植物光破坏防御机制研究进展

光破坏防御机制是植物为应对复杂多变的自然环境而产生的保护措施,这些措施从形态、生理和生化等方面反映了植物对环境的适应能力。本文根据光抑制的机理,对近年来植物的光破坏防御机制以及高等植物叶黄素循环机制的研究现状进行综述,认为叶黄素循环防御机制是植物光保护作用的重要措施之一。     

Photoprotection in a zeaxanthin- and lutein-deficient double mutant of Arabidopsis

When light absorption by a plant exceeds its capacity for light utilization, photosynthetic light harvesting is rapidly downregulated by photoprotective thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). To address the involvement of specific xanthophyll pigments in NPQ, we have analyzed mutants affecting xanthophyll metabolism in Arabidopsis thaliana. An npq1 lut2 double mutant was constructed, which lacks both zeaxanthin and lutein due to defects in the violaxanthin de-epoxidase and lycopene -cyclase genes. The npq1 lut2 strain had normal Photosystem II efficiency and nearly wild-type concentrations of functional Photosystem II reaction centers, but the rapidly reversible component of NPQ was completely inhibited. Despite the defects in xanthophyll composition and NPQ, the npq1 lut2 mutant exhibited a remarkable ability to tolerate high light.This revised version was published online in October 2005 with corrections to the Cover Date.     

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     

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.     

Mechanisms shaping the synergism of zeaxanthin and PsbS in photoprotective energy dissipation in the photosynthetic apparatus of plants

Safe operation of photosynthesis is vital to plants and is ensured by the activity of processes protecting chloroplasts against photo-damage. The harmless dissipation of excess excitation energy is considered to be the primary photoprotective mechanism and is most effective in the combined presence of PsbS protein and zeaxanthin, a xanthophyll accumulated in strong light as a result of the xanthophyll cycle. Here we address the problem of specific molecular mechanisms underlying the synergistic effect of zeaxanthin and PsbS. The experiments were conducted with Arabidopsis thaliana, using wild-type plants, mutants lacking PsbS (npq4), and mutants affected in the xanthophyll cycle (npq1), with the application of molecular spectroscopy and imaging techniques. The results lead to the conclusion that PsbS interferes with the formation of densely packed aggregates of thylakoid membrane proteins, thus allowing easy exchange and incorporation of xanthophyll cycle pigments into such structures. It was found that xanthophylls trapped within supramolecular structures, most likely in the interfacial protein region, determine their photophysical properties. The structures formed in the presence of violaxanthin are characterized by minimized dissipation of excitation energy. In contrast, the structures formed in the presence of zeaxanthin show enhanced excitation quenching, thus protecting the system against photo-damage.     

Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes,fluorescence and photosynthesis in leaves of Hedera canariensis

The role of the xanthophyll cycle in regulating the energy flow to the PS II reaction centers and therefore in photoprotection was studied by measurements of light-induced absorbance changes, Chl fluorescence, and photosynthetic O₂ evolution in sun and shade leaves of Hedera canariensis. The light-induced absorbance change at 510 nm (A₅₁₀) was used for continuous monitoring of zeaxanthin formation by de-epoxidation of violaxanthin. Non-radiative energy dissipation (NRD) was estimated from non-photochemical fluorescence quenching (NPQ).High capacity for zeaxanthin formation in sun leaves was accompanied by large NRD in the pigment bed at high PFDs as indicated by a very strong NPQ both when all PS II centers are closed (F'_m) and when all centers are open (F'_o). Such F_o quenching, although present, was less pronounced in shade leaves which have a much smaller xanthophyll cycle pool.Dithiothreitol (DTT) provided through the cut petiole completely blocked zeaxanthin formation. DTT had no detectable effect on photosynthetic O₂ evolution or the photochemical yield of PS II in the short term but fully inhibited the quenching of F_o and 75% of the quenching of F_m, indicating that NRD in the antenna was largely blocked. This inhibition of quenching was accompanied by an increased closure of the PS II reaction centers.In the presence of DTT a photoinhibitory treatment at a PFD of 200 mol m^-2 s^-1, followed by a 45 min recovery period at a low PFD, caused a 35% decrease in the photon yield of O₂ evolution, compared to a decrease of less than 5% in the absence of DTT. The F_v/F_m ratio, measured in darkness showed a much greater decrease in the presence than in the absence of DTT. In the presence of DTT F_o rose by 15–20% whereas no change was detected in control leaves.The results support the conclusion that the xanthophyll cycle has a central role in regulating the energy flow to the PS II reaction centers and also provide direct evidence that zeaxanthin protects against photoinhibitory injury to the photosynthetic system.Abbreviations F, F_m, F_o, F_v Fluorescence yield at actual degree of PS II center closure, when all centers are closed, when all centers are open, variable fluorescence - NPQ non-photochemical fluorescence quenching - NRD non-radiative energy dissipation - PFD photon flux density - Q_A primary acceptor PS II     

