Daminozide Alters Anthocyanin Metabolism in Ray Florets of Bronze Chrysanthemum (Chrysanthemum morifolium Ramat.)

Daminozide is a well-known chemical inhibitor of the gibberellic acid biosynthesis pathway regulating the vegetative growth of potted chrysanthemums (Chrysanthemum morifolium Ramat.). However, the precise mechanism underlying daminozide-related floral color loss is unknown. To investigate the latter, in two separate greenhouse experiments, bronze flowering chrysanthemum cultivars ‘Baton Rouge’ and ‘Pelee’ were treated weekly with consecutive (0 or 5,000 mg l^?1) foliar daminozide spray applications at early, intermediate, and late stages during the short-day photoperiod. The ray florets of both cultivars were sampled, and the effect of daminozide application on anthocyanins and their biosynthetic precursors were determined by HPLC. Daminozide applied to ‘Baton Rouge’ plants at early developmental stages was correlated with partial loss of red color, and HPLC analysis determined that this was associated with a 75 % reduction in ray floret anthocyanins. Conversely, a near complete loss of red coloration in daminozide-sprayed ‘Pelee’ relative to control plants was associated with as much as a 98 % decline in anthocyanins, irrespective of the time of application. HPLC analysis determined that daminozide application was associated with a 22–50 % increase in the flavones apigenin 7-O-rutinoside, acacetin 7-O-rutinoside, diosmetin 7-O-rutinoside, and eupatorin, and a 68 % increase in the flavonol quercetin 3-O-glucoside, in ray florets of ‘Pelee’ relative to control plants. There was no relative change in ‘Baton Rouge’ flavone and flavonol levels. The accumulation of bronze C. morifolium flavones and flavonols following foliar daminozide application suggests that red color loss is associated with inhibition of anthocyanidin synthase of ‘Pelee’ ray florets.     

Anthocyanin, Carotenoid, and Chlorophyll Changes in the Peel of Cox's Orange Pippin Apples during Ripening on and off the Tree

The concentrations of various peel pigments of Cox’s OrangePippin apples have been measured during ripening on the treeand during storage at 12 °C. Total chlorophyll decreased and total carotenoid increased atthe time of the respiration climacteric. These changes weremore pronounced in fruit maturing on the tree where a significantincrease of anthocyanin occurred; it did not occur in storedfruit. There was no consistent or marked difference in the ratesof destruction of chlorophylls a and b. The carotenoids found in the unripe fruit were those characteristicof photosynthetic tissue, ß-carotene, lutein, violaxanthin,and neoxanthin. These decreased to a greater or lesser extent,and at different rates, on and off the tree. Other carotenoidswhich increased greatly during ripening were identified as esters,mainly of violaxanthin. During the climacteric there is a transition from an assemblageof pigments associated with the chloroplast to that typicalof a chromoplast.     

Reorganization of the Photosystem II Unit in Developing Thylakoids of Higher Plants after Transfer to Darkness : Changes in Chlorophyll b, Light-Harvesting Chlorophyll Protein Content, and Grana Stacking

A light-dependent reversible grana stacking-unstacking process, paralleled by a reorganization of thylakoid components, has been noticed in greening etiolated bean (Phaseolus vulgaris, var. red kidney) leaves upon transfer to darkness. The reorganization, based on biochemical and biophysical criteria, involves mainly the photosystem II (PSII) unit components: upon transfer to darkness, the light-harvesting chlorophyll protein (LHCP), its 25 kilodalton polypeptide and chlorophyll b are decreased, while the CPa and its 42 kilodalton polypeptide are increased and new PSII units of smaller size are formed. This reorganization of components occurs only in thylakoids still in the process of development and not in those present in steady state conditions.

It is proposed that this process does not reflect the turnover of the LHCP component per se, but a regulatory process operating during development, by which the ratio of light-harvesting to PSII reaction center components, determined by the environmental conditions, controls the photosynthetic rate.

