Seasonal changes in leaf antioxidant systems and xanthophyll cycle characteristics in Taxus x media growing in sun and shade environments

We examined differences between summer and winter in xanthophyll cycle-dependent energy dissipation and leaf antioxidant systems in needles of the overwintering evergreen Taxus x media cv. Tauntonii (Taunton yew) growing in both sun and shade environments in Saint Paul, Minnesota. During the winter, both sun and shade plants exhibited increases in the capacity for, and utilization of, xanthophyll cycle-dependent thermal energy dissipation. Winter needles showed decreases (sun needles) or no change (shade needles) in superoxide dismutase activity (EC 1.15.1.1), no change in ascorbate peroxidase activity (EC 1.11.1.11) and no change (sun needles) or increases (shade needles) in reduced ascorbate levels. Both sun and shade needles showed large increases in glutathione reductase activity (EC 1.6.4.2) and total glutathione levels during the winter, in addition to increases in levels of α-tocopherol. These results suggest an important photoprotective role during the winter for xanthophyll cycle-dependent energy dissipation and for the antioxidants glutathione and α-tocopherol. They suggest a less important photoprotective function of the enzyme-based water–water cycle in winter acclimation in the seasonally very cold environment of Minnesota.     

Antioxidants and xanthophyll cycle-dependent energy dissipation in Cucurbita pepo L. and Vinca major L. acclimated to four growth PPFDs in the field

The acclimation of photochemistry, xanthophyll cycle-dependent energy dissipation, and antioxidants was characterized in leaves of Cucurbita pepo L. and Vinca major L. that developed under photosynthetic photon flux densities (PPFDs) ranging from deep shade to full sunlight in the field. The predominant acclimatory response of leaf pigment composition was an increase in the xanthophyll cycle pool size with increasing growth PPFD. In both species, the estimated rate of thermal energy dissipation at midday increased with increasing PPFD and midday levels of zeaxanthin and antheraxanthin per chlorophyll were closely correlated with the levels of non-photochemical fluorescence quenching under all growth PPFD regimes. However, at full sunlight there appeared to be considerably higher levels of xanthophyll cycle dependent energy dissipation in V. major compared with pumpkin while estimated rates of photochemistry exhibited the reverse trend. Leaf activities of the antioxidant enzymes ascorbate peroxidase and superoxide dismutase, as well as ascorbate content, increased with increasing growth PPFD in both plant species. Activities/contents were higher under 100% full sunlight and increased more strongly from intermediate growth PPFDs to 100% full sunlight in V. major than in C. pepo. These patterns of acclimation are similar to those exhibited by xanthophyll cycle-dependent energy dissipation. The patterns of acclimation of glutathione reductase are discussed in the context of the multiple roles for reduced glutathione. Catalase acclimated in a manner consistent with its role in scavenging H2O2 generated via photorespiration and/or mitochondrial respiration. Leaf -tocopherol did not exhibit growth PPFD-dependent trends.     

Dependence of photosynthesis and energy dissipation activity upon growth form and light environment during the winter

