De-epoxidation Of Violaxanthin Research Articles

Assessing leaf photoprotective mechanisms using terrestrial LiDAR: towards mapping canopy photosynthetic performance in three dimensions

Terrestrial laser scanning (TLS) data allow spatially explicit (x, y, z) laser return intensities to be recorded throughout a plant canopy, which could considerably improve our understanding of how physiological processes vary in three-dimensional space. However, the utility of TLS data for the quantification of plant physiological properties remains largely unexplored. Here, we test whether the laser return intensity of green (532-nm) TLS correlates with changes in the de-epoxidation state of the xanthophyll cycle and photoprotective non-photochemical quenching (NPQ), and compare the ability of TLS to quantify these parameters with the passively measured photochemical reflectance index (PRI). We exposed leaves from five plant species to increasing light intensities to induce NPQ and de-epoxidation of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z). At each light intensity, the green laser return intensity (GLRI), narrowband spectral reflectance, chlorophyll fluorescence emission and xanthophyll cycle pigment composition were recorded. Strong relationships between both predictor variables (GLRI, PRI) and both explanatory variables (NPQ, xanthophyll cycle de-epoxidation) were observed. GLRI holds promise to provide detailed (mm) information about plant physiological status to improve our understanding of the patterns and mechanisms driving foliar photoprotection. We discuss the potential for scaling these laboratory data to three-dimensional canopy space.

Changes in the Energy Transfer Pathways within Photosystem II Antenna Induced by Xanthophyll Cycle Activity

Energy transfer pathways between photosystem II (PSII) antenna complexes in intact thylakoid membranes have been studied using low-temperature (77 K) excitation fluorescence spectroscopy. The focus of this study was to see whether de-epoxidation of violaxanthin into zeaxanthin causes any alterations in the energetic couplings between the core antenna complexes CP43 and CP47 and the peripheral light-harvesting antenna (LHCII). It was discovered that the appearance of zeaxanthin caused characteristic alterations in the PSII excitation fluorescence spectra in the Soret-band region. While in the dark violaxanthin was found to be largely uncoupled from any emitting chlorophylls, its intensive de-epoxidation resulted in the appearance of two additional bands at 509 and 492 nm. The former was attributed to weak coupling of zeaxanthin to emitters in the CP43 and LHCII complexes and the latter to efficient coupling of violaxanthin of the CP29 complex to emitters in the CP43, CP47, and LHCII complexes. The role of CP29-bound violaxanthin is discussed in light of both its efficient energetic coupling and strong physical binding to this complex. The finding that zeaxanthin is energetically coupled to chlorophyll a emitters of the PSII antenna is discussed with respect to its suggested role as a quencher involved in photoprotective energy dissipation, or non-photochemical quenching (NPQ), in the photosynthetic membrane.

Three pools of zeaxanthin in Quercus coccifera leaves during light transitions with different roles in rapidly reversible photoprotective energy dissipation and photoprotection.

Under excess light, the efficient PSII light-harvesting antenna is switched into a photoprotected state in which potentially harmful absorbed energy is thermally dissipated. Changes occur rapidly and reversibly, enhanced by de-epoxidation of violaxanthin (V) to zeaxanthin (Z). This process is usually measured as non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence. Using instrumentation for instantaneous leaf freezing, NPQ, spectral reflectance, and interconversions within the xanthophyll cycle with time resolution of seconds were recorded from Quercus coccifera leaves during low light (LL) to high light (HL) transitions, followed by relaxation at LL. During the first 30 s of both the LL to HL and HL to LL transitions, no activity of the xanthophyll cycle was detected, whereas 70–75% of the NPQ was formed and relaxed, respectively, by that time, the latter being traits of a rapidly reversible photoprotective energy dissipation. Three different Z pools were identified, which play different roles in energy dissipation and photoprotection. In conclusion, ΔpH was crucial to NPQ formation and relaxation in Q. coccifera during light transitions. Only a minor fraction of Z was associated to quenching, whereas the largest Z pool was not related to thermal dissipation. The latter is proposed to participate in photoprotection acting as antioxidant.