     

Influence of Virus on the Chlorophyll, Carotenoid and Polyamine Contents in Grapevine Microcuttings

We have studied the effect of different types of virus infections on the content in chlorophylls, carotenoids and free polyamines in shoots of vine cv. Albariño cultured in vitro. The ratio of chlorophyll a/c hlorophyll b was maintained practically constant in the different cases studied. A significant increase was observed in the polyamines, especially putrescine, in shoots infected with the different types of virus, mainly in the cases of grapevine fleck disease+grapevine leafroll disease type I and grapevine stem pitting.     

Phytochrome Control of Chlorophyll and Carotenoid Accumulation in Sorghum bicolor

Etiolated Sorghum bicolor seedlings manifested a significantmorphological response to short term irradiations by red andfar-red light and to a continuous far-red light. Accumulationof chlorophylls in white light and carotenoids in darkness isunder red/far-red reversible control as well as along with theeffectiveness of ‘High Irradiance Reaction’. Phytochromeis also found to eliminate the lag phase during the accumulationof chlorophylls and carotenoids in white light. (Received March 11, 1981; Accepted May 2, 1981)     

Specificity of the Cyanobacterial Orange Carotenoid Protein: Influences of Orange Carotenoid Protein and Phycobilisome Structures

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome (PB), the extramembranous light-harvesting antenna. This mechanism is triggered by the photoactive Orange Carotenoid Protein (OCP), which acts both as the photosensor and the energy quencher. The OCP binds the core of the PB. The structure of this core differs in diverse cyanobacterial strains. Here, using two isolated OCPs and four classes of PBs, we demonstrated that differences exist between OCPs related to PB binding, photoactivity, and carotenoid binding. Synechocystis PCC 6803 (hereafter Synechocystis) OCP, but not Arthrospira platensis PCC 7345 (hereafter Arthrospira) OCP, can attach echinenone in addition to hydroxyechinenone. Arthrospira OCP binds more strongly than Synechocystis OCP to all types of PBs. Synechocystis OCP can strongly bind only its own PB in 0.8 m potassium phosphate. However, if the Synechocystis OCP binds to the PB at very high phosphate concentrations (approximately 1.4 m), it is able to quench the fluorescence of any type of PB, even those isolated from strains that lack the OCP-mediated photoprotective mechanism. Thus, the determining step for the induction of photoprotection is the binding of the OCP to PBs. Our results also indicated that the structure of PBs, at least in vitro, significantly influences OCP binding and the stabilization of OCP-PB complexes. Finally, the fact that the OCP induced large fluorescence quenching even in the two-cylinder core of Synechococcus elongatus PBs strongly suggested that OCP binds to one of the basal allophycocyanin cylinders.The cyanobacterial Orange Carotenoid Protein (OCP) is a photoactive soluble protein of 35 kD that binds a ketocarotenoid, 3′-hydroxyechinenone (hECN). It is present in the majority of phycobilisome (PB)-containing cyanobacterial strains (Kirilovsky and Kerfeld, 2012, 2013). The PBs are light-harvesting extramembrane complexes formed by a core from which rods radiate. The core and rods are constituted of water-soluble blue and red phycobiliproteins, which covalently attach bilins (for review, see Glazer, 1984; Grossman et al., 1993; MacColl, 1998; Tandeau de Marsac, 2003; Adir, 2005). The OCP was first described by Holt and Krogmann (1981), and its structure was determined in 2003 (Kerfeld et al., 2003). However, its function was discovered only in 2006 (Wilson et al., 2006) and its photoactivity in 2008 (Wilson et al., 2008). The OCP is essential in a photoprotective mechanism that decreases the energy arriving at the reaction centers under high irradiance. Strong light induces thermal dissipation of the energy absorbed by the PBs, resulting in a decrease of PB fluorescence emission and of energy transfer from the PBs to the reaction centers (Wilson et al., 2006). This process, which is light intensity dependent, is induced by blue or green light but not by orange or red light (Rakhimberdieva et al., 2004; Wilson et al., 2006). The absorption of strong blue-green light by the OCP induces changes in the conformation of the carotenoid, converting the inactive