Two very distinctive responses of photosynthesis to winter conditions have been identified. Mesophytic species that continue to exhibit growth during the winter typically exhibit higher maximal rates of photosynthesis during the winter or when grown at lower temperatures compared to individuals examined during the summer or when grown at warmer temperatures. In contrast, sclerophytic evergreen species growing in sun-exposed sites typically exhibit lower maximal rates of photosynthesis in the winter compared to the summer. On the other hand, shaded individuals of those same sclerophytic evergreen species exhibit similar or higher maximal rates of photosynthesis in the winter compared to the summer. Employment of the xanthophyll cycle in photoprotective energy dissipation exhibits similar characteristics in the two groups of plants (mesophytes and shade leaves of sclerophytic evergreens) that exhibit upregulation of photosynthesis during the winter. In both, zeaxanthin + antheraxanthin (Z + A) are retained and PS II remains primed for energy dissipation only on nights with subfreezing temperatures, and this becomes rapidly reversed upon exposure to increased temperatures. In contrast, Z + A are retained and PS II remains primed for energy dissipation over prolonged periods during the winter in sun leaves of sclerophytic evergreen species, and requires days of warming to become fully reversed. The rapid disengagement of this energy dissipation process in the mesophytes and shade sclerophytes apparently permits a rapid return to efficient photosynthesis and increased activity on warmer days during the winter. This may be associated with a decreasing opportunity for photosynthesis in source leaves relative to the demand for photosynthesis in the plant's sinks. In contrast, the sun-exposed sclerophytes – with a relatively high source to sink ratio – maintain PS II in a state primed for high levels of energy dissipation activity throughout much of the winter. Independent of whether photosynthesis was up- or downregulated, all species under all conditions exhibited higher levels of soluble carbohydrates during the winter compared to the summer. Thus downregulation of photosynthesis and of Photosystem II do not appear to limit carbohydrate accumulation under winter conditions. A possible signal communicating an altered source/sink balance, or that may be influencing the engagement of Z + A in energy dissipation, is phosphorylation of thylakoid proteins such as D1.This revised version was published online in October 2005 with corrections to the Cover Date.     

Both xanthophyll cycle-dependent thermal dissipation and the antioxidant system are up-regulated in grape (Vitis labrusca L cv Concord) leaves in response to N limitation

One-year-old grapevines (Vitis labrusca L. cv. Concord) were supplied with 0, 5, 10, 15, or 20 mM nitrogen (N) in a modified Hoagland's solution twice weekly for 4 weeks. As leaf N decreased in response to N limitation, leaf chlorophyll (Chl) decreased linearly whereas leaf absorptance declined curvilinearly. Compared with high N leaves, low N leaves had lower quantum efficiency of PSII as a result of both an increase in non-photochemical quenching (NPQ) and an increase in closure of PSII reaction centres at midday under high photon flux density (PFD). Both the xanthophyll cycle pool size on a Chl basis and the conversion of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) at noon increased with decreasing leaf N. NPQ was closely related to A+Z expressed either on a Chl basis or as a percentage of the xanthophyll cycle pool. As leaf N increased, superoxide dismutase (SOD) activity on a Chl basis decreased linearly; activities of catalase (CAT) and glutathione reductase (GR) on a Chl basis increased linearly; activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR) expressed on the basis of Chl decreased rapidly first, then gradually reached a low level. In response to N limitation, the contents of ascorbate (AsA), dehydroascorbate (DAsA), reduced glutathione (GSH), and oxidized glutathione (GSSG) increased when expressed on a Chl basis, whereas the ratios of both AsA to DAsA and GSH to GSSG decreased. It is concluded that, in addition to decreasing light absorption by lowering Chl concentration, both xanthophyll cycle-dependent thermal energy dissipation and the antioxidant system are up-regulated to protect low N leaves from photo-oxidative damage under high light.     

Resistance of cold-hardened winter rye leaves (Secale cereale L.) to photo-oxidative stress

Catalase and photosystem II (PSII) were strongly inactivated during exposure to 4 °C and moderate light in 22 °C-grown non-hardened leaves (NHL) of winter rye (Secale cereale L.), but highly resistant to photo-inactivation at low temperature in 4 °C-grown cold-hardened leaves (CHL). Resistance of CHL to chilling-induced photo-inactivation of catalase and PSII depended partially on more efficient de novo synthesis at 4 °C and partially on improved protection. Lower rates of chloroplast-mediated inactivation of catalase in vitro indicated that less reactive oxygen was released by chloroplasts from CHL than by chloroplasts from NHL. The contents of xanthophyll cycle carotenoids, α-tocopherol, ascorbate, glutathione, the activities of superoxide dismutase and glutathione reductase, and the tolerance against paraquat-induced photo-oxidative damage were greatly increased in CHL, relative to NHL. Zeaxanthin-related thermal energy dissipation was only of minor importance for paraquat-tolerance and protection of catalase in CHL. When CHL were transferred to a higher temperature of 22 °C the increased resistance to photo-inactivation of catalase and PSII and the increased paraquat-tolerance were largely lost within 3 d, whereas most non-enzymic and enzymic antioxidants retained higher levels than in NHL. The decline of resistance to photodamage during dehardening was not related to concomitant changes of antioxidants or antioxidative enzymes.     

