叶黄素循环及其在光保护中的分子机理研究

植物的生命活动离不开充足的光照 ,但是当光照过强时 ,叶片吸收的光能超过了光合电子传递所需 ,过剩的光能便会对光合器官产生潜在的危害 ,引起光合作用的光抑制或光破坏。依赖于叶黄素循环的热耗散被认为是光保护的主要途径。本文着重介绍近年来有关植物叶黄素循环在酶学方面的分子调控、它的主要功能以及依赖于叶黄素循环的热耗散在光保护中的分子机理等 ,并对需进一步研究的问题作了探讨     

Xanthophyil Cycle and Its Molecular Mechanism in Photoprotection

When planks absorb more light than that can be used for photosynthesis, the excessive energy can cause photooxidation and even photooxidation of photosynthetic apparatus. Xanthophyll cycle-dependent photo-protection is believed to be the main mechanism for plants to deal with excessive light energy. This review focuses on molecular biological aspects and regulations of violaxanthin de-epoxidase and zeaxanthin epoxidase involved in xanthophyll cycle. We will summarize the functions of xanthophyll cycle, especially recent advances in its thermal dissipation mechanism of photoprotection. Some interesting issues deserving further study will be discussed.     

Utilizing the effective xanthophyll cycle for blooming of Ochromonas smithii and O. itoi (Chrysophyceae) on the snow surface

Snow algae inhabit unique environments such as alpine and high latitudes, and can grow and bloom with visualizing on snow or glacier during spring-summer. The chrysophytes Ochromonas smithii and Ochromonas itoi are dominant in yellow-colored snow patches in mountainous heavy snow areas from late May to early June. It is considered to be effective utilizing the xanthophyll cycle and holding sunscreen pigments as protective system for snow algae blooming in the vulnerable environment such as low temperature and nutrients, and strong light, however the study on the photoprotection of chrysophytes snow algae has not been shown. To dissolve how the chrysophytes snow algae can grow and bloom under such an extreme environment, we studied with the object of light which is one point of significance to this problem. We collected the yellow snows and measured photosynthetically active radiation at Mt. Gassan in May 2008 when the bloom occurred, then tried to establish unialgal cultures of O. smithii and O. itoi, and examined their photosynthetic properties by a PAM chlorophyll fluorometer and analyzed the pigment compositions before and after illumination with high-light intensities to investigate the working xanthophyll cycle. This experimental study using unialgal cultures revealed that both O. smithii and O. itoi utilize only the efficient violaxanthin cycle for photoprotection as a dissipation system of surplus energy under prolonged high-light stress, although they possess chlorophyll c with diadinoxanthin.     

The peculiar NPQ regulation in the stramenopile Phaeomonas sp. challenges the xanthophyll cycle dogma

In changing light conditions, photosynthetic organisms develop different strategies to maintain a fine balance between light harvesting, photochemistry, and photoprotection. One of the most widespread photoprotective mechanisms consists in the dissipation of excess light energy in the form of heat in the photosystem II antenna, which participates to the Non Photochemical Quenching (NPQ) of chlorophyll fluorescence. It is tightly related to the reversible epoxidation of xanthophyll pigments, catalyzed by the two enzymes, the violaxanthin deepoxidase and the zeaxanthin epoxidase. In Phaeomonas sp. (Pinguiophyte, Stramenopiles), we show that the regulation of the heat dissipation process is different from that of the green lineage: the NPQ is strictly proportional to the amount of the xanthophyll pigment zeaxanthin and the xanthophyll cycle enzymes are differently regulated. The violaxanthin deepoxidase is already active in the dark, because of a low luminal pH, and the zeaxanthin epoxidase shows a maximal activity under moderate light conditions, being almost inactive in the dark and under high light. This light-dependency mirrors the one of NPQ: Phaeomonas sp. displays a large NPQ in the dark as well as under high light, which recovers under moderate light. Our results pinpoint zeaxanthin epoxidase activity as the prime regulator of NPQ in Phaeomonas sp. and therefore challenge the deepoxidase-regulated xanthophyll cycle dogma.     

