De-epoxidation Of Violaxanthin Research Articles

Molecular mechanisms of light harvesting in the minor antenna CP29 in near-native membrane lipidic environment.

CP29, a chlorophyll a/b-xanthophyll binding protein, bridges energy transfer between the major LHCII antenna complexes and photosystem II reaction centers. It hosts one of the two identified quenching sites, making it crucial for regulated photoprotection mechanisms. Until now, the photophysics of CP29 has been studied on the purified protein in detergent solutions since spectrally overlapping signals affect in vivo measurements. However, the protein in detergent assumes non-native conformations compared to its physiological state in the thylakoid membrane. Here, we report a detailed photophysical study on CP29 inserted in discoidal lipid bilayers, known as nanodiscs, which mimic the native membrane environment. Using picosecond time-resolved fluorescence and femtosecond transient absorption (TA), we observed shortening of the Chl fluorescence lifetime with a decrease of the carotenoid triplet formation yield for CP29 in nanodiscs as compared to the protein in detergent. Global analysis of TA data suggests a 1Chl* quenching mechanism dependent on excitation energy transfer to a carotenoid dark state, likely the proposed S*, which is believed to be formed due to a carotenoid conformational change affecting the S1 state. We suggest that the accessibility of the S* state in different local environments plays a key role in determining the quenching of Chl excited states. In vivo, non-photochemical quenching is activated by de-epoxidation of violaxanthin into zeaxanthin. CP29-zeaxanthin in nanodiscs further shortens the Chl lifetime, which underlines the critical role of zeaxanthin in modulating photoprotection activity.

ROS-Scavengers, Osmoprotectants and Violaxanthin De-Epoxidation in Salt-Stressed Arabidopsis thaliana with Different Tocopherol Composition.

To determine the role of α- and γ-tocopherol (TC), this study compared the response to salt stress (200 mM NaCl) in wild type (WT) Arabidopsis thaliana (L.) Heynh. And its two mutants: (1) totally TC-deficient vte1; (2) vte4 accumulating γ-TC instead of α-TC; and (3) tmt transgenic line overaccumulating α-TC. Raman spectra revealed that salt-exposed α-TC accumulating plants were more flexible in regulating chlorophyll, carotenoid and polysaccharide levels than TC deficient mutants, while the plants overaccumulating γ-TC had the lowest levels of these biocompounds. Tocopherol composition and NaCl concentration affected xanthophyll cycle by changing the rate of violaxanthin de-epoxidation and zeaxanthin formation. NaCl treated plants with altered TC composition accumulated less oligosaccharides than WT plants. α-TC deficient plants increased their oligosaccharide levels and reduced maltose amount, while excessive accumulation of α-TC corresponded with enhanced amounts of maltose. Salt-stressed TC-deficient mutants and tmt transgenic line exhibited greater proline levels than WT plants, lower chlorogenic acid levels, and lower activity of catalase and peroxidases. α-TC accumulating plants produced more methylated proline- and glycine- betaines, and showed greater activity of superoxide dismutase than γ-TC deficient plants. Under salt stress, α-TC demonstrated a stronger regulatory effect on carbon- and nitrogen-related metabolites reorganization and modulation of antioxidant patterns than γ-TC. This suggested different links of α- and γ-TCs with various metabolic pathways via various functions and metabolic loops.

Influence of the compatible solute sucrose on thylakoid membrane organization and violaxanthin de-epoxidation