Strategies providing success in a variable habitat: III. Dynamic control of photosynthesis in Cladophora glomerata

Diurnal patterns of photosynthesis were studied in July and April populations of Cladophora glomerata (L.) Kütz. from open and from shaded sites. Summer samples exposed to full sunlight showed decreased efficiency of open photosystem II at noon, and only slight differences were found between samples that had grown at open or at shaded sites. Electron transport rate was limited at highest fluence rates in shade plants, and non‐photochemical quenching (NPQ) revealed faster regulation in samples from open sites. Daily course of de‐epoxidation was not linearly correlated with the course of NPQ. The comparison of samples from open and from shaded sites revealed a higher capacity of thermal energy dissipation and an increase in the total amount of xanthophyll‐cycle pigments (21%) in samples from open sites. In April, down‐regulation of the efficiency of open photosystem II was related to lower water temperature, and hence, increased excitation pressure. In April the pool size of xanthophyll‐cycle pigments was increased by 21% in comparison with summer and suggested higher levels of thermal energy dissipation via de‐epoxidized xanthophylls. In both, summer and spring the amount of xanthophyll‐cycle pigments was 20% higher in samples from open sites. Acclimation of C. glomerata to growth light conditions was further shown by experimental induction of NPQ, indicating NPQ increases of 23%, and increases of 77% in the reversible component of NPQ in open site samples. The effect of temperature on photosynthetic rate was non‐linear, and different optimum temperatures of electron transport rate and oxygen evolution were exhibited.     

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.     

Winter acclimation of PsbS and related proteins in the evergreen Arctostaphylos uva-ursi as influenced by altitude and light environment

The evergreen groundcover bearberry (Arctostaphylos uva-ursi [L.] Sprengel) was characterized over two successive years (2002-2004) from both sun-exposed and shaded sites at a montane ponderosa pine and subalpine forest community of 1900- and 2800-m-high altitudes, respectively. During summer, photosynthetic capacities and pre-dawn photosystem II (PSII) efficiency were similarly high in all four populations, and in winter, only the sun-exposed and shaded populations at 2800 m exhibited complete down-regulation of photosynthetic oxygen evolution capacity and consistent sustained down-regulation of PSII efficiency. This photosynthetic down-regulation at high altitude involved a substantial decrease in PSII components [pheophytin, D1 protein, oxygen evolving complex ([OEC)], a strong up-regulation of several anti-early-light-inducible protein (Elip)- and anti-high-light-inducible protein (Hlip)-reactive bands and a warm-sustained retention of zeaxanthin and antheraxanthin (Z + A). PsbS, the protein modulating the rapid engagement and disengagement of Z +A in energy dissipation, exhibited its most pronounced winter increases in the shade at 1900 m, and thus apparently assumes a greater role in providing rapidly reversible zeaxanthin-dependent photoprotection during winter when light becomes excessive in the shaded population, which remains photosynthetically active. It is attractive to hypothesize that PsbS relatives (Elips/Hlips) may be involved in sustained zeaxanthin-dependent photoprotection under the more extreme winter conditions at 2800 m.     