orange dark form (OCP^o) into an active red form (OCP^r; Wilson et al., 2008). In OCP^o, the hECN is in an all-trans-configuration (Kerfeld et al., 2003; Polívka et al., 2005). In OCP^r, the apparent conjugation length of the carotenoid increases, resulting in a less distorted, more planar structure (Wilson et al., 2008). Fourier transform infrared spectra showed that conformational changes in the protein are also induced (Wilson et al., 2008) that are essential for the induction of the photoprotective mechanism. Only OCP^r is able to bind to the core of PBs and to induce thermal energy dissipation (Wilson et al., 2008; Punginelli et al., 2009; Gorbunov et al., 2011; Gwizdala et al., 2011). Since the photoactivation of the OCP has a very low quantum yield (0.03; Wilson et al., 2008), the concentration of activated protein is zero in darkness and very low under low-light conditions (Wilson et al., 2008; Gorbunov et al., 2011). Thus, the photoprotective mechanism functions only under high-light conditions.The crystal structures of the Arthrospira maxima OCP and of the Synechocystis PCC 6803 (hereafter Synechocystis) OCP were solved in 2003 and 2010, respectively (Kerfeld et al., 2003; Wilson et al., 2010). These structures, assumed to correspond to the dark OCP^o form, are essentially identical. The OCP consists of an all-α-helical N-terminal domain (residues 1–165), unique to cyanobacteria, and an α-helical/β-sheet C-terminal domain that is a member of the Nuclear Transport Factor2 superfamily (residues 191–320; Synechocystis numbering). Both domains are joined by a linker (residues 166–190; Synechocystis numbering) that appears to be flexible. The hECN molecule spans the N- and C-terminal domains of the protein, with its carbonyl end embedded in and hydrogen bonded to two absolutely conserved residues (Tyr-201 and Trp-288) in the C-terminal domain. The carotenoid is almost entirely buried; only 3.4% of the 3′ hECN is solvent exposed (Kerfeld et al., 2003). Synechocystis OCP can also bind with high-affinity echinenone (ECN) and zeaxanthin. While the ECN OCP is photoactive, the zeaxanthin OCP is photoinactive (Punginelli et al., 2009), indicating the importance of the carotenoid carbonyl group for photoactivity. The largest interface through which the two domains interact and through which the carotenoid passes is stabilized by a small number of hydrogen bonds, including one formed between Arg-155 and Glu-244 (Wilson et al., 2010). This salt bridge stabilizes the closed structure of OCP^o. Upon illumination, protein conformational changes cause the breakage of this bond and the opening of the protein (Wilson et al., 2012). Arg-155, which becomes more exposed upon the separation of the two domains, is essential for the OCP^r binding to the PBs (Wilson et al., 2012).After exposure to high irradiance, when the light intensity decreases, recovery of full antenna capacity and fluorescence requires another protein, the Fluorescence Recovery Protein (FRP; Boulay et al., 2010). The active form of this soluble 13-kD protein is a dimer (Sutter et al., 2013). It interacts with the OCP^r C-terminal domain (Boulay et al., 2010; Sutter et al., 2013). This accelerates the red-to-orange OCP conversion and helps the OCP to detach from the PB (Boulay et al., 2010; Gwizdala et al., 2011).Genes encoding the full-length OCP are found in the vast majority of cyanobacteria but not in all; 90 of 127 genomes recently surveyed contain at least one gene for a full-length OCP (Kirilovsky and Kerfeld, 2013). The genomes of Synechococcus elongatus and Thermosynechococcus elongatus, two cyanobacterial strains used as model organisms in photosynthesis and stress studies, do not contain a full-length ocp gene. These strains also lack FRP and β-carotene ketolase (involved in ketocarotenoid synthesis). As a consequence, these strains lack the OCP-related photoprotective mechanism and are more sensitive to fluctuating light intensities (Boulay et al., 2008).The core of the hemidiscoidal PBs of Synechocystis, the model organism routinely used for the study of the OCP-related photoprotective mechanism, consists of three cylinders, each one formed by four trimers of allophycocyanin (APC; Fig. 1; for review, see Glazer, 1984; Bryant, 1991; Grossman et al., 1993; MacColl, 1998; Adir, 2005). The APC trimers are predominantly assembled from a two-subunit heterodimer, αAPC-βAPC, which binds two