Main conclusionThe compatible solute sucrose reduces the efficiency of the enzymatic de-epoxidation of violaxanthin, probably by a direct effect on the protein parts of violaxanthin de-epoxidase which protrude from the lipid phase of the thylakoid membrane.The present study investigates the influence of the compatible solute sucrose on the violaxanthin cycle of higher plants in intact thylakoids and in in vitro enzyme assays with the isolated enzyme violaxanthin de-epoxidase at temperatures of 30 and 10 °C, respectively. In addition, the influence of sucrose on the lipid organization of thylakoid membranes and the MGDG phase in the in vitro assays is determined. The results show that sucrose leads to a pronounced inhibition of violaxanthin de-epoxidation both in intact thylakoid membranes and the enzyme assays. In general, the inhibition is similar at 30 and 10 °C. With respect to the lipid organization only minor changes can be seen in thylakoid membranes at 30 °C in the presence of sucrose. However, sucrose seems to stabilize the thylakoid membranes at lower temperatures and at 10 °C a comparable membrane organization to that at 30 °C can be observed, whereas control thylakoids show a significantly different membrane organization at the lower temperature. The MGDG phase in the in vitro assays is not substantially affected by the presence of sucrose or by changes of the temperature. We conclude that the presence of sucrose and the increased viscosity of the reaction buffers stabilize the protein part of the enzyme violaxanthin de-epoxidase, thereby decreasing the dynamic interactions between the catalytic site and the substrate violaxanthin. This indicates that sucrose interacts with those parts of the enzyme which are accessible at the membrane surface of the lipid phase of the thylakoid membrane or the MGDG phase of the in vitro enzyme assays.

Proton motive force in plant photosynthesis dominated by ΔpH in both low and high light.

The proton motive force (pmf) across the thylakoid membrane couples photosynthetic electron transport and ATP synthesis. In recent years, the electrochromic carotenoid and chlorophyll absorption band shift (ECS), peaking ∼515 nm, has become a widely used probe to measure pmf in leaves. However, the use of this technique to calculate the parsing of the pmf between the proton gradient (ΔpH) and electric potential (Δψ) components remains controversial. Interpretation of the ECS signal is complicated by overlapping absorption changes associated with violaxanthin de-epoxidation to zeaxanthin (ΔA505) and energy-dependent nonphotochemical quenching (qE; ΔA535). In this study, we used Arabidopsis (Arabidopsis thaliana) plants with altered xanthophyll cycle activity and photosystem II subunit S (PsbS) content to disentangle these overlapping contributions. In plants where overlap among ΔA505, ΔA535, and ECS is diminished, such as npq4 (lacking ΔA535) and npq1npq4 (also lacking ΔA505), the parsing method implies the Δψ contribution is virtually absent and pmf is solely composed of ΔpH. Conversely, in plants where ΔA535 and ECS overlap is enhanced, such as L17 (a PsbS overexpressor) and npq1 (where ΔA535 is blue-shifted to 525 nm) the parsing method implies a dominant contribution of Δψ to the total pmf. These results demonstrate the vast majority of the pmf attributed by the ECS parsing method to Δψ is caused by ΔA505 and ΔA535 overlap, confirming pmf is dominated by ΔpH following the first 60 s of continuous illumination under both low and high light conditions. Further implications of these findings for the regulation of photosynthesis are discussed.

Advances in Understanding of Desiccation Tolerance of Lichens and Lichen-Forming Algae.

Lichens are symbiotic associations (holobionts) established between fungi (mycobionts) and certain groups of cyanobacteria or unicellular green algae (photobionts). This symbiotic association has been essential in the colonization of terrestrial dry habitats. Lichens possess key mechanisms involved in desiccation tolerance (DT) that are constitutively present such as high amounts of polyols, LEA proteins, HSPs, a powerful antioxidant system, thylakoidal oligogalactolipids, etc. This strategy allows them to be always ready to survive drastic changes in their water content. However, several studies indicate that at least some protective mechanisms require a minimal time to be induced, such as the induction of the antioxidant system, the activation of non-photochemical quenching including the de-epoxidation of violaxanthin to zeaxanthin, lipid membrane remodeling, changes in the proportions of polyols, ultrastructural changes, marked polysaccharide remodeling of the cell wall, etc. Although DT in lichens is achieved mainly through constitutive mechanisms, the induction of protection mechanisms might allow them to face desiccation stress in a better condition. The proportion and relevance of constitutive and inducible DT mechanisms seem to be related to the ecology at which lichens are adapted to.

Shedding light on the dark side of xanthophyll cycles.