Mechanistic aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts and leaves

Higher plants must dissipate absorbed light energy that exceeds the photosynthetic capacity to avoid molecular damage to the pigments and proteins that comprise the photosynthetic apparatus. Described in this minireview is a current view of the biochemical, biophysical and bioenergetic aspects of the primary photoprotective mechanism responsible for dissipating excess excitation energy as heat from photosystem II (PSII). The photoprotective heat dissipation is measured as nonphotochemical quenching (NPQ) of the PSII chlorophyll a (Chl a) fluorescence. The NPQ mechanism is controlled by the trans-thylakoid membrane pH gradient (ΔpH) and the special xanthophyll cycle pigments. In the NPQ mechanism, the de-epoxidized endgroup moieties and the trans-thylakoid membrane orientations of antheraxanthin (A) and zeaxanthin (Z) strongly affect their interactions with protonated chlorophyll binding proteins (CPs) of the PSII inner antenna. The CP protonation sites and steps are influenced by proton domains sequestered within the proteo-lipid core of the thylakoid membrane. Xanthophyll cycle enrichment around the CPs may explain why changes in the peripheral PSII antenna size do not necessarily affect either the concentration of the xanthophyll cycle pigments on a per PSII unit basis or the NPQ mechanism. Recent time-resolved PSII Chi a fluorescence studies suggest the NPQ mechanism switches PSII units to an increased rate constant of heat dissipation in a series of steps that include xanthophyll de-epoxidation, CP-protonation and binding of the xanthophylls to the protonated CPs; the concerted process can be described with a simple two-step, pH-activation model. The xanthophyll cycle-dependent NPQ mechanism is profoundly influenced by temperatures suboptimal for photosynthesis via their effects on the trans-thylakoid membrane energy coupling system. Further, low temperature effects can be grouped into either short term (minutes to hours) or long term (days to seasonal) series of changes in the content and composition of the PSII pigment-proteins. This minireview concludes by briefly highlighting primary areas of future research interest regarding the NPQ mechanism.     

The shaded side of apple fruit becomes more sensitive to photoinhibition with fruit development

Developmental changes of photochemical and non-photochemical processes and the antioxidant system in the shaded peel vs the sun-exposed peel of 'Gala' apple and their responses to sudden exposure of high light were determined to understand the susceptibility of the shaded peel to high light damage with fruit development. As fruit developed, actual PSII efficiency of the shaded peel decreased, whereas non-photochemical quenching (mainly the slow component) increased at any given PFD. Photochemical quenching coefficient of the shaded peel decreased at any given PFD with fruit development. As fruit developed, the activity of superoxide dismutase, ascorbate peroxidase and dehydroascorbate reductase and the level of reduced ascorbate and total ascorbate decreased; the activity of monodehydroascorbate reductase and glutathione reductase remained low, whereas catalase activity and the level of reduced glutathione and total glutathione increased in the shaded peel. Exposure to high light (1500 micromol m(-2) s(-1)) for 2 h significantly decreased the maximum quantum efficiency of PSII (F(V)/F(M)) in the shaded peel at each developmental stage, with the decrease being larger with fruit development. The F(V)/F(M) of the sun-exposed peel was also decreased by the high light treatment, but the decrease was much smaller than that in the shaded peel at each developmental stage. We conclude that the shaded peel of apple fruit becomes more sensitive to photoinhibition with fruit development, and this increased sensitivity is apparently related to the decease in the overall capacity for photosynthesis and photoprotection of the shaded peel with fruit development.     

Antioxidants and xanthophyll cycle-dependent energy dissipation in Cucurbita pepo L. and Vinca major L. upon a sudden increase in growth PPFD in the field

Portions of shade-acclimated shoots of Cucurbita pepo L. (pumpkin) and Vinca major L. were rapidly transferred to full sunlight exposure and chlorophyll fluorescence emission, pigment composition, antioxidant enzyme activities, ascorbate contents, and the content of thiobarbituric acid-reactive substances (TBARS) were measured at regular intervals for 17 d. The most notable response of leaf pigment composition was a pronounced increase in the pool size of xanthophyll cycle carotenoids that occurred over a period of 4 d (pumpkin) or 11-18 d (V. major). On day 1 after the transfer midday efficiencies of open PSII units decreased to levels similar to or below those observed in full sun-acclimated leaves. Efficiencies of open PSII units were inversely correlated with xanthophyll cycle conversion states in both species on all dates of sampling. A rapid increase in thermal energy dissipation on day 1 may thus have contributed to the absence of either an increase in the content of TBARS (a measure of lipid damage) or pronounced depressions in pre-dawn Fv/Fm. Activities of the antioxidant enzymes ascorbate peroxidase, superoxide dismutase, glutathione reductase, and catalase as well as ascorbate content increased in both species upon transfer, with superoxide dismutase exhibiting the most dramatic increase. Pumpkin, but not V. major, developed new leaves during the study which possessed the attributes of sun-acclimated leaves.     

