to integrate the multiple features that are required for a highly sensitized phytochrome

Phytochrome is a red (R)/far-red (FR) light-sensing photoreceptor that regulates various aspects of plant development. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates atypical phytochrome responses, such as the FR high irradiance response (FR-HIR), which is elicited under prolonged FR. A proteasome-based degradation pathway rapidly eliminates active Pfr (the FR-absorbing form of phyA) under R. To elucidate the structural basis for the phyA-specific properties, we systematically constructed 16 chimeric phytochromes in which each of four parts of the phytochrome molecule, namely, the N-terminal extension plus the Per/Arnt/Sim domain (N-PAS), the cGMP phosphodiesterase/adenyl cyclase/FhlA domain (GAF), the phytochrome domain (PHY), and the entire C-terminal half, was occupied by either the phyA or phytochrome B sequence. These phytochromes were expressed in transgenic Arabidopsis thaliana to examine their physiological activities. Consequently, the phyA N-PAS sequence was shown to be necessary and sufficient to promote nuclear accumulation under FR, whereas the phyA sequence in PHY was additionally required to exhibit FR-HIR. Furthermore, the phyA sequence in PHY alone substantially increased the light sensitivity to R. In addition, the GAF phyA sequence was important for rapid Pfr degradation. In summary, distinct structural modules, each of which confers different properties to phyA, are assembled on the phyA molecule.     

Mapping of antigenic domains on phytochrome from etiolated Avena sativa L. by immunoblot analysis of proteolytically derived peptides

Several monoclonal antibodies to phytochrome that interact with putative functionally important domains have been previously identified. The locations of some of these domains are determined here by epitope mapping experiments that utilize immunoblot analyses of proteolytically degraded phytochrome. Seven independent epitopes are identified. An epitope that is recognized by monoclonal antibody Oat-25 is confirmed to be wholly located near the N terminus of phytochrome. This domain undergoes a conformational change when phytochrome is interconverted between its red- and far-red-absorbing forms and is recognized by Oat-25 better in the red-absorbing form. A second domain that also undergoes a photointerconvertible conformation change and that contains the epitope for Oat-16 is localized near the site of chromophore attachment, which is about 36 kDa from the N terminus. A third domain, which contains the most highly conserved epitope on phytochrome that has so far been identified, is recognized by Pea-25 and is located about 85 kDa from the N terminus. Other epitopes and their approximate distances from the N terminus are those recognized by Oat-22 (36 kDa), Oat-13 (65 kDa), and Oat-8 and Oat-28 (70-75 kDa). Even though epitopes for Oat-16 and Oat-22, as well as for Oat-8 and Oat-28, are close together, competitive binding assays indicate that they are different. Immunoblot analyses also indicate that the epitope for Oat-28 is further from the N terminus of phytochrome than is that for Oat-8.     

Dominant negative suppression of arabidopsis photoresponses by mutant phytochrome A sequences identifies spatially discrete regulatory domains in the photoreceptor.

We used the exaggerated short hypocotyl phenotype induced by oat phytochrome A overexpression in transgenic Arabidopsis to monitor the biological activity of mutant phytochrome A derivatives. Three different mutations, which were generated by removing 52 amino acids from the N terminus (delta N52), the entire C-terminal domain (delta C617), or amino acids 617-686 (delta 617-686) of the oat molecule, each caused striking dominant negative interference with the ability of endogenous Arabidopsis phytochrome A to inhibit hypocotyl growth in continuous far-red light ("far-red high irradiance response" conditions). By contrast, in continuous white or red light, delta N52 was as active as the unmutagenized oat phytochrome A protein in suppressing hypocotyl elongation, while delta C617 and delta 617-686 continued to exhibit dominant negative behavior under these conditions. These data suggest that at least three spatially discrete molecular domains coordinate the photoregulatory activities of phytochrome A in Arabidopsis seedlings. The first is the chromophore-bearing N-terminal domain between residues 53 and 616 that is apparently sufficient for the light-induced initiation but not the completion of productive interactions with transduction chain components. The second is the C-terminal domain between residues 617 and 1129 that is apparently necessary for completion of productive interactions under all irradiation conditions. The third is the N-terminal 52 amino acids that are apparently necessary for completion of productive interactions only under far-red high irradiance conditions and are completely dispensable under white and red light regimes.     

Spectral Characterization and Proteolytic Mapping of Native 120-Kilodalton Phytochrome from Cucurbita pepo L

A spectral, immunochemical, and proteolytic characterization of native 120-kilodalton (kD) phytochrome from Cucurbita pepo L. is presented and compared with that previously reported for native 124-kD phytochrome from Avena sativa. The molecule was partially purified (~200-fold) in the phytochrome—far red-absorbing form (Pfr) in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride, using a modification of the procedure initially developed to purify 124-kD Avena phytochrome. The spectral properties of the preparations obtained are indistinguishable from those described for 124-kD Avena phytochrome, including a Pfr λ_max at 730 nanometers, a spectral change ratio (ΔA_r/ΔA_fr) of 1.05, and negligible dark reversion of Pfr to the red-absorbing form (Pr) in the presence or absence of sodium dithionite. This lack of dark reversion in vitro contrasts with observations that Cucurbita phytochrome, like phytochrome from most other dicotyledons, exhibits substantial dark reversion in vivo. Ouchterlony double immunodiffusion analysis with polyclonal antibodies indicates that 120-kD Cucurbita phytochrome is immunologically dissimilar to 124-kD Avena phytochrome. However, despite this dissimilarity, immunoblot analyses of proteolytic digests have identified at least three spatially separate epitopes that are common to both phytochromes. Using endogeneous protease(s), a peptide map for Cucurbita phytochrome has been constructed and the role that specific domains play in the overall structure of the photoreceptor has been examined. One domain near the NH₂ terminus is critical to the spectral integrity of the molecule indicating that this domain plays a structural role analogous to that of a domain near the NH₂ terminus of Avena phytochrome. Proteolytic removal of this domain occurs preferentially in Pr and its removal shifts the Pfr λ_max to 722 nm, increases the spectral change ratio to 1.3, and substantially enhances the dark reversion rate. The apparent conservation of this domain among evolutionarily divergent plant species and its involvement in a conformational change upon photoconversion makes it potentially relevant to the mechanism(s) of phytochrome action. Preliminary evidence from gel filtration studies suggests that the 55-kD chromophoreless COOH-terminal region of the polypeptide contains a domain responsible for dimerization of phytochrome monomers.