Photoprotection mutants of Arabidopsis thaliana acclimate to high light by increasing photosynthesis and specific antioxidants

Biochemical and physiological acclimation to different light environments is crucial for plant growth and survival. In high light (HL), feedback de-excitation (qE) is a well-known photoprotective mechanism that dissipates excess excitation energy in the light-harvesting antenna of photosystem II (PSII) and relieves excitation pressure in the photosynthetic electron transport chain. The xanthophylls zeaxanthin (Z) and lutein (L) function in qE, but also have roles as antioxidants. Although several studies have shown that qE is important during short-term fluctuations in light intensity, here we show that it is not required for the growth of Arabidopsis thaliana in prolonged HL conditions in the laboratory. Mutants that are deficient in qE alone, qE and Z synthesis, or in qE, Z synthesis and also L synthesis were able to grow at 1800 micromol photons m(-2) s(-1) and exhibited no major symptoms of photooxidative stress. The mutants (and wild type) acclimated to HL by increasing photosynthetic capacity and decreasing light harvesting, which together rendered qE less important for photoprotection. At a metabolite level, the HL-grown mutants appeared to compensate for their remaining qE deficit with increased alpha-tocopherol and ascorbate levels compared to the wild type. The specificity of this response provides insight into the relationship between qE and the antioxidant network in plants.     

Nocturnally retained zeaxanthin does not remain engaged in a state primed for energy dissipation during the summer in two Yucca species growing in the Mojave Desert

Differently oriented leaves of Yucca schidigera and Yucca brevifolia were characterized in the Mojave Desert with respect to photosystem II and xanthophyll cycle activity during three different seasons, including the hot and dry summer, the relatively cold winter, and the mild spring season. Photosynthetic utilization of a high percentage of the light absorbed in PSII was observed in all leaves only during the spring, whereas very high levels of photoprotective, thermal energy dissipation were employed both in the summer and the winter season in all exposed leaves of both species. Both during the summer and the winter season, when energy dissipation levels were high diurnally, xanthophyll cycle pools (relative to either Chl or other carotenoids) were higher relative to the spring, and a nocturnal retention of high levels of zeaxanthin and antheraxanthin (Z + A) occurred in all exposed leaves of both species. Although this nocturnal retention of Z + A was associated with nocturnal maintenance of a low PSII efficiency (F_v/F_m) on a cold winter night, pre‐dawn F_v/F_m was high in (Z + A)‐retaining leaves following a warm summer night. This indicates nocturnal engagement of Z + A in a state primed for energy dissipation throughout the cold winter night – while high levels of retained Z + A were not engaged for energy dissipation prior to sunrise on a warm summer morning. Possible mechanisms for a lack of sustained engagement of retained Z + A for energy dissipation at elevated temperatures are discussed.     

Photoinhibition of Photosynthesis: Role of Carotenoids in Photoprotection of Chloroplast Constituents

Exposure of plants to irradiation, in excess to saturate photosynthesis, leads to reduction in photosynthetic capacity without any change in bulk pigment content. This effect is known as photoinhibition. Photoinhibition is followed by destruction of carotenoids (Cars), bleaching of chlorophylls (Chls), and increased lipid peroxidation due to formation of reactive oxygen species if the excess irradiance exposure continues. Photoinhibition of photosystem 2 (PS2) in vivo is often a photoprotective strategy rather than a damaging process. For sustainable maintenance of chloroplast function under high irradiance, the plants develop various photoprotective strategies. Cars perform essential photoprotective roles in chloroplasts by quenching the triplet Chl and scavenging singlet oxygen and other reactive oxygen species. Recently photoprotective role of xanthophylls (zeaxanthin) for dissipation of excess excitation energy under irradiance stress has been emphasised. The inter-conversion of violaxanthin (Vx) into zeaxanthin (Zx) in the light-harvesting complexes (LHC) serves to regulate photon harvesting and subsequent energy dissipation. De-epoxidation of Vx to Zx leads to changes in structure and properties of these xanthophylls which brings about significant structural changes in the LHC complex. This ultimately results in (1) direct quenching of Chl fluorescence by singlet-singlet energy transfer from Chl to Zx, (2) trans-thylakoid membrane mediated, pH-dependent indirect quenching of Chl fluorescence. Apart from these, other processes such as early light-inducible proteins, D1 turnover, and several enzymatic defence mechanisms, operate in the chloroplasts, either for tolerance or to neutralise the harmful effect of high irradiance.     

Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation

This review places photoprotection into the context of ecology and species diversity. The focus is on photoprotection via the safe removal - as thermal energy - of excess solar energy absorbed by the light collecting system, which counteracts the formation of reactive oxygen species. An update on the surprisingly complex, multiple variations of thermal energy dissipation is presented, placing these different forms into ecological and genetic contexts. Zeaxanthin-facilitated, flexible thermal dissipation associated with the PsbS protein and controlled by the trans-thylakoid pH gradient apparently occurs ubiquitously in plants, and can become sustained (and thus less flexible) at low temperatures. Long-lived, slow-growing plants with low intrinsic capacities for photosynthesis have greater capacities for this flexible dissipation than short-lived, fast-growing species. Furthermore, potent, but inflexible (zeaxanthin-facilitated) thermal dissipation, prominent in evergreen species under prolonged environmental stress, is characterized with respect to the involvement of photosystem II core rearrangement and/or degradation as well as the absence of control by trans-thylakoid pH and, possibly, PsbS. A role of PsbS-related proteins in photoprotection is discussed.     

Differences in the response of carbon assimilation to summer stress (water deficits, high light and temperature) in four Mediterranean tree species

Daily changes in photoprotective mechanisms were studied in sun leaves of Quercus suber L., Quercus ilex L., Olea europaea L. and Eucalyptus globulus Labill. trees during the summer in Portugal. Even though stomatal closure explained most of the diurnal variation in carbon assimilation along the summer, a decline in the photochemical yield of photosystem II (F'v/F'm) also occurred, as a result of an excess of intercepted solar radiation when carbon assimilation is limited by stomatal closure due to high vapour pressure deficits and/or soil water deficits. These changes were accompanied by the conversion of violaxanthin to antheraxanthin and zeaxanthin which were correlated with thermal dissipation of excess photon energy. In spite of a common general response, differences between species were observed Olea europaea , which is a slowgrowing tree, had the lowest net photosynthetic rates, the highest proportion of carotenoids in relation to chlorophyll and the highest rates of deepoxidation of violaxanthin. This enabled a large thermal dissipation of the excess intercepted radiation but led to rather small values of light utilisation for photochemistry (ca 20%). In contrast, in E. globulus , a fastgrowing tree, photosynthetic rates were the highest, thermal dissipation of absorbed radiation the lowest and maximal values of light utilisation for photochemistry reached ca 50%. The two Quercus species exhibited an intermediate response. A high degree of coordination is apparent between stomatal behaviour, photosynthetic capacity and photoprotection mechanisms.     

Rapid changes in xanthophyll cycle-dependent energy dissipation and photosystem II efficiency in two vines, Stephania japonica and Smilax australis, growing in the understory of an open Eucalyptus forest

Leaves of Stephania japonica and Smilax australis were characterized in situ on the coast of north-eastern New South Wales, Australia, where they were growing naturally in three different light environments: deep shade, in the understory of an open Eucalyptus forest where they received frequent sunflecks of high intensity, and in an exposed site receiving full sunlight. In deep shade the xanthophyll cycle remained epoxidized during the day and the vast majority of absorbed light was utilized for photosynthesis. In the exposed site both deepoxidation and epoxidation of the xanthophyll cycle and changes in the level of xanthophyll-dependent thermal energy dissipation largely tracked the diurnal changes in photon flux density (PFD). In the understory the xanthophyll cycle became largely deepoxidized to zeaxanthin and antheraxanthin upon exposure of the leaves to the first high intensity sunfleck and this high level of deepoxidation was maintained throughout the day both during and between subsequent sunflecks. In contrast, thermal energy dissipation activity, and the efficiency of photosystem II, fluctuated rapidly in response to the changes in incident PFD. These findings suggest a fine level of control over the engagement of zeaxanthin and antheraxanthin in energy dissipation activity, presumably through rapid changes in thylakoid acidification, such that they became rapidly engaged for photoprotection during the sunflecks and rapidly disengaged upon return to low light when continued engagement might limit carbon gain.     