The xanthophyll cycle in green algae (chlorophyta): its role in the photosynthetic apparatus

Light-dependent conversion of violaxanthin to zeaxanthin, the so-called xanthophyll cycle, was shown to serve as a major, short-term light acclimation mechanism in higher plants. The role of xanthophylls in thermal dissipation of surplus excitation energy was deduced from the linear relationship between zeaxanthin formation and the magnitude of non-photochemical quenching. Unlike in higher plants, the role of the xanthophyll cycle in green algae (Chlorophyta) is ambiguous, since its contribution to energy dissipation can significantly vary among species. Here, we have studied the role of the xanthophyll cycle in the adaptation of several species of green algae (Chlorella, Scenedesmus, Haematococcus, Chlorococcum, Spongiochloris) to high irradiance. The xanthophyll cycle has been found functional in all tested organisms; however its contribution to non-photochemical quenching is not as significant as in higher plants. This conclusion is supported by three facts: (i) in green algae the content of zeaxanthin normalized per chlorophyll was significantly lower than that reported from higher plants, (ii) antheraxanthin + zeaxanthin content displayed different diel kinetics from NPQ and (iii) in green algae there was no such linear relationship between NPQ and Ax + Zx, as found in higher plants. We assume that microalgae rely on other dissipation mechanism(s), which operate along with xanthophyll cycle-dependent quenching.     

Mechanistic aspects of the xanthophyll dynamics in higher plant thylakoids

Plant thylakoids have a highly conserved xanthophyll composition, consisting of β-carotene, lutein, neoxanthin and a pool of violaxanthin that can be converted to antheraxanthin and zeaxanthin in excess light conditions. Recent work has shown that xanthophylls undergo dynamic changes, not only in their composition but also in their distribution among Lhc proteins. Xanthophylls are released from specific binding site in the major trimeric LHCII complex of photosystem II and are subsequently bound to different sites into monomeric Lhcb proteins and dimeric Lhca proteins. In this work we review available evidence from in vivo and in vitro studies on the structural determinants that control xanthophyll exchange in Lhc proteins. We conclude that the xanthophyll exchange rate is determined by the structure of individual Lhc gene products and it is specifically controlled by the lumenal pH independently from the activation state of the violaxanthin de-epoxidase enzyme. The xanthophyll exchange induces important modifications in the organization of the antenna system of Photosystem II and, possibly of Photosystem I. Major changes consist into a modulation of the light harvesting efficiency and an increase of the protection from lipid peroxidation. The xanthophyll cycle thus appears to be a signal transduction system for co-ordinated regulation of the photoprotection mechanisms under persistent stress from excess light.     

Lhc proteins and the regulation of photosynthetic light harvesting function by xanthophylls

Photoprotection of the chloroplast is an important component of abiotic stress resistance in plants. Carotenoids have a central role in photoprotection. We review here the recent evidence, derived mainly from in vitro reconstitution of recombinant Lhc proteins with different carotenoids and from carotenoid biosynthesis mutants, for the existence of different mechanisms of photoprotection and regulation based on xanthophyll binding to Lhc proteins into multiple sites and the exchange of chromophores between different Lhc proteins during exposure of plants to high light stress and the operation of the xanthophyll cycle. The use of recombinant Lhc proteins has revealed up to four binding sites in members of Lhc families with distinct selectivity for xanthophyll species which are here hypothesised to have different functions. Site L1 is selective for lutein and is here proposed to be essential for catalysing the protection from singlet oxygen by quenching chlorophyll triplets. Site L2 and N1 are here proposed to act as allosteric sites involved in the regulation of chlorophyll singlet excited states by exchanging ligand during the operation of the xanthophyll cycle. Site V1 of the major antenna complex LHC II is here hypothesised to be a deposit for readily available substrate for violaxanthin de-epoxidase rather than a light harvesting pigment. Moreover, xanthophylls bound to Lhc proteins can be released into the lipid bilayer where they contribute to the scavenging of reactive oxygen species produced in excess light. This revised version was published online in June 2006 with corrections to the Cover Date.     