phycocyanobilins, one in each subunit. Of the 12 total APC trimers in the PB core, eight are trimers of αAPC-βAPC. These trimers have a maximal emission at 660 nm (APC₆₆₀). The upper cylinder contains only APC₆₆₀ trimers. In contrast, each basal cylinder contains only two APC₆₆₀ trimers. Each basal cylinder also contains the following: (1) a trimer in which one αAPC subunit is replaced by a special αAPC-like subunit called ApcD, and (2) a trimer in which one β-subunit is replaced by ApcF, a βAPC-like subunit, and one α-subunit is replaced by the N-terminal domain of ApcE, an αAPC-like domain (Fig. 1). The trimers containing one or two of these special subunits have a maximal emission at 680 nm (APC₆₈₀). In each cylinder, the two external trimers are stabilized by an 8.7-kD linker protein.Open in a separate windowFigure 1.Schematic orthogonal projections of the various PB cores. In the PBs containing three or five cylinders, the top complete cylinder is formed by four αAPC-βAPC trimers emitting at 660 nm. Each of the basal cylinders of three types of PBs contains two αAPC-βAPC trimers emitting at 660 nm and two trimers emitting at 683 nm. In one of them, one αAPC is replaced by ApcD, and in the other one, αAPC-βAPC is replaced by the dimer ApcF-ApcE. In the five cylinder PBs, two additional semicylinders formed by two αAPC-βAPC trimers are present. In all the cylinders, the two external trimers include an 8.7-kD linker protein (ApcC).The C-terminal part of Synechocystis ApcE contains three interconnected repeated domains of about 120 residues (called Rep domains) that are similar to the conserved domains of rod linkers. Each Rep domain interacts with an APC trimer situated in different cylinders, which stabilizes the core of PB (Zhao et al., 1992; Shen et al., 1993; Ajlani et al., 1995; Ajlani and Vernotte, 1998). The ApcE protein also determines the number of APC cylinders that form the PB core (Capuano et al., 1991, 1993). Indeed, there are PBs containing only the two basal cylinders, as in S. elongatus (ex S. elongatus PCC 7942) and Synechococcus PCC 6301. In these strains, the approximately 72-kD ApcE possesses only two Rep domains. There also exist pentacylindrical cores in which, in addition to the three cylinders existing in Synechocystis PBs, there are two other cylinders, each formed by two APC₆₆₀ trimers, for example in Anabaena variabilis, Anabaena PCC 7120, and Mastigocladus laminosus (Glauser et al., 1992; Ducret et al., 1998). In the pentacylindrical PBs, ApcE (approximately 125 kD) contains four Rep domains (Capuano et al., 1993). Finally, ApcE is also involved in the interaction between the PB and the thylakoids.The bicylindric and tricylindric cores are surrounded by six rods formed generally by three hexamers of the blue phycocyanin (PC) or two PC hexamers and one hexamer containing phycoerythrin or phycoerythrincyanonin. The rods and the hexamers are stabilized by nonchromophorylated linker proteins. A linker protein, L^RC also stabilizes the binding of the rods to the core. The pentacylindric PBs can contain up to eight rods. The quantity and length of rods and the presence of phycoerythrin or phycoerythrocyanin at the periphery of the rods depends on environmental conditions like light intensity or quality (Kipe-Nolt et al., 1982; Glauser et al., 1992).The OCP probably binds to one of the APC₆₆₀ trimers (Tian et al., 2011, 2012; Jallet et al., 2012), and the presence of the rods stabilizes this binding to Synechocystis PBs (Gwizdala et al., 2011). The different structures of PBs in other strains could affect the binding of the OCP. Thus, we undertook a study about the relationship between the structure of PBs and OCP binding in preparation for introducing the OCP-related photoprotective mechanism into S. elongatus using Synechocystis genes. In this study, we used the in vitro reconstitution system developed by Gwizdala et al. (2011) with three different types of isolated PBs: Arthrospira platensis PCC 7345 (hereafter Arthrospira) PBs, having a tricylindrical core like Synechocystis PBs; Anabaena variabilis (hereafter Anabaena) PBs, having a pentacylindrical core; and S. elongatus PCC 7942 (hereafter Synechococcus) PBs, having a bicylindrical core. We also used two different OCPs, the Synechocystis OCP and the Arthrospira OCP. Each OCP was isolated from mutant Synechocystis cells overexpressing one or the other ocp gene with a C-terminal His tag.     