植物光破坏防御机制的研究进展

植物在长期进化过程中,形成了一系列保护光合机构免受强光破坏的光破坏防御机制,本文仅就近年来光破坏防御机制中^3Chl途径、能量耗散和叶绿体呼吸作了简要综述。     

Acclimation of leaf carotenoid composition and ascorbate levels to gradients in the light environment within an Australian rainforest

The influence of the growth photon flux density (PFD) on the size and composition of the carotenoid pool and the size of the reduced ascorbate pool was determined across a light gradient from the forest floor to the canopy and the forest edge of a sub-tropical rainforest in New South Wales, Australia. Nineteen plant species (most collected from multiple sites) representing a broad taxonomic range consistently possessed larger total carotenoid pools when found growing in more exposed sites. There was a significant positive correlation between β-carotene content and growth PFD and a significant negative correlation between α-carotene content and growth PFD. Neoxanthin content exhibited no significant trend while the trend in lutein content varied with mode of expression. The pigments of the xanthophyll cycle (violaxanthin, antheraxanthin and zeaxanthin) exhibited the most pronounced response to growth PFD; they comprised a much greater portion of the total carotenoid pool in high light-acclimated plants. The pool of reduced ascorbate was also several-fold greater in high light-acclimated plants. These acclimatory changes in carotenoid and ascorbate content are consistent with a need for a greater capacity to dissipate excessive absorbed light energy in high light-acclimated plants.     

Up-regulation of a photosystem II core protein phosphatase inhibitor and sustained D1 phosphorylation in zeaxanthin-retaining, photoinhibited needles of overwintering Douglas fir

Overwintering needles of the evergreen conifer Douglas fir exhibited an association between arrest of the xanthophyll cycle in the dissipating state (as zeaxanthin + antheraxanthin; Z + A) with a strongly elevated predawn phosphorylation state of the D1 protein of the photosystem II (PSII) core. Furthermore, the high predawn phosphorylation state of PSII core proteins was associated with strongly increased levels of TLP40, the cyclophilin-like inhibitor of PSII core protein phosphatase, in winter versus summer. In turn, decreases in predawn PSII efficiency, F_v/F_m, in winter were positively correlated with pronounced decreases in the non-phosphorylated form of D1. In contrast to PSII core proteins, the light-harvesting complex of photosystem II (LHCII) did not exhibit any nocturnally sustained phosphorylation. The total level of the D1 protein was found to be the same in summer and winter in Douglas fir when proteins were extracted in a single step from whole needles. In contrast, total D1 protein levels were lower in thylakoid preparations of overwintering needles versus needles collected in summer, indicating that D1 was lost during thylakoid preparation from overwintering Douglas fir needles. In contrast to total D1, the ratio of phosphorylated to non-phosphorylated D1 as well as the levels of the PsbS protein were similar in thylakoid versus whole needle preparations. The level of the PsbS protein, that is required for pH-dependent thermal dissipation, exhibited an increase in winter, whereas LHCII levels remained unchanged.     

Relaxation of the non-photochemical chlorophyll fluorescence quenching in diatoms: kinetics,components and mechanisms

Diatoms are especially important microorganisms because they constitute the larger group of microalgae. To survive the constant variations of the light environment, diatoms have developed mechanisms aiming at the dissipation of excess energy, such as the xanthophyll cycle and the non-photochemical chlorophyll (Chl) fluorescence quenching. This contribution is dedicated to the relaxation of the latter process when the adverse conditions cease. An original nonlinear regression analysis of the relaxation of non-photochemical Chl fluorescence quenching, qN, in diatoms is presented. It was used to obtain experimental evidence for the existence of three time-resolved components in the diatom Phaeodactylum tricornutum: qNf, qNi and qNs. qNf (s time-scale) and qNs (h time-scale) are exponential in shape. By contrast, qNi (min time-scale) is of sigmoidal nature and is dominant among the three components. The application of metabolic inhibitors (dithiothreitol, ammonium chloride, cadmium and diphenyleneiodonium chloride) allowed the identification of the mechanisms on which each component mostly relies. qNi is linked to the relaxation of the ΔpH gradient and the reversal of the xanthophyll cycle. qNs quantifies the stage of photoinhibition caused by the high light exposure, qNf seems to reflect fast conformational changes within thylakoid membranes in the vicinity of the photosystem II complexes.     