Xanthophyll cycles are broadly important in photoprotection, and the reversible de-epoxidation of xanthophylls typically occurs in excess light conditions. However, as presented in this review, compiling evidence in a wide range of photosynthetic eukaryotes shows that xanthophyll de-epoxidation also occurs under diverse abiotic stress conditions in darkness. Light-driven photochemistry usually leads to the pH changes that activate de-epoxidases (e.g. violaxanthin de-epoxidase), but in darkness alternative electron transport pathways and luminal domains enriched in monogalactosyl diacyl glycerol (which enhance de-epoxidase activity) likely enable de-epoxidation. Another 'dark side' to sustaining xanthophyll de-epoxidation is inactivation and/or degradation of epoxidases (e.g. zeaxanthin epoxidase). There are obvious benefits of such activity regarding stress tolerance, and indeed this phenomenon has only been reported in stressful conditions. However, more research is required to unravel the mechanisms and understand the physiological roles of dark-induced formation of zeaxanthin. Notably, the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in darkness is still a frequently ignored process, perhaps because it questions a previous paradigm. With that in mind, this review seeks to shed some light on the dark side of xanthophyll de-epoxidation, and point out areas for future work.

Lipid Dependence of Xanthophyll Cycling in Higher Plants and Algae.

The xanthophyll cycles of higher plants and algae represent an important photoprotection mechanism. Two main xanthophyll cycles are known, the violaxanthin cycle of higher plants, green and brown algae and the diadinoxanthin cycle of Bacillariophyceae, Xanthophyceae, Haptophyceae, and Dinophyceae. The forward reaction of the xanthophyll cycles consists of the enzymatic de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin or diadinoxanthin to diatoxanthin during periods of high light illumination. It is catalyzed by the enzymes violaxanthin or diadinoxanthin de-epoxidase. During low light or darkness the back reaction of the cycle, which is catalyzed by the enzymes zeaxanthin or diatoxanthin epoxidase, restores the epoxidized xanthophylls by a re-introduction of the epoxy groups. The de-epoxidation reaction takes place in the lipid phase of the thylakoid membrane and thus, depends on the nature, three dimensional structure and function of the thylakoid lipids. As the xanthophyll cycle pigments are usually associated with the photosynthetic light-harvesting proteins, structural re-arrangements of the proteins and changes in the protein-lipid interactions play an additional role for the operation of the xanthophyll cycles. In the present review we give an introduction to the lipid and fatty acid composition of thylakoid membranes of higher plants and algae. We introduce the readers to the reaction sequences, enzymes and function of the different xanthophyll cycles. The main focus of the review lies on the lipid dependence of xanthophyll cycling. We summarize the current knowledge about the role of lipids in the solubilization of xanthophyll cycle pigments. We address the importance of the three-dimensional lipid structures for the enzymatic xanthophyll conversion, with a special focus on non-bilayer lipid phases which are formed by the main thylakoid membrane lipid monogalactosyldiacylglycerol. We additionally describe how lipids and light-harvesting complexes interact in the thylakoid membrane and how these interactions can affect the structure of the thylakoids. In a dedicated chapter we offer a short overview of current membrane models, including the concept of membrane domains. We then use these concepts to present a model of the operative xanthophyll cycle as a transient thylakoid membrane domain which is formed during high light illumination of plants or algal cells.

Zeaxanthin independence of photophysics in light-harvesting complex II in a membrane environment

Green plants protect against photodamage by dissipating excess energy in a process called non-photochemical quenching (NPQ). In vivo, NPQ is activated by a drop in the luminal pH of the thylakoid membrane that triggers conformational changes of the antenna complexes, which activate quenching channels. The drop in pH also triggers de-epoxidation of violaxanthin, one of the carotenoids bound within the antenna complexes, into zeaxanthin, and so the amplitude of NPQ in vivo has been shown to increase in the presence of zeaxanthin. In vitro studies on light-harvesting complex II (LHCII), the major antenna complex in plants, compared different solubilization environments, which give rise to different levels of quenching and so partially mimic NPQ in vivo. However, in these studies both completely zeaxanthin-independent and zeaxanthin-dependent quenching have been reported, potentially due to the multiplicity of solubilization environments. Here, we characterize the zeaxanthin dependence of the photophysics in LHCII in a near-physiological membrane environment, which produces slightly enhanced quenching relative to detergent solubilization, the typical in vitro environment. The photophysical pathways of dark-adapted and in vitro de-epoxidized LHCIIs are compared, representative of the low-light and high-light conditions in vivo, respectively. The amplitude of quenching as well as the dissipative photophysics are unaffected by zeaxanthin at the level of individual LHCIIs, suggesting that zeaxanthin-dependent quenching is independent of the channels induced by the membrane. Furthermore, our results demonstrate that additional factors beyond zeaxanthin incorporation in LHCII are required for full development of NPQ.