Time Dependence of Phytochrome-mediated Carotenoid and Chlorophyll Synthesis in Sorghum bicolor L

In 5-day-old etiolated Sorghum seedlings, red light irradiationfor 1 s enhanced carotenoid and chlorophyll accumulation, and5 min of red light treatment saturated the photoresponse. Thedegree of red/far-red photoreversibility of carotenoid accumulationwas dependent on the age of the plant. No significant escapefrom far-red reversibility was observed up to 30 min after theonset of a saturating red light pulse in 5-day-old etiolatedseedlings. Thereafter, the escape was relatively fast and completedwithin 180 min. Sorghum bicolor L, carotenogenesis, phytochrome, time dependence, chlorophyll synthesis     

植物花青素合成中的MYB蛋白

概述不同植物中花青素合成调控因子MYB蛋白的研究进展。     

Difference Analysis of ClCYC2-Like Genes Expression and DNA Methylation Between the Two Types of Florets in Chrysanthemum lavandulifolium

DNA methylation is an important epigenetic modification, that is involved in the regulation of gene expression and cell differentiation, and plays an important regulatory role in flower development in higher plants. There are two types of florets on the capitulum in the genus Chrysanthemum, the flower symmetry factor CYCLOIDEA (CYC) 2-like genes may be important candidate genes for determining the identity of the two types of florets. In this study, the diploid plant Chrysanthemum lavandulifolium was used as the research material, and qRT-PCR and bisulfite sequencing polymerase chain reaction (BSP) were used to identify the expression and DNA methylation pattern of CYC2-like genes in the two types of florets. Gene expression analysis showed that the six ClCYC2-like genes were significantly different in the two types of florets, and the expression levels of ClCYC2c, ClCYC2d, ClCYC2e and ClCYC2f in the ray florets were significantly higher than those in the disc florets. For the DNA methylation analysis of the three genes ClCYC2c, ClCYC2d, and ClCYC2e, it was found that the DNA methylation levels of these three genes were negative correlated with their expression levels, and the ways in which the three genes were regulated by the DNA methylation were different. It is speculated that the different DNA methylation of ClCYC2-like genes in the two types of florets may affect the differentiation and development of the two types of florets. This study provides new clues about epigenetics for the analysis of capitulum formation in Asteraceae.

     

切花寒菊小花对低温胁迫的生理响应及其抗寒性分析

以切花寒菊品种'寒紫'和'寒黄'为材料,比较了低温下(<0℃)2个品种舌状花和管状花的相对电导率(REC)变化及半致死温度(LT50)差异,并测定了从温室温度(14.4℃～16.2℃)至3℃的降温过程中2个品种舌状花和管状花的SOD活性和MDA、可溶性糖、可溶性蛋白含量及花粉生活力变化.结果表明:低于-6℃条件下,2个品种电导率均随温度下降而显著上升,且低温下'寒黄'舌状花、管状花的相对电导率均低于'寒紫'.两品种半致死温度均在-9℃左右或更低,且'寒黄'低于'寒紫',管状花低于舌状花.在室温至3℃的降温过程中,2个品种SOD活性、可溶性蛋白含量及花粉萌发率先升后降,MDA含量则先降后升,且均在6℃或9℃时出现拐点,而可溶性糖含量表现为持续增加,且'寒紫'花粉萌发率下降较'寒黄'早且快.结果发现,'寒黄'抗寒性强于'寒紫'、管状花强于舌状花,2品种小花出现冻害的极限低温在-6℃以下,而9℃甚至6℃是寒菊花期出现生理障碍的转折点;切花寒菊可通过提高自身SOD活性及可溶性糖、可溶性蛋白含量来抵抗低温胁迫,且调节能力'寒黄'强于'寒紫'、管状花强于舌状花.     