The xanthophyll cycle and NPQ in diverse desert and aquatic green algae

It has long been suspected that photoprotective mechanisms in green algae are similar to those in seed plants. However, exceptions have recently surfaced among aquatic and marine green algae in several taxonomic classes. Green algae are highly diverse genetically, falling into 13 named classes, and they are diverse ecologically, with many lineages including members from freshwater, marine, and terrestrial habitats. Genetically similar species living in dramatically different environments are potentially a rich source of information about variations in photoprotective function. Using aquatic and desert-derived species from three classes of green algae, we examined the induction of photoprotection under high light, exploring the relationship between nonphotochemical quenching and the xanthophyll cycle. In liquid culture, behavior of aquatic Entransia fimbriata (Klebsormidiophyceae) generally matched patterns observed in seed plants. Nonphotochemical quenching was lowest after overnight dark adaptation, increased with light intensity, and the extent of nonphotochemical quenching correlated with the extent of deepoxidation of xanthophyll cycle pigments. In contrast, overnight dark adaptation did not minimize nonphotochemical quenching in the other species studied: desert Klebsormidium sp. (Klebsormidiophyceae), desert and aquatic Cylindrocystis sp. (Zygnematophyceae), and desert Stichococcus sp. (Trebouxiophyceae). Instead, exposure to low light reduced nonphotochemical quenching below dark-adapted levels. De-epoxidation of xanthophyll cycle pigments paralleled light-induced changes in nonphotochemical quenching for species within Klebsormidiophyceae and Trebouxiophyceae, but not Zygnematophyceae. Inhibition of violaxanthin–zeaxanthin conversion by dithiothreitol reduced high-light-associated nonphotochemical quenching in all species (Zygnematophyceae the least), indicating that zeaxanthin can contribute to photoprotection as in seed plants but to different extents depending on taxon or lineage.     

Xanthophyll synthesis in diatoms: quantification of putative intermediates and comparison of pigment conversion kinetics with rate constants derived from a model

Recently, we reported the presence of the violaxanthin-antheraxanthin-zeaxanthin cycle in diatoms, and showed that violaxanthin is the putative precursor of both diadinoxanthin and fucoxanthin in the diatom Phaeodactylum tricornutum Bohlin (M. Lohr and C. Wilhelm, 1999, Proc. Natl. Acad. Sci. USA 96: 8784–8789). In the present study, two possible intermediates in the synthesis of violaxanthin from β-carotene were identified in P. tricornutum, namely β-cryptoxanthin and β-cryptoxanthin epoxide. In low light, the latter pigment prevails, but in high light β-cryptoxanthin accumulates, probably as the result of an increased activity of the xantophyll-cycle de-epoxidase. The apparent kinetics of several xanthophyll conversion steps were determined for P. tricornutum and Cyclotella meneghiniana Kützing. The experimentally determined conversion rates were used to evaluate the hypothetical pathway of xanthophyll synthesis in diatoms. For this purpose a mathematical model was developed which allows the calculation of theoretical rates of pigment conversion for microalgae under steady-state growth conditions. A comparison between measured and calculated conversion rates agreed well with the proposal of a sequential synthesis of fucoxanthin via violaxanthin and diadinoxanthin. The postulation of zeaxanthin as an obligatory intermediate in the synthesis of violaxanthin, however, resulted in large discrepancies between the measured and calculated rates of its epoxidation. Instead of zeaxanthin, β-cryptoxanthin epoxide may be involved in the biosynthesis of violaxanthin in diatoms. Received: 16 March 2000 / Accepted: 30 June 2000     

Characterization of the Xanthophyll Cycle and Other Photosynthetic Pigment Changes Induced by Iron Deficiency in Sugar Beet (Beta vulgaris L.)

In this work we characterize the changes induced by iron deficiency in the pigment composition of sugar beet (Beta vulgaris L.) leaves. When sugar beet plants were grown hydroponically under limited iron supply, neoxanthin and β-carotene decreased concomitantly with chlorophyll a, whereas lutein and the carotenoids within the xanthophyll cycle were less affected. Iron deficiency caused major increases in the lutein/chlorophyll a and xanthophyll cycle pigments/chlorophyll a molar ratios. Xanthophyll cycle carotenoids in Fe-deficient plants underwent epoxidations and de-epoxidations in response to ambient light conditions. In dark adapted Fe-deficient plants most of the xanthophyll cycle pigment pool was in the epoxidated form violaxanthin. We show, both by HPLC and by in vivo 505 nanometers absorbance changes, that in Fe deficient plants and in response to light, the de-epoxidated forms antheraxanthin and zeaxanthin were rapidly formed at the expense of violaxanthin. Several hours after returning to dark, the xanthophyll cycle was shifted again toward violaxanthin. The ratio of variable to maximum chlorophyll fluorescence from intact leaves was decreased by iron deficiency. However, in iron deficient leaves this ratio was little affected by light conditions which displace the xanthophyll cycle toward epoxidation or de-epoxidation. This suggests that the functioning of the xanthophyll cycle is not necessarily linked to protection against excess light input.     