An extreme heatwave enhanced the xanthophyll de-epoxidation state in leaves of Eucalyptus trees grown in the field.

Heatwaves are becoming more frequent with climate warming and can impact tree growth and reproduction. Eucalyptus parramattensis can cope with an extreme heatwave in the field via transpiratory cooling and enhanced leaf thermal tolerance that protected foliar tissues from photo-inhibition and photo-oxidation during natural midday irradiance. Here, we explored whether changes in foliar carotenoids and/or the xanthophyll cycle state can facilitate leaf acclimation to long-term warming and/or an extreme heatwave event. We found that leaves had similar carotenoid levels when grown for one year under ambient and experimental long-term warming (+ 3°C) conditions in whole tree chambers. Exposure to a 4-day heatwave (> 43°C) significantly altered the xanthophyll de-epoxidation state of carotenoids revealing one mechanism by which trees could minimise foliar photo-oxidative damage. The levels of zeaxanthin were significantly higher in both young and old leaves during the heatwave, revealing that violaxanthin de-epoxidation and perhaps de novo zeaxanthin synthesis contributed to enhancement of the xanthophyll cycle state. In a future climate of long-term warming and increased heatwave events, leaves of E. parramattensis will be able to utilise biochemical strategies to alter the xanthophyll cycle state and cope with extreme temperatures under natural solar irradiation.

Seasonal responses to cold and light stresses by two elevational ecotypes of Rhododendron catawbiense: A comparative study of overwintering strategies

For winter survival, evergreens not only need cold acclimation (CA), but also photoprotection. This study was conducted to examine how two elevational ecotypes of Rhododendron catawbiense respond to cold and light stresses in a common garden. Seasonal changes in leaf freezing tolerance (LFT), photosystem II efficiency, photosynthesis, carotenoids (xanthophyll cycle pigments, lutein, beta-carotene, neoxanthin) were investigated from summer to winter. Parallel changes in the expression of violaxanthin de-epoxidase (VDE) gene were examined to explore its association with violaxanthin de-epoxidation. High elevation (HE) ecotype attained overall greater LFT while low elevation (LE) ecotype was more responsive to short-days for the first stage of CA. In summer, both ecotypes predominantly employed reversible xanthophyll cycle (RXC) to dissipate excess light energy, but LE used it more robustly. In autumn, only HE exhibited slight damage to the reaction centers (RCs), i.e. photosystem II degradation (PSD). In winter, both ecotypes suspended photosynthesis, were photoinhibited, and employed primarily sustained de-epoxidation (SD) for photoprotection; but LE still used RXC more effectively than HE. Overall, LE employed xanthophyll cycle more robustly in summer or winter. Results suggest that microclimates of R. catawbiense ecotypes inform their differential physiological response to cold and light stresses. VDE expression data indicate violaxanthin de-epoxidation in R. catawbiense may not be solely transcriptionally regulated.

The Photosystem II Subunit S under Stress

Nonphotochemical quenching is the protective mechanism against overexcitation of photosystem II, triggered by excess ΔpH in photosynthetic membranes. The light-harvesting complexes (LHCs), the de-epoxidation of violaxanthin to zeaxanthin, and the photosystem II subunit S (PsbS) work in synergy for an optimized multilevel response. Understanding the fine details of this synergy has proven challenging to scientific research. Here, we employ large-scale, all-atom molecular simulations and beyond experimental insight, we proceed a step further in identifying the PsbS dynamics that could possibly be associated with this synergy. For the first time, to our knowledge, we probe the distinct behavior of PsbS under ΔpH that probes the details of the potential dimer-to-monomer transition, and in a violaxanthin/zeaxanthin-rich membrane, at an all-atom resolution. We propose that the lumen-exposed residues, threonine 162 and glutamic acid 173, form stabilizing hydrogen bonds between the PsbS monomers only at high lumen pH, whereas at low pH (excess ΔpH) this interaction is lost, and leads to higher flexibility of the protein and potentially to the dimer-to-monomer transition. Lastly, we discuss how conformational changes under the presence of ΔpH/zeaxanthin are related to the PsbS role in the current nonphotochemical quenching model in the literature. For the latter, we probe a PsbS-monomeric LHCII association. The association is proposed to potentially alter the monomeric LHCII sensitivity to ΔpH by changing the pKa values of interacting LHCII residues. This serves as an example where protonation-ligation events enhance protein-protein interactions fundamental to many life processes.