A kinetic model of rapidly reversible nonphotochemical quenching

Oxygen-evolving photosynthetic organisms possess nonphotochemical quenching (NPQ) pathways that protect against photo-induced damage. The majority of NPQ in plants is regulated on a rapid timescale by changes in the pH of the thylakoid lumen. In order to quantify the rapidly reversible component of NPQ, called qE, we developed a mathematical model of pH-dependent quenching of chlorophyll excitations in Photosystem II. Our expression for qE depends on the protonation of PsbS and the deepoxidation of violaxanthin by violaxanthin deepoxidase. The model is able to simulate the kinetics of qE at low and high light intensities. The simulations suggest that the pH of the lumen, which activates qE, is not itself affected by qE. Our model provides a framework for testing hypothesized qE mechanisms and for assessing the role of qE in improving plant fitness in variable light intensity.

The investigation of violaxanthin de-epoxidation in the primitive green alga Mantoniella squamata (Prasinophyceae) indicates mechanistic differences in xanthophyll conversion to higher plants

Schaller S., Latowski D., JemioŁa-Rzemińska M., Quaas T., Wilhelm C., StrzaŁka K. and Goss R. 2012. The investigation of violaxanthin de-epoxidation in the primitive green alga Mantoniella squamata (Prasinophyceae) indicates mechanistic differences in xanthophyll conversion to higher plants. Phycologia 51: 359–370. DOI: 10.2116/11-127.1The violaxanthin (Vx) de-epoxidation of the green alga Mantoniella squamata (Prasinophyceae) was investigated in thylakoid membranes and in enzyme assays with the isolated Vx de-epoxidase (VDE), where pure Vx or purified light-harvesting complexes of M. squamata (MLHC) or spinach (LHCII) were used. The detailed analysis of the VDE amino acid sequences of prasinophycean algae and higher plants suggests that structural differences of the M. squamata VDE are responsible for the slow conversion of Ax to Zx that is typical for this alga. The present experiments further demonstrate that the availability of the substrate Vx has a strong impact on the overall de-epoxidation reaction and the ratio of the second de-epoxidation rate (de-epoxidation from Ax to Zx) to the first de-epoxidation rate (conversion from Vx to Ax). High concentrations of available Vx, as found in enzyme assays with pure Vx, lead to saturation of the VDE and a strong competition with the intermediate reaction product Ax, thus decreasing the ratio of the second de-epoxidation rate to the first de-epoxidation rate. In de-epoxidation assays with isolated LHCs, where the availability of Vx is limited by the detachment of Vx from the protein, an increased ratio of the second de-epoxidation rate to the first de-epoxidation rate is observed. The present study also shows that the interaction between the isolated LHC and the VDE can influence the ratio of the two de-epoxidation rates. Mantoniella squamata VDE, which exhibits a very low ratio of the second de-epoxidation rate to the first de-epoxidation rate in thylakoids or in enzyme assays with the MLHC, shows a high ratio when it is incubated with the LHCII of spinach.

Acclimatory responses of Arabidopsis to fluctuating light environment: comparison of different sunfleck regimes and accessions