Acclimation of Foliar Antioxidant Systems to Growth Irradiance in Three Broad-Leaved Evergreen Species

The protective role of leaf antioxidant systems in the mechanism of plant acclimation to growth irradiance was studied in Vinca major, Schefflera arboricola, and Mahonia repens, which were grown for several months at 20, 100, and 1200 [mu]mol photons m-2 s-1. As growth irradiance increased, several constituents of the "Mehler-peroxidase" pathway also increased: superoxide dismutase, ascorbate peroxidase, glutathione reductase, ascorbate, and glutathione. This occurred concomitantly with increases in the xanthophyll cycle pool size and in the rate of nonphotochemical energy dissipation under steady-state conditions. There was no evidence for photosystem II overreduction in plants grown at high irradiance, although the reduction state of the stromal NADP pool, estimated from measurements of NADP-malate dehydrogenase activity, was greater than 60% in V. major and S. arboricola. Ascorbate, which removes reactive O2 species generated by O2 photoreduction in the chloroplast and serves as a reductant for the conversion of the xanthophyll cycle pigments to the de-epoxidized forms A plus Z, generally exhibited the most dramatic increases in response to growth irradiance. We conclude from these results that O2 photoreduction occurs at higher rates in leaves acclimated to high irradiance, despite increases in xanthophyll cycle-dependent energy dissipation, and that increases in leaf antioxidants protect against this potential oxidative stress.     

Effects of aluminum on light energy utilization and photoprotective systems in citrus leaves

* BACKGROUND AND AIMS: Under high photon flux, excitation energy may be in excess in aluminum (Al)-treated leaves, which use a smaller fraction of the absorbed light in electron transport due to decreased CO2 assimilation compared with normal leaves. The objectives of this study were to test the hypothesis that the antioxidant systems are up-regulated in Al-treated citrus leaves and correlate with protection from photoxidative damage, and to test whether xanthophyll cycle-dependent thermal energy dissipation is involved in dissipating excess excitation energy. * METHODS: 'Cleopatra' tangerine seedlings were fertilized and irrigated daily for 8 weeks with quarter-strength Hoagland's nutrient solution containing Al at a concentration of 0 or 2 mM from Al2(SO4)3.18H2O. Thereafter, leaf absorptance, chlorophyll (Chl) fluorescence, Al, pigments, antioxidant enzymes and metabolites were measured on fully expanded leaves. * KEY RESULTS: Compared with control leaves, energy was in excess in Al-treated leaves, which had smaller thermal energy dissipation, indicated by non-photochemical quenching (NPQ). In contrast, conversion of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) at midday increased in both treatments, but especially in Al-treated leaves, although A + Z accounted for less 40 % of the total xanthophyll cycle pool in them. Activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and catalase (CAT), and concentrations of ascorbate (AsA), dehydroascorbate (DASA), reduced glutathione (GSH) and oxidized glutathione (GSSG) were higher in Al-treated than in control leaves. * CONCLUSIONS: These results corroborate the hypothesis that, compared with control leaves, antioxidant systems are up-regulated in Al-treated citrus leaves and protect from photoxidative damage, whereas thermal energy dissipation was decreased. Thus, antioxidant systems are more important than thermal energy dissipation in dissipating excess excitation energy in Al-treated citrus leaves.     