Linear models relating xanthophylls and lumen acidity to non-photochemical fluorescence quenching. Evidence that antheraxanthin explains zeaxanthin-independent quenching

Zeaxanthin has been correlated with high-energy non-photochemical fluorescence quenching but whether antheraxanthin, the intermediate in the pathway from violaxanthin to zeaxanthin, also relates to quenching is unknown. The relationships of zeaxanthin, antheraxanthin and pH to fluorescence quenching were examined in chloroplasts ofPisum sativum L. cv. Oregon andLactuca sativa L. cv. Romaine. Data matrices as five levels of violaxanthin de-epoxidation against five levels of light-induced lumen-proton concentrations were obtained for both species. The matrices included high levels of antheraxanthin as well as lumen-proton concentrations induced by subsaturating to saturation light levels. Analyses of the matrices by simple linear and multiple regression showed that quenching is predicted by models where the major independent variable is the product of lumen acidity and de-epoxidized xanthophylls, the latter as the sum of zeaxanthin and antheraxanthin. The interactions of lumen acidity and xanthophyll concentration are shown in three-dimensional plots of the best-fit multiple regression models. Antheraxanthin apparently contributes to quenching as effectively as zeaxanthin and explains quenching previously not accounted for by zeaxanthin. Hence, we propose that all high-energy dependent quenching is xanthophyll dependent. Quenching requires a threshold lumen pH that varies with xanthophyll composition. After the threshold, quenching is linear with lumen acidity or xanthophyll composition.     

Carotenoid composition in sun and shade leaves of plants with different life forms

The carotenoid composition of sun leaves of nine species of annual crop plants (some with several varieties) was compared with sun and shade leaves of several other groups of plants, among those sun and shade leaves of several species of perennial shrubs and vines and deep-shade leaves of seven rainforest species. All sun leaves contained considerably greater amounts of the components of the xanthophyll cycle violaxanthin, antheraxanthin and zeaxanthin as well as of β-carotene than the shade leaves, as had previously been reported for a variety of other species by Thayer & Björkman (Photosynthesis Research, 1990, 23, 331–343). Therefore, high light specifically stimulated β,β-carotenoid synthesis. The sun leaves of these crop species did not contain α-carotene which was, however, present in large amounts in all shade leaves and in smaller amounts in sun leaves of three of the four species of perennial shrubs and vines. There was no difference in neoxanthin content on a chlorophyll basis between sun and shade leaves, and there was no consistent general difference in the lutein content between all sun and all shade leaves. The zeaxanthin (and antheraxanthin) content at peak irradiance and the xanthophyll cycle pool size were compared for sun leaves from the different groups of plants with different life forms and different metabolic activities. When growing in full sunlight the annual crop species and a perennial mesophyte had high rates of photosynthesis whereas the perennial shrubs and vines had relatively low photosynthesis rates. More zeaxanthin (and antheraxanthin) were accumulated at noon in full sunlight in those species with the lower photosynthesis rates. However, it was not such that those species also possessed the larger pools of violaxanthin + antheraxanthin + zeaxanthin. Instead, the xanthophyll cycle pools of sun leaves of the annual crop species and the perennial mesophyte were not smaller, and were even possibly larger, than those of sun leaves of the perennial shrubs and vines with low photosynthesis rates. This was so in spite of the fact that the crop species experienced much lesser degrees of excessive light at full sun than the shrubs and vines. Thus, many of the crop species converted only about 30–50% of their xanthophyll cycle pool to zeaxanthin at noon, whereas the shrubs and vines typically converted more than 80% of their pool into zeaxanthin. The crop species also had larger pools of β-carotene than the shrubs and vines but smaller pools of lutein than the majority of the latter species.     

Winter down-regulation of intrinsic photosynthetic capacity coupled with up-regulation of Elip-like proteins and persistent energy dissipation in a subalpine forest

Overwintering, sun-exposed and photosynthetically inactive evergreens require powerful photoprotection. The goal of this study was to seasonally characterize photosynthesis and key proteins/components involved in electron transport and photoprotection. Maximal photosystem II (PSII) efficiency and photosynthetic capacity, amounts of zeaxanthin (Z), antheraxanthin (A), pheophytin and proteins (oxygen-evolving 33 kDa protein (OEC), PSII core protein D1 and subunit S (PsbS) protein, and members of the early light-inducible protein (Elip) family) were assessed in five conifer species at high altitude and in ponderosa pine (Pinus ponderosa) at moderate altitude during summer and winter. Relative to summer, winter down-regulation of photosynthetic capacity and loss of PSII efficiency at the high-altitude sites were paralleled by decreases in OEC, D1, and pheophytin; massive nocturnal retention of (Z + A) and up-regulation of two to four proteins cross-reactive with anti-Elip antibodies; and no change in PsbS amount. By contrast, ponderosa pine at moderate altitude exhibited no down-regulation of photosynthetic capacity, smaller depressions in PSII efficiency, and less up-regulation of Elip family members. These results support a function for members of the Elip family in the acclimation of sun-exposed needles that down-regulate photosynthesis during winter. A possible role in sustained photoprotection is considered.