Acclimation to fluctuating light environment with short (lasting 20 s, at 650 or 1,250 μmol photons m−2 s−1, every 6 or 12 min) or long (for 40 min at 650 μmol photons m−2 s−1, once a day at midday) sunflecks was studied in Arabidopsis thaliana. The sunfleck treatments were applied in the background daytime light intensity of 50 μmol photons m−2 s−1. In order to distinguish the effects of sunflecks from those of increased daily irradiance, constant light treatments at 85 and 120 μmol photons m−2 s−1, which gave the same photosynthetically active radiation (PAR) per day as the different sunfleck treatments, were also included in the experiments. The increased daily total PAR in the two higher constant light treatments enhanced photosystem II electron transport and starch accumulation in mature leaves and promoted expansion of young leaves in Columbia-0 plants during the 7-day treatments. Compared to the plants remaining under 50 μmol photons m−2 s−1, application of long sunflecks caused upregulation of electron transport without affecting carbon gain in the form of starch accumulation and leaf growth or the capacity of non-photochemical quenching (NPQ). Mature leaves showed marked enhancement of the NPQ capacity under the conditions with short sunflecks, which preceded recovery and upregulation of electron transport, demonstrating the initial priority of photoprotection. The distinct acclimatory responses to constant PAR, long sunflecks, and different combinations of short sunflecks are consistent with acclimatory adjustment of the processes in photoprotection and carbon gain, depending on the duration, frequency, and intensity of light fluctuations. While the responses of leaf expansion to short sunflecks differed among the seven Arabidopsis accessions examined, all plants showed NPQ upregulation, suggesting limited ability of this species to utilize short sunflecks. The increase in the NPQ capacity was accompanied by reduced chlorophyll contents, higher levels of the xanthophyll-cycle pigments, faster light-induced de-epoxidation of violaxanthin to zeaxanthin and antheraxanthin, increased amounts of PsbS protein, as well as enhanced activity of superoxide dismutase. These acclimatory mechanisms, involving reorganization of pigment–protein complexes and upregulation of other photoprotective reactions, are probably essential for Arabidopsis plants to cope with photo-oxidative stress induced by short sunflecks without suffering from severe photoinhibition and lipid peroxidation.

Violaxanthin de-epoxidase is rate-limiting for non-photochemical quenching under subsaturating light or during chilling in Arabidopsis

In response to conditions of excess light energy, plants induce non-photochemical quenching (NPQ) as a protective mechanism to prevent over reduction of photosystem II and the generation of reactive oxygen species (ROS). The xanthophyll cycle, which contributes significantly to reversible NPQ to thermally dissipate excess absorbed light energy, involves de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin in response to excess light energy. The activation of violaxanthin de-epoxidase (VDE), which catalyzes the de-epoxidation reaction, requires the generation of a light-induced, transthylakoid pH gradient. In this work, we overexpressed or repressed the expression of VDE in Arabidopsis (Arabidopsis thaliana) to examine whether VDE is rate-limiting for the induction of NPQ. Increasing VDE expression increased the de-epoxidation state of xanthophyll pigments, the rate of NPQ induction, and the level of NPQ achieved under subsaturating light. In saturating light, however, overexpression of VDE did not increase the xanthophyll pigment de-epoxidation state, the level of NPQ achieved following its initial induction, or substantially improve tolerance to high light. Only under chilling, which reduces VDE activity, did an increase in VDE expression provide slightly greater phototolerance. Repression of VDE expression impaired violaxanthin de-epoxidation, reduced the generation of NPQ, and lowered the level of NPQ achieved while increasing photosensitivity. These results demonstrate that the endogenous level of VDE is rate-limiting for NPQ in Arabidopsis under subsaturating but not saturating light and can become rate-limiting under chilling conditions. These results also show that increasing VDE expression confers greater phototolerance mainly under conditions which limit endogenous VDE activity.

A Theoretical Investigation of Xanthophyll–Protein Hydrogen Bonding in the Photosystem II Antenna

Photoprotective nonphotochemical quenching (NPQ) in higher plants is the result of the formation of energy-quenching traps in the light-harvesting antenna of photosystem II (PSII). The primary driving forces behind NPQ are the protonation of the thylakoid lumen and the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin in the antenna. There is currently some disagreement over whether de-epoxidation occurs only at the peripheral, V1, binding site of the major LHCII or also at the internal, L2, site of the minor antenna CP29 complex of PSII. We have used density functional theory (DFT) to study of hydrogen bonding between xanthophylls and the protein scaffold of LHCII and CP29. We argue that a lack of hydrogen bonding for violaxanthin in LHCII is consistent with it being weakly bound and accessible for de-epoxidation. Conversely, the strong violaxanthin-protein hydrogen bonding at the L2 site of CP29 is consistent with evidence that it is not readily accessible for de-epoxidation and therefore quenching by zeaxanthin at the L2 of CP29 is an unlikely candidate for in vivo NPQ.