Light-induced Changes of the Carotenoid Levels in Chloroplast Envelopes

The carotenoid content of thylakoids and envelopes isolated from dark-or light-treated spinach (Spinacia oleracea L.) chloroplasts was compared. In thylakoids, light induced a decrease of violaxanthin parallel with a stoichiometric increase of zeaxanthin due to violaxanthin deepoxidation. In envelopes, violaxanthin was also decreased and the relative decrease was similar to thylakoids, but zeaxanthin increase was small resulting in an over-all decrease of the amount of envelope carotenoids. When violaxanthin deepoxidation in thylakoids was partly inhibited by 10 nm nigericin, violaxanthin decrease in the envelope was inhibited to a similar degree.     

不同花色菊花品种花色素结构基因的表达特性

对红色、黄色、粉紫色和白色菊花品种不同开放度的花序舌状花中CHS、CHI、DFR、F3H、F3′H和3GT基因的表达量进行了相对定量分析。结果表显示:6个基因的表达因不同花色、不同发育阶段而异。‘钟山红鹰’(红色)中各基因的表达量均较高,且均在Ⅱ(松蕾期)或Ⅲ(半开期)期达到峰值,其中DFR、3GT基因的表达量远高于其他花色品种。‘金陵娇黄’(黄色)中CHS、CHI基因表达量较高,且Ⅰ(紧蕾期)、Ⅱ期表达量高于Ⅲ、Ⅳ(盛开期)期;3GT、DFR基因表达量分别高或低于‘金陵笑靥’(粉紫色)品种中相应基因的表达量,但均比红色品种低;F3H在4个品种中表达量最低,F3′H表达量接近或略低于红色或粉紫色品种,且各阶段表达水平较稳定。‘金陵笑靥’中DFR表达量仅次于‘钟山红鹰’,3GT和CHS表达量低于红色与黄色品种。‘钟山雪桂’(白色)中各基因仅有微量表达,除F3H外各基因的表达量明显低于其他花色品种。研究表明,花色素结构基因DFR、3GT是菊花花色素合成的关键基因,DFR很可能是限速关键基因,一定表达水平的CHS、CHI也是菊花花色素合成所必须的,F3H基因与花色素合成不存在直接相关。     

Stage-Specific Changes in Calcium-Regulated Protein Phosphorylation in Developing Tomato Fruits

The role of calcium and calmodulin in the in vitro phosphorylationof soluble and membrane proteins was studied in relation togrowth and development of tomato fruits. Calcium at micromolarconcentrations promoted the phosphorylation of both solubleand membrane proteins. The calmodulin antagonists, chlorpromazineand trifluoperazine, inhibited the phosphorylation of severalproteins. Qualitative changes were observed in the pattern ofprotein phosphorylation at different developmental stages. Therewas a general decrease in protein phosphorylation towards ripening.These results indicated that calcium may be involved in theregulation of phosphorylation of different proteins at differentstages of fruit development. ¹ Scientific Paper No. 7149, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321. ² Supported in part by a grant from the National Science Foundation.DCB-8502215. (Received May 14, 1985; Accepted September 12, 1985)     

Partial Purification and Characterization of Red Chlorophyll Catabolite Reductase,a Stroma Protein Involved in Chlorophyll Breakdown

Red chlorophyll (Chl) catabolite (RCC) reductase, which catalyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was characterized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction depended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family.     