The current concepts of functional role of carotenoids in the eukaryotic chloroplasts

Current state of knowledge of functional role of carotenoids in algal and higher plant chloroplasts is reviewed. Basic functions of carotenoids are shown to be light-protective, light-absorbing, and structural, as well as participating in photochemical processes of photosystems I and II. Such xanthophylls as neoxanthin, fucoxanthin, peridinin and alloxanthin, which have allenic or acetylenic bond, mostly function as light-absorbers. They transmit absorbed energy to chlorophyll b. Other xanthophylls occupying certain strictly specified loci in light-absorbing chlorophyll-a/b-protein complexes of photosystems have either structural function (lutein) or light-protective function (zeaxanthin, antheraxanthin, violaxanthin). Carotenoids of xanthophyll cycles preserve chlorophylls and lipids of photosynthetic membranes from photodestruction at overlighting in the presence of oxygen. In eukaryotic chloroplasts, three types of xanthophyll cycles were found: violaxanthin, lutein-5,6-epoxide, and diadinoxanthin. The similarities and dissimilarities between epoxidation and de-epoxidation reactions of these cycles are discussed in detail in the present work. The pattern of occurrence of xanthophyll cycles among higher plants and freshwater and marine algae is outlined.     

Epoxidation of zeaxanthin and antheraxanthin reverses non-photochemical quenching of photosystem II chlorophyll a fluorescence in the presence of trans-thylakoid ΔpH

The xanthophyll cycle apparently aids the photoprotection of photosystem II by regulating the nonradiative dissipation of excess absorbed light energy as heat. However, it is a controversial question whether the resulting nonphotochemical quenching is soley dependent on xanthophyll cycle activity or not. The xanthophyll cycle consists of two enzymic reactions, namely deepoxidation of the diepoxide violaxanthin to the epoxide-free zeaxanthin and the much slower, reverse process of epoxidation. While deepoxidation requires a transthylakoid pH gradient (ΔpH), epoxidation can proceed irrespective of a ΔpH. Herein, we compared the extent and kinetics of deepoxidation and epoxidation to the changes in fluorescence in the presence of a light-induced thylakoid ΔpH. We show that epoxidation reverses fluorescence quenching without affecting thylakoid ΔpH. These results suggest that epoxidase activity reverses quenching by removing deepoxidized xanthophyll cycle pigments from quenching complexes and converting them to a nonquenching form. The transmembrane organization of the xanthophyll cycle influences the localization and the availability of deepoxidized xanthophylls is to support nonphotochemical quenching capacity. The results confirm the view that rapidly reversible nonphotochemical quenching is dependent on deepoxidized xanthophyll.     

The role of xanthophylls in the supramolecular organization of the photosynthetic complex LHCII in lipid membranes studied by high-resolution imaging and nanospectroscopy

The xanthophyll cycle is a regulatory mechanism operating in the photosynthetic apparatus of plants. It consists of the conversion of the xanthophyll pigment violaxanthin to zeaxanthin, and vice versa, in response to light intensity. According to the current understanding, one of the modes of regulatory activity of the cycle is associated with the influence on a molecular organization of pigment-protein complexes. In the present work, we analyzed the effect of violaxanthin and zeaxanthin on the molecular organization of the LHCII complex, in the environment of membranes formed with chloroplast lipids. Nanoscale imaging based on atomic force microscopy (AFM) showed that the presence of exogenous xanthophylls promotes the formation of the protein supramolecular structures. Nanoscale infrared (IR) absorption analysis based on AFM-IR nanospectroscopy suggests that zeaxanthin promotes the formation of LHCII supramolecular structures by forming inter-molecular β-structures. Meanwhile, the molecules of violaxanthin act as “molecular spacers” preventing self-aggregation of the protein, potentially leading to uncontrolled dissipation of excitation energy in the complex. This latter mechanism was demonstrated with the application of fluorescence lifetime imaging microscopy. The intensity-averaged chlorophyll a fluorescence lifetime determined in the LHCII samples without exogenous xanthophylls at the level of 0.72 ns was longer in the samples containing exogenous violaxanthin (2.14 ns), but shorter under the presence of zeaxanthin (0.49 ns) thus suggesting a role of this xanthophyll in promotion of the formation of structures characterized by effective excitation quenching. This mechanism can be considered as a representation of the overall photoprotective activity of the xanthophyll cycle.     