Photoprotection in Plants Involves a Change in Lutein 1 Binding Domain in the Major Light-harvesting Complex of Photosystem II

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).

Non-photochemical fluorescence quenching in Chromera velia is enabled by fast violaxanthin de-epoxidation

Non-photochemical quenching (NPQ) is a mechanism protecting photosynthetic organisms against excessive irradiation. Here, we analyze a unique NPQ mechanism in the alga Chromera velia, a recently discovered close relative of apicomplexan parasites. NPQ in C. velia is enabled by an operative and fast violaxanthin de-epoxidation to zeaxanthin without accumulation of antheraxanthin. In C. velia violaxanthin also serves as a main light-harvesting pigment. Therefore, in C. velia violaxanthin acts as a key factor in both light harvesting and photoprotection. This is in contrast to a similar alga, Nannochloropsis limnetica, where violaxanthin has only light-harvesting function.

Photoprotective Energy Dissipation Involves the Reorganization of Photosystem II Light-Harvesting Complexes in the Grana Membranes of Spinach Chloroplasts

Plants must regulate their use of absorbed light energy on a minute-by-minute basis to maximize the efficiency of photosynthesis and to protect photosystem II (PSII) reaction centers from photooxidative damage. The regulation of light harvesting involves the photoprotective dissipation of excess absorbed light energy in the light-harvesting antenna complexes (LHCs) as heat. Here, we report an investigation into the structural basis of light-harvesting regulation in intact spinach (Spinacia oleracea) chloroplasts using freeze-fracture electron microscopy, combined with laser confocal microscopy employing the fluorescence recovery after photobleaching technique. The results demonstrate that formation of the photoprotective state requires a structural reorganization of the photosynthetic membrane involving dissociation of LHCII from PSII and its aggregation. The structural changes are manifested by a reduced mobility of LHC antenna chlorophyll proteins. It is demonstrated that these changes occur rapidly and reversibly within 5 min of illumination and dark relaxation, are dependent on ΔpH, and are enhanced by the deepoxidation of violaxanthin to zeaxanthin.

Lutein from deepoxidation of lutein epoxide replaces zeaxanthin to sustain an enhanced capacity for nonphotochemical chlorophyll fluorescence quenching in avocado shade leaves in the dark.

Leaves of avocado (Persea americana) that develop and persist in deep shade canopies have very low rates of photosynthesis but contain high concentrations of lutein epoxide (Lx) that are partially deepoxidized to lutein (L) after 1 h of exposure to 120 to 350 μmol photons m(-2) s(-1), increasing the total L pool by 5% to 10% (ΔL). Deepoxidation of Lx to L was near stoichiometric and similar in kinetics to deepoxidation of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z). Although the V pool was restored by epoxidation of A and Z overnight, the Lx pool was not. Depending on leaf age and pretreatment, the pool of ΔL persisted for up to 72 h in the dark. Metabolism of ΔL did not involve epoxidation to Lx. These contrasting kinetics enabled us to differentiate three states of the capacity for nonphotochemical chlorophyll fluorescence quenching (NPQ) in attached and detached leaves: ΔpH dependent (NPQ(ΔpH)) before deepoxidation; after deepoxidation in the presence of ΔL, A, and Z (NPQ(ΔLAZ)); and after epoxidation of A+Z but with residual ΔL (NPQ(ΔL)). The capacity of both NPQ(ΔLAZ) and NPQ(ΔL) was similar and 45% larger than NPQ(ΔpH), but dark relaxation of NPQ(ΔLAZ) was slower. The enhanced capacity for NPQ was lost after metabolism of ΔL. The near equivalence of NPQ(ΔLAZ) and NPQ(ΔL) provides compelling evidence that the small dynamic pool ΔL replaces A+Z in avocado to "lock in" enhanced NPQ. The results are discussed in relation to data obtained with other Lx-rich species and in mutants of Arabidopsis (Arabidopsis thaliana) with increased L pools.