Effect of Salicylic Acid on Chlorophyll and Carotenoid Contents of Wheat and Moong Seedlings

With the increase in concentration of applied salicylic acid (SA), chlorophyll (Chl) content decreased significantly in both wheat and moong seedlings. Chl a/b ratio decreased significantly only in wheat and remained constant in moong. On the other hand, total carotenoid (Car) content, size of xanthophyll pool, and de-epoxidation rate increased significantly with an increase in SA concentration in both plant species. Hence SA treatment may induce Car biosynthesis in these plant species, but the increase in the xanthophyll pool and de-epoxidation rate indicates that SA may create oxidative stress the degree of which is different in various plants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Regulation of Orange Carotenoid Protein Activity in Cyanobacterial Photoprotection

Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism. Absorption of strong blue-green light by the OCP induces carotenoid and protein conformational changes converting the orange (inactive) OCP into a red (active) OCP. Only the red orange carotenoid protein (OCP^r) is able to bind to phycobilisomes, the cyanobacterial antenna, and to quench excess energy. In this work, we have constructed and characterized several OCP mutants and focused on the role of the OCP N-terminal arm in photoactivation and excitation energy dissipation. The N-terminal arm largely stabilizes the closed orange OCP structure by interacting with its C-terminal domain. This avoids photoactivation at low irradiance. In addition, it slows the OCP detachment from phycobilisomes by hindering fluorescence recovery protein interaction with bound OCP^r. This maintains thermal dissipation of excess energy for a longer time. Pro-22, at the beginning of the N-terminal arm, has a key role in the correct positioning of the arm in OCP^r, enabling strong OCP binding to phycobilisomes, but is not essential for photoactivation. Our results also show that the opening of the OCP during photoactivation is caused by the movement of the C-terminal domain with respect to the N-terminal domain and the N-terminal arm.Full sunlight is dangerous for plants, algae, and cyanobacteria. It can cause oxidative damages leading to the destruction of the photosynthetic apparatus and to cell death. A short-term photoprotective mechanism developed by oxygenic photosynthetic organisms is the reduction of excitation energy being funneled into the photochemical reaction centers by dissipating excess energy as heat at the level of the antennae (Niyogi and Truong, 2013). In plants and green algae, this mechanism involves the membrane chlorophyll antennae, the light-harvesting complex (for review, see Horton et al., 1996; Horton and Ruban, 2005; Jahns and Holzwarth, 2012), and in cyanobacteria, the extramembrane phycobiliprotein-containing antennae, the phycobilisomes (PBSs; for review, see Kirilovsky and Kerfeld, 2012; Kirilovsky, 2014). Despite these differences in composition and structure of their antennae, carotenoids have an essential role in both plants and cyanobacteria. In plants, high irradiance leads to acidification of the lumen that triggers conformational changes in the light-harvesting complexes and in their organization in the membrane, switching the light-harvesting complex into an effective energy-dissipating form. In cyanobacteria, high irradiance photoactivates a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), that acts as the stress sensor and the energy quencher. In both cases, changes in pigment-pigment interactions (carotenoid-chlorophyll, carotenoid-bilin, chlorophyll-chlorophyll) enable thermal dissipation of excitation energy via three different possible mechanisms: excitation energy transfer (Ruban et al., 2007; Berera et al., 2013), charge transfer (Holt et al., 2005; Tian et al., 2011), or excitonic interactions between the pigments (Bode et al., 2009).The study of the photoactivation of the OCP and its interaction with the phycobilisome is essential to elucidate the mechanism of energy quenching in cyanobacterial photoprotection. The OCP is a soluble 35-kD protein constituted by an α-helical N-terminal domain (residues1–165) and an α-helix/β-sheet C-terminal domain (residues 190–317; Kerfeld et al., 2003; Wilson et al., 2010; Fig. 1A). A flexible linker of 25 amino acids connects both domains. The ketocarotenoid 3′-hydroxyechinenone (3′-hECN), having a carbonyl (keto) group in one of the rings and a hydroxyl group in the other one, spans both domains of the protein, with the carbonyl group residing in a hydrophobic