Arachidonic acid is important for efficient use of light by the microalga Lobosphaera incisa under chilling stress

The oleaginous microalga Lobosphaera incisa (Trebouxiophyceae, Chlorophyta) contains arachidonic acid (ARA, 20:4 n−6) in all membrane glycerolipids and in the storage lipid triacylglycerol. The optimal growth temperature of the wild-type (WT) strain is 25°C; chilling temperatures (≤15°C) slow its growth. This effect is more pronounced in the delta-5-desaturase ARA-deficient mutant P127, in which ARA is replaced with dihomo-γ-linolenic acid (DGLA, 20:3 n−6). In nutrient-replete cells grown at 25°C, the major chloroplast lipid monogalactosylglycerol (MGDG) was dominated by C18/C16 species in both strains. Yet ARA constituted over 10% of the total fatty acids in the WT MGDG as a component of C20/C18 and C20/C20 species, whereas DGLA was only a minor component of MGDG in P127. Both strains increased the percentage of 18:3 n−3 in membrane lipids under chilling temperatures. The temperature downshift led to a dramatic increase in triacylglycerol at the expense of chloroplast lipids. WT and P127 showed a similarly high photochemical quantum yield of photosystem II, whereas non-photochemical quenching (NPQ) and violaxanthin de-epoxidation were drastically higher in P127, especially at 15°C. Fluorescence anisotropy measurements indicated that ARA-containing MGDG might contribute to sustaining chloroplast membrane fluidity upon dropping to the chilling temperature. We hypothesize that conformational changes in chloroplast membranes and increased rigidity of the ARA-deficient MGDG of P127 at chilling temperatures are not compensated by trienoic fatty acids. This might ‘lock’ violaxanthin de-epoxidase in the activated state causing high constitutive NPQ and alleviate the risk of photodamage under chilling conditions in the mutant.

The xanthophyll cycle affects reversible interactions between PsbS and light-harvesting complex II to control non-photochemical quenching.

To maintain high photosynthetic rates, plants must adapt to their light environment on a timescale of seconds to minutes. Therefore, the light-harvesting antenna system of photosystem II in thylakoid membranes, light-harvesting complex II (LHCII), has a feedback mechanism, which determines the proportion of absorbed energy dissipated as heat: non-photochemical chlorophyll fluorescence quenching (NPQ). This is crucial to prevent photo-oxidative damage to photosystem II (PSII) and is controlled by the transmembrane pH differences (ΔpH). High ΔpH activates NPQ by protonation of the protein PsbS and the enzymatic de-epoxidation of LHCII-bound violaxanthin to zeaxanthin. But the precise role of PsbS and its interactions with different LHCII complexes remain uncertain. We have investigated PsbS-LHCII interactions in native thylakoid membranes using magnetic-bead-linked antibody pull-downs. The interaction of PsbS with the antenna system is affected by both ΔpH and the level of zeaxanthin. In the presence of ΔpH alone, PsbS is found to be mainly associated with the trimeric LHCII protein polypeptides, Lhcb1, Lhcb2 and Lhcb3. However, a combination of ΔpH and zeaxanthin increases the proportion of PsbS bound to the minor LHCII antenna complex proteins Lhcb4, Lhcb5 and Lhcb6. This pattern of interaction is not influenced by the presence of PSII reactions centres. Similar to LHCII particles in the photosynthetic membrane, PsbS protein forms clusters in the NPQ state. NPQ recovery in the dark requires uncoupling of PsbS. We suggest that PsbS acts as a 'seeding' centre for the LHCII antenna rearrangement that is involved in NPQ.