Mechanism and regulation of the violaxanthin cycle: The role of antenna proteins and membrane lipids

The violaxanthin cycle describes the reversible conversion of violaxanthin to zeaxanthin via the intermediate antheraxanthin. This light-dependent xanthophyll conversion is essential for the adaptation of plants and algae to different light conditions and allows a reversible switch of photosynthetic light-harvesting complexes between a light-harvesting state under low light and a dissipative state under high light. The photoprotective functions of zeaxanthin have been intensively studied during the last decade, but much less attention has been directed to the mechanism and regulation of xanthophyll conversion. In this review, an overview is given on recent progress in the understanding of the role of (i) xanthophyll binding by antenna proteins and of (ii) the lipid properties of the thylakoid membrane in the regulation of xanthophyll conversion. The consequences of these findings for the mechanism and regulation of xanthophyll conversion in the thylakoid membrane will be discussed.     

The xanthophyll cycle, its regulation and components

During the last few years much interest has been focused on the photoprotective role of zeaxanthin. In excessive light zeaxanthin is rapidly formed in the xanthophyll cycle from violaxanthin, via the intermediate antheraxanthin, a reaction reversed in the dark. The role of zeaxanthin and the xanthophyll cycle in photoprotection, is based on fluorescence quenching measurements, and in many studies a good correlation to the amount of zeaxanthin (and antheraxanthin) has been found. Other suggested roles for the xanthophylls involve, protection against oxidative stress of lipids, participation in the blue light response, modulation of the membrane fluidity and regulation of abscisic acid synthesis. The enzyme violaxanthin de-epoxidase has recently been purified from spinach and lettuce as a 43-kDa protein. It was found as 1 molecule per 20–100 electron-transport chains. The gene has been cloned and sequenced from Lactuca sativa, Nicotiana tabacum and Arabidopsis thaliana. The transit peptide was characteristic of nuclear-encoded and lumen-localized proteins. The activity of violaxanthin de-epoxidase is controlled by the lumen pH. Thus, below pH 6.6 the enzyme binds to the thylakoid membrane. In addition ascorbate becomes protonated to ascorbic acid (pK_a= 4.2) the true substrate (K_m= 0.1 m M ) for the violaxanthin de-epoxidase. We present arguments for an ascorbate transporter in the thylakoid membrane. The enzyme zeaxanthin epoxidase requires FAD as a cofactor and appears to use ferredoxin rather than NADPH as a reductant. The zeaxanthin epoxidase has not been isolated but the gene has been sequenced and a functional protein of 72.5 kDa has been expressed. The xanthophyll cycle pigments are almost evenly distributed in the thylakoid membrane and at least part of the pigments appears to be free in the lipid matrix where we conclude that the conversion by violaxanthin de-epoxidase occurs.     

The chloroplast pigments of the algal classes Eustigmatophyceae and Xanthophyceae. II. Xanthophyceae

The chloroplast pigments of Pleurochloris meiringensis Vischer, Mischococcus sphaerocephalus Vischer and Tribonema aequale Pascher have been analysed by a variety of chromatographic methods. There are significant differences between these xanthophycean algae and the eustigmatophycean algae formerly classified with them. In particular the former contain diadinoxanthin as the major xanthophyll whereas diadinoxanthin is absent from the latter and violaxanthin occurs as the major xanthophyll pigment. The other pigments of the Xanthophyceae sensu stricto include chlorophyll a, β-carotene, vaucheriaxanthin-ester, heteroxanthin, diatoxanthin, cryptoxanthin epoxide and a neoxanthin-like pigment.     