pocket of the C-terminal domain. Tyr-201 and Trp-288 interact via hydrogen bonds to the carotenoid keto group. In the dark, the OCP is orange (OCP^o). Absorption of blue-green light by the carotenoid induces conformational changes in the carotenoid and in the protein converting the orange form into the active red form (OCP^r; Wilson et al., 2008; Fig. 1C). The photoconversion reaction has a very low quantum yield, and the rate of OCP^r accumulation largely depends on light intensity (Wilson et al., 2008). Thus, accumulation of the red form occurs only under high irradiance (Wilson et al., 2008). Both OCP^o and OCP^r are energetically suitable to quench PBS fluorescence and excitation energy (Polívka et al., 2013; Niedzwiedzki et al., 2014), but only OCP^r is able to bind to the PBS and dissipate most of the excess energy as heat (Gwizdala et al., 2011). In OCP^o, strong interactions exist between the N- and C-terminal globular domains, including salt bridges between residues Trp-277-Asn-104 and Arg-155-Glu-244 (Kerfeld et al., 2003; Wilson et al., 2010). Upon photoactivation, these bonds are broken, leading to the solvent exposure of Arg-155, which plays an essential role in OCP binding to PBS (Wilson et al., 2012; Fig. 1C). The PBSs from Synechocystis sp. PCC 6803 (used in this work and hereafter simply referred to as Synechocystis) are formed by a core of allophycocyanin (APC) trimers. These trimers are organized in three cylinders from which rods containing phycocyanin hexamers radiate (for review, see Grossman et al., 1993; MacColl, 1998; Adir, 2005). OCP^r binds to one APC trimer, and its open structure allows the interaction between the OCP carotenoid and one APC bilin (Wilson et al., 2012). The first site of energy and fluorescence quenching is an APC trimer emitting at 660 nm (Tian et al., 2011, 2012, 2013; Takahashi et al., 2013). Once OCP^r is attached to PBS, thermal dissipation increases and less energy arrives at both photosystems (Wilson et al., 2006; Rakhimberdieva et al., 2010; Gorbunov et al., 2011). When the light becomes less intense, full antenna capacity is required. The Fluorescence Recovery Protein (FRP) is essential for this process. FRP accelerates the OCP^r to OCP^o dark conversion and facilitates OCP detachment from PBS (Boulay et al., 2010; Gwizdala et al., 2011; Sutter et al., 2013). The active FRP is a nonchromophorylated dimer that interacts with the C-terminal domain of the OCP^r (Sutter et al., 2013).Open in a separate windowFigure 1.A and B, Structure of the OCP from Synechocystis sp. PCC 6803 (Protein Data Bank identifier: 3MG1). The OCP monomer is represented in the orange state. The N-terminal arm (residues 1–22; red) interacts with the C-terminal domain (residues 196–315; sky blue). The Pro-22 and the Asp-6 are marked in blue. The N-terminal domain (residues 22–165) is green in the figure, and the linker between N-terminal and C-terminal domains is colored in violet. C, Model of photoactivation. Upon light absorption, the orange OCP^o is converted into the active red OCP^r. Changes in the carotenoid conformation induce conformational changes in the C-terminal domain, leading to the breakage of the interactions between the N-terminal and C-terminal domains and the opening of the protein.Previously, it has been demonstrated that the N-terminal globular domain of the OCP (green in Fig. 1, A and B) is a constitutively active energy quencher (Leverenz et al., 2014). Thus, its interaction with the C-terminal globular domain is essential for inhibiting OCP binding to PBS and energy quenching under low irradiance. This process must be tightly regulated. Little is known about this regulation. One possibility is that the N-terminal arm of the protein (red in Fig. 1, A and B), which in OCP^o interacts with the C-terminal globular domain, could have a role in this regulation.According to the OCP structure Asp-6, could form a hydrogen bond with Arg-229, which could stabilize the closed form of OCP^o. Pro-22 is located at the bent junction between the N-terminal arm and the N-terminal globular domain. It has been proposed that a cis-trans Pro isomerization could be involved in OCP photoactivation (Gorbunov et al., 2011), suggesting that Pro-22 isomerization could help the movement of the N-terminal arm and its detachment from the C-terminal domain during OCP photoactivation. In this work, we studied the effect of deleting the N-terminal arm and the mutations Asp-6-Leu and Pro-22-Val on photoactivity and OCP interaction with PBS and FRP (for the position of the N-terminal arm in the structure of OCP^o, see in Fig. 1, A and B).