Direct isolation of a functional violaxanthin cycle domain from thylakoid membranes of higher plants.

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

Adjustment of photoprotection to tidal conditions in intertidal seagrasses

Light adaptive strategies were studied in a comparative analysis of the congeneric seagrass species Zostera muelleri and Zostera marina at two case study areas in New Zealand and Germany. Surveys in intertidal seagrass meadows were conducted from pre-dawn until sunset on days when either low or high tide coincided with noon. The results show marked fluctuations of photophysiology (optimum and effective quantum yield, non-photochemical quenching, cycling of xanthophyll cycle (XC) pigments) over daily and tidal cycles. At both locations, we observed a near complete conversion (de-epoxidation) of violaxanthin to zeaxanthin at times with high irradiance and a rapid and complete re-epoxidation under subsequent lower light conditions. At the New Zealand site we also observed significantly larger XC-pigment pool sizes in seagrass leaves sampled in a week when low tide coincided with noon (larger daily fluence and higher maximum irradiance), compared with leaves sampled in a week when high tide was at noon. This dynamic adjustment of xanthophyll pool size has not been previously reported for intertidal seagrasses. It adds to our understanding of an important adaptive feature in a highly dynamic light environment and to the general ecology and adaptability of seagrasses.

Molecular studies on structural changes and oligomerisation of violaxanthin de-epoxidase associated with the pH-dependent activation.

Violaxanthin de-epoxidase (VDE) is a conditionally soluble enzyme located in the thylakoid lumen and catalyses the conversion of violaxanthin to antheraxanthin and zeaxanthin, which are located in the thylakoid membrane. These reactions occur when the plant or algae are exposed to saturating light and the zeaxanthin formed is involved in the process of non-photochemical quenching that protects the photosynthetic machinery during stress. Oversaturation by light results in a reduction of the pH inside the thylakoids, which in turn activates VDE and the de-epoxidation of violaxanthin. To elucidate the structural events responsible for the pH-dependent activation of VDE, full length and truncated forms of VDE were studied at different pH using circular dichroism (CD) spectroscopy, crosslinking and small angle X-ray scattering (SAXS). CD spectroscopy showed the formation of α-helical coiled-coil structure, localised in the C-terminal domain. Chemical crosslinking of VDE showed that oligomers were formed at low pH, and suggested that the position of the N-terminal domain is located near the opening of lipocalin-like barrel, where violaxanthin has been predicted to bind. SAXS was used to generate models of monomeric VDE at high pH and also a presumably dimeric structure of VDE at low pH. For the dimer, the best fit suggests that the interaction is dominated by one of the domains, preferably the C-terminal domain due to the lost ability to oligomerise at low pH, shown in earlier studies, and the predicted formation of coiled-coil structure.

Functional and structural characterization of domain truncated violaxanthin de‐epoxidase

Photosynthetic organisms need protection against excessive light. By using non-photochemical quenching, where the excess light is converted into heat, the organism can survive at higher light intensities. This process is partly initiated by the formation of zeaxanthin, which is achieved by the de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin. This reaction is catalyzed by violaxanthin de-epoxidase (VDE). VDE consists of three domains of which the central lipocalin-like domain has been the most characterized. By truncating the domains surrounding the lipocalin-like domain, we show that VDE activity is possible without the C-terminal domain but not without the N-terminal domain. The N-terminal domain shows no VDE activity by itself but when separately expressed domains are mixed, VDE activity is possible. This shows that these domains can be folded separately and could therefore be studied separately. An increase of the hydrodynamic radius of wild-type VDE was observed when pH was lowered toward the pH required for activity, consistent with a pH-dependent oligomerization. The C-terminally truncated VDE did not show such an oligomerization, was relatively more active at higher pH but did not alter the KM for ascorbate. Circular dichroism measurements revealed the presence of α-helical structure in both the N- and C-terminal domains. By measuring the initial formation of the product, VDE was found to convert a large number of violaxanthin molecules to antheraxanthin before producing any zeaxanthin, favoring a model where violaxanthin is bound non-symmetrically in VDE.