The lipid dependence of diadinoxanthin de-epoxidation presents new evidence for a macrodomain organization of the diatom thylakoid membrane

The present study shows that thylakoid membranes of the diatom Cyclotella meneghiniana contain much higher amounts of negatively charged lipids than higher plant or green algal thylakoids. Based on these findings, we examined the influence of SQDG on the de-epoxidation reaction of the diadinoxanthin cycle and compared it with results from the second negatively charged thylakoid lipid PG. SQDG and PG exhibited a lower capacity for the solubilization of the hydrophobic xanthophyll cycle pigment diadinoxanthin than the main membrane lipid MGDG. Although complete pigment solubilization took place at higher concentrations of the negatively charged lipids, SQDG and PG strongly suppressed the de-epoxidation of diadinoxanthin in artificial membrane systems. In in vitro assays employing the isolated diadinoxanthin cycle enzyme diadinoxanthin de-epoxidase, no or only a very weak de-epoxidation reaction was observed in the presence of SQDG or PG, respectively. In binary mixtures of the inverted hexagonal phase forming lipid MGDG with the negatively charged bilayer lipids, comparable suppression took place. This is in contrast to binary mixtures of MGDG with the neutral bilayer lipids DGDG and PC, where rapid and efficient de-epoxidation was observed. In complex lipid mixtures resembling the lipid composition of the native diatom thylakoid membrane, we again found strong suppression of diadinoxanthin de-epoxidation due to the presence of SQDG or PG. We conclude that, in the native thylakoids of diatoms, a strict separation of the MGDG and SQDG domains must occur; otherwise, the rapid diadinoxanthin de-epoxidation observed in intact cells upon illumination would not be possible.     

Photo- and antioxidant protection and salicylic acid accumulation during post-anthesis leaf senescence in Salvia lanigera grown under Mediterranean climate

Post-anthesis leaf senescence is a key developmental process in the life of plants as it is the time during which material built up by the plant during its growth phase is mobilized into reproductive tissues. Here we aimed to study the extent of photo- and antioxidant protection and salicylic acid (SA) accumulation during post-anthesis leaf senescence in a perennial plant, Salvia lanigera Poir. grown under Mediterranean field conditions. SA levels increased sharply (up to 2.7-fold) during early stages of leaf senescence until fruit and seed formation occurred (i.e. 4 weeks after anthesis). Later on, SA levels kept at constant high levels until leaf abscission occurred (i.e. 7 weeks after anthesis). Reductions in chlorophyll and carotenoid (lutein, violaxanthin and β-carotene) levels occurred progressively during leaf senescence. In contrast, xanthophyll cycle de-epoxidation increased during early stages of leaf senescence and remained constant later, similar to SA accumulation. Indeed, xanthophyll cycle de-epoxidation strongly positively correlated with SA levels (r² = 0.92). The maximum efficiency of PSII (F_v/F_m ratio) kept around 0.80 throughout the experiment, except during the latest stage of leaf senescence (i.e. after fruit and seed formation), when this ratio decreased to 0.72, thus indicating damage to PSII. It is concluded that endogenous SA levels increase sharply during early stages of post-anthesis leaf senescence and concomitantly with activation of photoprotection mechanisms, such as xanthophyll cycle-dependent excess energy dissipation, thus avoiding damage to PSII until fruit and seed formation have been accomplished.     

The xanthophyll cycle - molecular mechanism and physiological significance

The light-dependent, cyclic changes of xanthophyll pigments: violaxanthin, antheraxanthin and zeaxanthin, called the xanthophyll cycle, have been known for about fifty years. This process was characterised for higher plants, several fern and moss species and in some algal groups. Two enzymes, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE), belonging to the lipocalin protein family, are engaged in the xanthophyll cycle. VDE requires for its activity ascorbic acid and reversed hexagonal structure formed by monogalactosyldiacylglycerol. ZE, postulated to be a flavoprotein, has not been purified yet and it is known from its gene sequence only. Zeaxanthin epoxidation is dependent on the reducing power of NADPH and presence of additional proteins. The xanthophyll cycle is postulated to play a role in many important physiological processes. Zeaxanthin, formed from violaxanthin under high light conditions, is thought to be a main photoprotector in autotrophic cells due to its ability to dissipate excess of absorbed light energy that can be measured as a non-photochemical quenching. In addition the zeaxanthin formation is important in protection of the thylakoid membranes against lipid peroxidation. Other postulated functions of the xanthophyll cycle, which include regulation of membrane physical properties, blue light reception and regulation of abscisic acid synthesis, are also discussed.