140 Ecophysiology of The Red Alga Porphyra Umbilicalis Kützing (Rhodophyta,Bangalies): Responses to Abiotic Stress

The effects of tidal elevation, emersion, sun exposure, and season on several antioxidant enzymes (ascorbate peroxidase, glutathione reductase, and catalase), pigments (phycoerythrin, phycocyanin, chlorophyll a and total carotene) and photosynthetic efficiency of photosystem II (Fv/Fm) in Porphyra umbilicalis were evaluated. Plants were collected monthly from sun‐exposed and shaded locations in the high, mid, and low intertidal following periods of tidal emersion ranging from 0–6 hours. Glutathione reductase activity was greatly affected by emersion during summer months, while ascorbate peroxidase and catalase activities showed no seasonal patterns. Differences in glutathione reductase and catalase levels occurred between sun‐exposed and shaded plants in the high and mid intertidal during summer. At all elevations, photosynthetic pigments showed a strong seasonal trend, with the effect of sun exposure being most apparent during summer. While total carotene increased with emersion during summer months, the combined effects of emersion and season were inconsistent for phycoerythrin, phycocyanin and chl a. Photosynthetic efficiency (Fv/Fm) decreased following emersion in summer and fall. During most months, sun exposed plants had lower Fv/Fm values compared to plants growing in the shade. This study emphasizes the importance of examining the effects of abiotic stresses simultaneously in order to reveal interactive relationships.     

141 New Entities in the Order Chaetopeltidales

The effects of tidal elevation, emersion, sun exposure, and season on several antioxidant enzymes (ascorbate peroxidase, glutathione reductase, and catalase), pigments (phycoerythrin, phycocyanin, chlorophyll a and total carotene) and photosynthetic efficiency of photosystem II (Fv/Fm) in Porphyra umbilicalis were evaluated. Plants were collected monthly from sun-exposed and shaded locations in the high, mid, and low intertidal following periods of tidal emersion ranging from 0–6 hours. Glutathione reductase activity was greatly affected by emersion during summer months, while ascorbate peroxidase and catalase activities showed no seasonal patterns. Differences in glutathione reductase and catalase levels occurred between sun-exposed and shaded plants in the high and mid intertidal during summer. At all elevations, photosynthetic pigments showed a strong seasonal trend, with the effect of sun exposure being most apparent during summer. While total carotene increased with emersion during summer months, the combined effects of emersion and season were inconsistent for phycoerythrin, phycocyanin and chl a. Photosynthetic efficiency (Fv/Fm) decreased following emersion in summer and fall. During most months, sun exposed plants had lower Fv/Fm values compared to plants growing in the shade. This study emphasizes the importance of examining the effects of abiotic stresses simultaneously in order to reveal interactive relationships.     

The xanthophyll cycle and energy dissipation in differently oriented faces of the cactus Opuntia macrorhiza

  Diurnal changes in titratable acidity, photosynthesis, energy dissipation activity, and the carotenoid composition of differently oriented cladodes of the cactus Opuntia macrorhiza were characterized during exposure to full sunlight in the field. Four cladode faces were chosen such that each was exposed to maximum photon flux densities (PFD) at different times of the day in addition to receiving different daily integrated PFDs. The sum of all carotenoids per chlorophyll was found to increase with increasing exposure to PFD, with the carotenoids of the xanthophyll cycle present in the most exposed face at more than twice the concentration found in the least exposed face. All faces exhibited large increases in xanthophyll cycle-dependent energy dissipation as the sun rose in the morning, even those receiving only minimal levels of diffuse radiation. The transient high levels of energy dissipation in those faces that did not receive direct sunlight in the morning may have been due to low temperature inhibition of photosynthesis (predawn low of 2°C). For the two faces receiving peak PFDs in the morning hours (north and east faces), the level of energy dissipation activity increased rapidly during exposure to direct sunlight in the early morning, gradually declining in the late morning under warm temperatures, and was negligible during the afternoon low light conditions. Changes in the xanthophyll cycle paralleled the changes in energy dissipation with the majority of the cycle present as violaxanthin (V) prior to sunrise, largely de-epoxidized to zeaxanthin (Z) and antheraxanthin (A) during exposure to direct sunlight, and reconverted to V during the afternoon. For the two faces receiving peak PFDs in the afternoon (south and west faces), energy dissipation activity increased dramatically during the early morning low light period, subsequently decreasing during midday as decarboxylation of malic acid proceeded maximally (providing a high concentration of CO₂ for photosynthesis), and then increased to the highest level in the late afternoon as the supply of malic acid was depleted and rates of photosynthetic electron transport declined. The xanthophyll cycle, largely present as Z and A prior to sunrise in the south and west faces, was de-epoxidized to the greatest extent in the late afternoon, followed by epoxidation back to the predawn level by sunset. In all cladode faces high levels of energy dissipation activity were accompanied by decreases in the intrinsic efficiency of photosystem II (PSII), indicative of a regulatory process that diverted the excess energy away from the reaction centers during periods of excess light. Furthermore, the overnight retention of Z and A by the south and west faces was accompanied by a sustained reduction in PSII efficiency (i.e., “photoinhibition”). We suggest that this “photoinhibition” represents the sustained engagement of nocturnally retained Z and A in the photoprotective down-regulation of PSII. Received: 8 May 1996 / Accepted: 9 September 1996     