Origin of Absorption Changes Associated with Photoprotective Energy Dissipation in the Absence of Zeaxanthin

To prevent photo-oxidative damage to the photosynthetic membrane in strong light, plants dissipate excess absorbed light energy as heat in a mechanism known as non-photochemical quenching (NPQ). NPQ is triggered by the trans-membrane proton gradient (ΔpH), which causes the protonation of the photosystem II light-harvesting antenna (LHCII) and the PsbS protein, as well as the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin. The combination of these factors brings about formation of dissipative pigment interactions that quench the excess energy. The formation of NPQ is associated with certain absorption changes that have been suggested to reflect a conformational change in LHCII brought about by its protonation. The light-minus-dark recovery absorption difference spectrum is characterized by a series of positive and negative bands, the best known of which is ΔA(535). Light-minus-dark recovery resonance Raman difference spectra performed at the wavelength of the absorption change of interest allows identification of the pigment responsible from its unique vibrational signature. Using this technique, the origin of ΔA(535) was previously shown to be a subpopulation of red-shifted zeaxanthin molecules. In the absence of zeaxanthin (and antheraxanthin), a proportion of NPQ remains, and the ΔA(535) change is blue-shifted to 525 nm (ΔA(525)). Using resonance Raman spectroscopy, it is shown that the ΔA(525) absorption change in Arabidopsis leaves lacking zeaxanthin belongs to a red-shifted subpopulation of violaxanthin molecules formed during NPQ. The presence of the same ΔA(535) and ΔA(525) Raman signatures in vitro in aggregated LHCII, containing zeaxanthin and violaxanthin, respectively, leads to a new proposal for the origin of the xanthophyll red shifts associated with NPQ.

Characterization of pigment apparatus in winter-green and summer-green leaves of a shade-tolerant plant Ajuga reptans

Comparative study was performed to assess the content and proportions of photosynthetic pigments and the violaxanthin cycle (VXC) activity in winter-green and summer-green leaves of bugleweed (Ajuga reptans L.) plants grown in shaded (photosynthetically active radiation, PAR 150 μmol/(m2 s)) and sunny (PAR 1200 μmol/(m2 s)) habitats in the Botanic Garden of Jagiellonian University (Krakow, Poland). In overwintered and newly formed leaves of shade plants, the content of green and yellow pigments was two times higher than in leaves of sun plants. The shade plants were distinguished by accumulation of β-carotene, while lutein was predominant in leaves of sun plants. Under the action of strong light (2000 μmol/(m2s)), the level of violaxanthin deepoxidation in winter-green leaves of shade and sun plants increased five- to sixfold, whereas it changed insignificantly in summer-green leaves of shade plants. It is concluded that, in a shadetolerant species A. reptans, the photosynthetic apparatus of winter-green leaves in sun and shade plants and of summer-green leaves in sun plants is protected against excess insolation by high activity of VXC. The carotenoids of summer-green leaves in shade plants are supposed to function mainly as light-harvesting pigments.