Ethylene Regulates Energy-Dependent Non-Photochemical Quenching in Arabidopsis through Repression of the Xanthophyll Cycle.

Energy-dependent (qE) non-photochemical quenching (NPQ) thermally dissipates excess absorbed light energy as a protective mechanism to prevent the over reduction of photosystem II and the generation of reactive oxygen species (ROS). The xanthophyll cycle, induced when the level of absorbed light energy exceeds the capacity of photochemistry, contributes to qE. In this work, we show that ethylene regulates the xanthophyll cycle in Arabidopsis. Analysis of eto1-1, exhibiting increased ethylene production, and ctr1-3, exhibiting constitutive ethylene response, revealed defects in NPQ resulting from impaired de-epoxidation of violaxanthin by violaxanthin de-epoxidase (VDE) encoded by NPQ1. Elevated ethylene signaling reduced the level of active VDE through decreased NPQ1 promoter activity and impaired VDE activation resulting from a lower transthylakoid membrane pH gradient. Increasing the concentration of CO2 partially corrected the ethylene-mediated defects in NPQ and photosynthesis, indicating that changes in ethylene signaling affect stromal CO2 solubility. Increasing VDE expression in eto1-1 and ctr1-3 restored light-activated de-epoxidation and qE, reduced superoxide production and reduced photoinhibition. Restoring VDE activity significantly reversed the small growth phenotype of eto1-1 and ctr1-3 without altering ethylene production or ethylene responses. Our results demonstrate that ethylene increases ROS production and photosensitivity in response to high light and the associated reduced plant stature is partially reversed by increasing VDE activity.

Non-photochemical quenching and xanthophyll cycle activities in six green algal species suggest mechanistic differences in the process of excess energy dissipation

In the present study the non-photochemical quenching (NPQ) of four biofilm-forming and two planktonic green algae was investigated by fluorescence measurements, determinations of the light-driven proton gradient and determination of the violaxanthin cycle activity by pigment analysis. It was observed that, despite the common need for efficient photoprotection, the structural basis of NPQ was heterogeneous in the different species. Three species, namely Chlorella saccharophila, Chlorella vulgaris and Bracteacoccus minor, exhibited a zeaxanthin-dependent NPQ, while in the three other species, Tetracystis aeria, Pedinomonas minor and Chlamydomonas reinhardtii violaxanthin de-epoxidation was absent or unrelated to the establishment of NPQ. Acclimation of the algae to high light conditions induced an increase of the NPQ activity, suggesting that a significant part of the overall NPQ was rather inducible than constitutively present in the green algae. Comparing the differences in the NPQ mechanisms with the phylogenetic position of the six algal species led to the conclusion that the NPQ heterogeneity observed in the present study was not related to the phylogeny of the algae but to the environmental selection pressure. Finally, the difference in the NPQ mechanisms in the different species is discussed within the frame of the current NPQ models.

Leaf chloroplast ultrastructure and photosynthetic properties of a chlorophyll-deficient mutant of rice

Leaf chloroplast ultrastructure and photosynthetic properties of a natural, yellow-green leaf mutant (ygl1) of rice were characterized. Our results showed that chloroplast development was significantly delayed in the mutant leaves compared with the wild-type rice (WT). As leaves matured, more grana stacks formed concurrently with increasing leaf chlorophyll (Chl) content. Except for the lower intercellular CO2 concentration, the ygl1 plants had a higher leaf net photosynthetic rate, stomatal conductance, and transpiration rate than those of the WT plants. Under equal amounts of Chl, the excitation energy of PSI and PSII was much stronger in the mutant than that in the WT. The ygl1 plants showed higher nonphotochemical quenching and lower photochemical quenching. They also exhibited higher actual photochemical efficiency of PSII with a higher electron transport rate. Under the light of 200 μmol(photon) m-2 s-1, the ygl1 mutant showed lesser deepoxidation of violaxanthin in the xanthophyll cycle than WT, but it increased substantially under strong light conditions. In conclusion, the photosynthetic machinery of the ygl1 remained stable during leaf development. The plants were less sensitive to photoinhibition compared with WT due to the active xanthophyll cycle. The ygl1 plants were efficient in both light harvesting and conversion of solar energy.