Two forms of sustained xanthophyll cycle-dependent energy dissipation in overwintering Euonymus kiautschovicus

Seasonal differences in PSII efficiency (F_v/F_m), the conversion state of the xanthophyll cycle (Z + A)/ (V + A + Z), and leaf adenylate status were investigated in Euonymus kiautschovicus. On very cold days in winter, F_v/F_m assessed directly in the field remained low and Z + A high throughout day and night in both sun and shade leaves. Pre-dawn transfer of leaves from subfreezing temperatures in the field to room temperature revealed that recovery (increases in F_v/F_m and conversion of Z + A to violaxanthin) consisted of one, rapid phase in shade leaves, whereas in sun leaves a rapid phase was followed by a slow phase requiring days. The pre-dawn ATP/ADP ratio, as well as that determined at midday, was similar when comparing overwintering leaves with those sampled in the summer, although pre-dawn levels of ATP + ADP were elevated in all leaves during winter relative to summer. After a natural transition to warmer days during the winter, pre-dawn F_v/F_m and Z + A in shade leaves had returned to values typical for summer, whereas in sun leaves F_v/F_m and Z + A levels remained intermediate between the cold day in winter and the summer day. Thus two distinct forms of sustained (Z + A)-dependent energy dissipation were identified based upon their differing characteristics. The form that was sustained on cold days but relaxed rapidly upon warming occurred in all leaves and may result from maintenance of a low lumenal pH responsible for the nocturnal engagement of (Z + A)-dependent thermal dissipation exclusively on very cold days in the winter. The form that was sustained even upon warming and correlated with slow Z + A to violaxanthin conversion occurred only in sun leaves and may represent a sustained engagement of (Z + A)-dependent energy dissipation associated with an altered PSII protein composition. In the latter, warm-sustained form, uncoupler or cycloheximide infiltration had no effect on the slow phase of recovery, but lincomycin infiltration inhibited the slow increase in F_v/F_m and the conversion of Z + A to violaxanthin.     

Close relationship between the state of the xanthophyll cycle pigments and photosystem II efficiency during recovery from winter stress