The photoprotective mechanisms in Secale cereale leaves under Cu and high light stress condition

The influence of excess Cu ions and high light treatment on the function of photosystem II was investigated in order to examine how this heavy metal modifies the photoprotective mechanisms operating at the molecular level in Secale cereale plants. Thus, non-treated plants and those treated with 5 or 50 μM Cu, simultaneously illuminated with 150 μmol m −2 s −1 or 1200 μmol m −2 s −1 light intensity, were studied. To analyze the PSII reaction to the stress conditions, Chl a fluorescence induction was applied. An increase in the value of Φ PSII and R fd parameters indicated that the photosynthetic apparatus adapted to the high light condition by effective utilization of excitation energy in the light and dark phases of photosynthesis. This phenomenon was accompanied by dissipation of excitation energy within the antenna complexes. The xanthophyll cycle pigments in Secale cereale leaves were separated and quantified by the HPLC technique. The results showed that, under high light irradiance, both 5 and 50 μM Cu induced the process of violaxanthin de-epoxidation and zeaxanthin accumulation. The significant zeaxanthin accumulation was found to be involved in photoprotective energy dissipation as heat, which was supported by correlation between the rate of violaxanthin de-epoxidation and the value of SV parameters. Interestingly, Cu treatment caused violaxanthin isomerization from its trans to 15-, 13- and 9- cis forms in proportional correlation to the metal concentration. This phenomenon was confirmed by a study of Cu-induced violaxanthin isomerization in vitro, which suggests a direct metal–pigment molecule interaction. We also observed that the violaxanthin trans– cis isomerization increased simultaneously with anteraxanthin content. On the basis of these findings, it can be speculated that violaxanthin isomerization is the basic process responsible for the xanthophyll cycle operation.

Low‐light‐induced Violaxanthin De‐epoxidation in Shortly Preheated Leaves: Uncoupling from ΔpH‐dependent Nonphotochemical Quenching

Plants protect themselves against excessive light by the induction of Delta pH-dependent nonphotochemical quenching (qE) that is associated with de-epoxidation of violaxanthin (V) to zeaxanthin (Z) in thylakoid membranes. In this work, we report that low light (12 micromol photons m(-2) s(-1)) is sufficient for a marked stimulation of the V to Z conversion in shortly preheated wheat leaves (5 min, 40 degrees C), but without a substantial increase in qE. Re-irradiation of these leaves with high light led to a rapid induction of nonphotochemical quenching, implying a potential photoprotective role of low-light-induced Z in preheated leaves. On the contrary to low light conditions, preheated leaves exposed to high light behaved similar to nonheated leaves with respect to the V to Z conversion and qE induction. The obtained results indicate that low-light-induced lumen acidification in preheated leaves is high enough to activate V de-epoxidation, but not sufficiently high to induce the formation of quenching centers.

Unravelling the roles of desiccation-induced xanthophyll cycle activity in darkness: a case study in Lobaria pulmonaria

Desiccation-tolerance ability in photosynthetic organisms is largely based on a battery of photoprotective mechanisms. Xanthophyll cycle operation induced by desiccation in the absence of light has been previously proven in the desiccation-tolerant fern Ceterach officinarum. To understand the physiological function of xanthophyll cycle induction in darkness and its implication in the desiccation tolerance in more detail, we studied its triggering factors and its photochemical effects in the lichen Lobaria pulmonaria. We found that both the drying rate and the degree of desiccation play a crucial role in the violaxanthin de-epoxidase activation. De-epoxidation of violaxanthin to zeaxanthin (Z) occurs when the tissue has lost most of its water and only after slow dehydration, suggesting that a minimum period of time is required for the enzyme activity induction. Fluorescence analysis showed that Z, synthesised during tissue dehydration in the absence of light, prevents photoinhibition when rewatered tissues are illuminated. This is probably due to Z implication in both non-photochemical quenching and/or antioxidative responses.

Light-induced decrease of reflectance provides an insight in the photoprotective mechanisms of ripening apple fruit