The potential involvement of the xanthophyll cycle in photoprotection of overwintering evergreen plants was investigated. Leaves from five evergreen species. Pseudotsuga menziesii, Pinus panderosa, Euonyums kiautschovicus. Mahonia repens and Malva neglecta, were collected from the field predawn during winter and transferred to the laboratory where chlorophyll fluorescence emission as well as the chlorophyll and carotenoid composition were ascertained periodically for 4.5 days. Leaves and needles from all species were found to have retained large amounts of the xanthophyll cycle pigments zeaxanthin and antheraxanthin, and they exhibited sustained low values of the intrinsic efficiency of photosystem II (PSII; measured as the ratio of variable to maximal fluorescence, F_v/F_m) upon collection. The increase in PSII efficiency was biphasic, with a rapid phase (requiring several hours) and a slow phase (requiring several days). Changes in the conversion state of the xanthophyll cycle were found to correlate with increases in PSII efficiency in both phases, with the latter phase involving large increases in both F_m (maximal fluorescence) and F_o (minimal fluorescence) throughout the period of recovery. The relationship between F_m quenching (expressed as nonphotochemical or Stern-Volmer quenching [NPQ] of F_m, i.e. F_m/ F_m–1) and F_o quenching (F_o/F_o–1) was linear, as expected for changes in xanthophyll cycle-dependent energy dissipation in the antenna complexes. Furthermore, the relationship between F_v/F_m and NPQ during recovery followed the theoretical relationship predicted for changes in the rate constant for energy dissipation in the antenna complexes. This fit between the theoretical relationship and the actual data indicates that all changes in NPQ or F_v/F_m can be accounted for by changes in this rate constant. The results suggest a role for the photoprotective xanthophyll cycle-dependent dissipation process in the lowered efficiency of PSII observed in coldstressed evergreen plants in the field.     

Short-term changes in free radical scavengers and chloroplast pigments in Pinus canariensis needles as affected by mild drought stress

Potted Pinus canariensis seedlings were subjected to mild drought by withholding irrigation for one week. This treatment induced a reduction in maximum stomatal conductance (50 mmol m⁻² s⁻¹) compared to irrigated controls (130 mmol m⁻² s⁻¹). Needle water potentials of non-irrigated trees were maintained at control level (-0.44 MPa). Such a mild drought is a potential oxidative stressor due to the production of active oxygen species (AOS) in illuminated chloroplasts which lack CO₂ due to stomatal closure. Photoprotective pigments (e.g. the xanthophyll cycle) may avoid this situation through light energy dissipation, and antioxidants such as ascorbic acid, tocopherols, and glutathione, may detoxify AOS. Concentrations of ascorbate, glutathione, chlorophyll, and the xanthophyll cycle carotenoids were minimal in the evening (under low light) compared to light-saturated conditions. α-Carotene was highest in the evening. These short-term changes were not affected by drought. The xanthophyll cycle pool tended to be more de-epoxidized in strongly illuminated needles of non-irrigated trees at the beginning of the experiment, but this effect was transient. The glutathione pool was more oxidized in needles of non-irrigated trees (up to 20 percnt; of total vs. 10 percnt; at control) after the xanthophyll changes took place, whereas the redox state of ascorbate remained stable.     

Significance of antioxidants and electron sinks for the cold-hardening-induced resistance of winter rye leaves to photo-oxidative stress

The contents of ascorbate and glutathione and the activities of superoxide dismutase and glutathione reductase were increased to levels as high as those in cold-hardened leaves (CHL) by incubating non-hardened leaves (NHL) of winter rye (Secale cereale L.) with the precursor substrates L -galactonic acid-γ-lactone and 2-oxothiazolidine-4-carboxylate. Reduced glutathione was rapidly depleted from NHL after application of D , L -buthionine sulfoximine, an inhibitor of its biosynthesis. In spite of greatly divergent antioxidant contents the rates of photo-inactivation of photosystem II (PSII) and catalase observed in the presence of translation inhibitors did not differ greatly. The paraquat-induced catalase inactivation and chlorophyll degradation in light were reduced in NHL with increased antioxidant levels. Paraquat-induced photo-inactivation of PSII was, however, not mitigated. The CHL had a higher capacity to prevent paraquat-induced oxidation of ascorbate and glutathione than NHL with increased antioxidant contents. Increased antioxidant contents did not establish resistance to low temperature-induced photo-inactivation of PSII and catalase in NHL. The resistance of CHL to low temperature-induced photo-inactivation of PSII and catalase required repair at low temperature and active carbon assimilation but was only little affected when photorespiration was suppressed by phosphinothricin. Protection of PSII depended also on non-photochemical quenching of excitation energy.