The magnitude of real-time changes in reflectance of ripening apple ( Malus × domestica Borkh., cv. Antonovka) fruit induced by PAR irradiation (500 μE m −2 s −1) was monitored together with pigment composition and non-photochemical dissipation of absorbed light energy. In fruit with high chlorophyll content irradiation induced, within 180 s, a notable decrease of reflectance in the bands centred at 520, 692 and 740 nm. These changes were strongly inter-correlated and exhibited tight relationships with chlorophyll fluorescence intensity, violaxanthin de-epoxidation, NPQ, qN and qP. The decrease of the reflectance in the blue-green is suggested to be related mainly with irradiation-induced conversion of violaxanthin to zeaxanthin whereas the decrease of the reflectance in the red and NIR was obviously due to the quenching of chlorophyll fluorescence. Based on these findings, spectral index AVI ( Apple Violaxanthin cycle Index) for non-destructive assessment of violaxanthin cycle operation in apple was developed. According to the results of violaxanthin cycle monitoring with the use of AVI, its activity as well as non-photochemical quenching declined in the course of apple ripening on the background of the increase in the proportion of extrathylakoid carotenoids (mainly fatty acid-esterified xanthophylls) together with the relative amount of light intercepted by these pigments. Comparative importance of violaxanthin cycle and light screening by extrathylakoid carotenoids for photoprotection at different stages of fruit development is discussed.

The main thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) promotes the de-epoxidation of violaxanthin associated with the light-harvesting complex of photosystem II (LHCII)

In higher plants, the major part of the xanthophyll cycle pigment violaxanthin (Vx) is non-covalently bound to the main light-harvesting complex of PSII (LHCII). Under saturating light conditions Vx has to be released from its binding site into the surrounding lipid phase, where it is converted to zeaxanthin (Zx) by the enzyme Vx de-epoxidase (VDE). In the present study we investigated the influence of thylakoid lipids on the de-epoxidation of Vx, which was still associated with the LHCII. We isolated LHCII with different concentrations of native, endogenous lipids and Vx by sucrose gradient centrifugation or successive cation precipitation. Analysis of the different LHCII preparations showed that the concentration of LHCII-associated Vx was correlated with the concentration of the main thylakoid lipid monogalactosyldiacylglycerol (MGDG) associated with the complexes. Decreases in the MGDG content of the LHCII led to a diminished Vx concentration, indicating that a part of the total Vx pool was located in an MGDG phase surrounding the LHCII, whereas another part was bound to the LHCII apoproteins. We further studied the convertibility of LHCII-associated Vx in in-vitro enzyme assays by addition of isolated VDE. We observed an efficient and almost complete Vx conversion in the LHCII fractions containing high amounts of endogenous MGDG. LHCII preparations with low concentrations of MGDG exhibited a strongly reduced Vx de-epoxidation, which could be increased by addition of exogenous, pure MGDG. The de-epoxidation of LHCII-associated Vx was saturated at a much lower concentration of native, endogenous MGDG compared with the concentration of isolated, exogenous MGDG, which is needed for optimal VDE activity in in-vitro assays employing pure isolated Vx.

Dark induction of the photoprotective xanthophyll cycle in response to dehydration

Some plants tolerate tissue dehydration. Dehydration conditions suppress photosynthesis, exacerbating photooxidative stress. In this study, fern samples were collected from the field, desiccated in darkness, and subsequently re-watered. During dark dehydration, zeaxanthin (Z) was formed and maximal photochemical efficiency of PS II was strongly reduced. Rehydration in the dark reversed these effects. Violaxanthin de-epoxidase was responsible for the dark formation of Z as illustrated by its complete inhibition by DTT. Nonetheless, its activity was not affected by nigericin, indicating that Z formation in the dark could be a process independent of the transmembrane pH-gradient into the thylakoids. Synthesis de novo of Z was rejected after blocking carotenogenesis with norfluorazon. Dark formation of Z was also observed in dehydrating leaves of desiccation-intolerant plants, which seems to indicate that this is a phenomenon scattered among different taxa within the plant kingdom. Plants may trigger this mechanism during dehydration, for chlorophyll protection during desiccation, and for faster acclimation when rehydrating conditions return. Violaxanthin de-epoxidation to form Z is typically a light-dependent process, but the formation induced solely by dehydration might represent an anticipatory mechanism for preventing early morning photodamage in desiccation-tolerant plants such as the fern Ceterach officinarum.