Xanthophyll Cycle-dependent Energy Dissipation Research Articles

Thioredoxin-like protein CDSP32 alleviates Cd-induced photosynthetic inhibition in tobacco leaves by regulating cyclic electron flow and excess energy dissipation

Thioredoxin-like protein CDSP32 (Trx CDSP32), a thioredoxin-like (Trx-like) protein located in the chloroplast, can regulate photosynthesis and the redox state of plants under stress. In order to examine the role of Trx CDSP32 in the photosynthetic apparatus of plants exposed to cadmium (Cd), the effects of Trx CDSP32 on photosynthetic function and photoprotection in tobacco leaves under Cd exposure were studied using a proteomics approach with wild-type (WT) and Trx CDSP32 overexpression (OE) tobacco plants. Cd exposure reduced stomatal conductance, blocked PSII photosynthetic electron transport, and inhibited carbon assimilation. Increased water use efficiency (WUE), cyclic electron flow (CEF) of the proton gradient regulation 5 pathway (PGR5-CEF), and regulated energy dissipation [Y(NPQ)] are important mechanisms of Cd adaptation. However, CEF of the NAD(P)H dehydrogenase pathway (NDH-CEF) was inhibited by Cd exposure. Relative to control conditions, the expression levels of violaxanthin de-epoxidase (VDE) and photosystem II 22 kDa protein (PsbS) in OE leaves were significantly increased under Cd exposure, but those in WT leaves did not change significantly. Moreover, the expression of zeaxanthin epoxidase (ZE) under Cd exposure was significantly higher than that in WT leaves. Thus, Trx CDSP32 increased Y(NPQ) and alleviated PSII photoinhibition under Cd exposure. Trx CDSP32 not only increased PGR5-like protein 1A and 1B expression, but also alleviated the down-regulation of NAD(P)H-quinone oxidoreductase subunits induced by Cd exposure. Thus, Trx CDSP32 promotes CEF in Cd-exposed tobacco leaves. Thus, Trx CDSP32 alleviates the Cd-induced photoinhibition in tobacco leaves by regulating two photoprotective mechanisms: CEF and xanthophyll cycle-dependent energy dissipation.

Photosynthetic and Photoprotective Responses to Steady-State and Fluctuating Light in the Shade-Demanding Crop Amorphophallus xiei Grown in Intercropping and Monoculture Systems.

Photosynthetic and photoprotective responses to simulated sunflecks were examined in the shade-demanding crop Amorphophallus xiei intercropped with maize (intercropping condition) or grown in an adjacent open site (monoculture condition). Both intercropping leaves and monoculture leaves exhibited very fast induction responses. The times taken to achieve 90% maximum net photosynthetic rate in intercropping leaves and monoculture leaves were 198.3 ± 27.4 s and 223.7 ± 20.5 s during the photosynthetic induction, respectively. During an 8-min simulated sunfleck, the proportion of excess excited energy dissipated through the xanthophyll cycle-dependent pathway (ΦNPQ) and dissipated through constitutive thermal dissipation and the fluorescence (Φf, d) pathway increased quickly to its maximum, and then plateaued slowly to a steady state in both intercropping and monoculture leaves. When the illumination was gradually increased within photosystem II (PSII), ΦNPQ increased quicker and to a higher level in monoculture leaves than in intercropping leaves. Relative to their monoculture counterparts, intercropping leaves exhibited a significantly lower accumulation of oxygen free radicals, a significantly higher content of chlorophyll, and a similar content of malondialdehyde. Although monoculture leaves exhibited a larger mass-based pool size of xanthophyll cycle [V (violaxanthin) + A (antheraxanthin) + Z (zeaxanthin)] than intercropping leaves, intercropping leaves had a higher ratio of (Z + A)/(V + Z + A) than monoculture leaves. intercropping leaves had markedly higher glutathione content and ascorbate-peroxidase activity than their monoculture counterparts. Similar activities of catalase, peroxidase, dehydroascorbate reductase, and monodehydroascorbate were found in both systems. Only superoxide dismutase activity and ascorbate content were lower in the intercropping leaves than in their monoculture counterparts. Overall, the xanthophyll cycle-dependent energy dissipation and the enzymatic antioxidant defense system are important for protecting plants from photooxidation in an intercropping system with intense sunflecks.

Physiological Mechanisms Underlying Fruit Sunburn

Sunburn is a physiological disorder that can be observed in fruits of several crops growing in areas with warm climates, as a result of photodamage due to an excess of heat and/or light irradiance (visible and ultraviolet light). The main cause is thought to be an increase in reactive oxygen species production which causes oxidative damage due to the incapacity of the fruit to recover from stress. This can result in a characteristic morphological and structural phenotype unacceptable to consumers, leading to severe losses in productivity for farmers. Fruits have a great array of mechanisms to mitigate or reduce reactive oxygen species production and the inactivation of photosynthetic apparatus, such as enhanced xanthophyll cycle-dependent energy dissipation, accumulation of photoprotective pigments and heat-shock proteins, and the biosynthesis of antioxidants, among others. Nevertheless, these mechanisms become inefficient when the stress factors altering the fruit surface exceed a certain threshold (of both duration and intensity). Although this disorder has been studied in detail and previous efforts have provided significant advances in understanding the underlying mechanisms causing sunburn in a number of fruits, further research is still needed. This will undoubtedly provide new approaches and tools for improving current mitigation strategies.

Peanut violaxanthin de-epoxidase alleviates the sensitivity of PSII photoinhibition to heat and high irradiance stress in transgenic tobacco.

This is the first study on peanut VDE, which led to multiple biochemical and physiological changes to heat and HI stress by improving de-epoxidation of the xanthophylls cycle. A peanut (Arachis hypogaea L.) violaxanthin de-epoxidase gene (AhVDE) was isolated by RT-PCR and RACE methods. The deduced amino acid sequence of AhVDE showed high identities with violaxanthin de-epoxidase of other plant species. The expression of AhVDE was obviously upregulated by 4, 40°C and high light, NaCl, and abscisic acid. Sense and RNAi transgenic tobaccos were further used to investigate the physiological effects and functional mechanism of AhVDE. Compared with WT, the content of Z, the ratio of (A+Z)/(V+A+Z) and the non-photochemical quenching were higher in sense plants, and lower in the RNAi lines under heat and high irradiance (HI) stress, respectively. Additionally, photoinhibition of photosystem II (PSII) reflected by the maximal photochemical efficiency in WT lines was more severe, and in the RNAi lines was the most severe compared with that in the sense lines. Meanwhile, overexpressing AhVDE also led to multiple biochemical and physiological changes under heat and HI stress. Higher activities of superoxide dismutase and ascorbate peroxidase, lower content of reactive oxygen species and slighter membrane damage were observed in sense lines after heat and HI stress. These results suggested that, peanut VDE can alleviate PSII photoinhibition to heat and HI stress by improving the xanthophyll cycle-dependent energy dissipation.

Leaf chlorophyll fluorescence, reflectance, and physiological response to freshwater and saltwater flooding in the evergreen shrub, Myrica cerifera

Photosynthesis, water relations, chlorophyll fluorescence, and leaf reflectance were used to evaluate stress due to freshwater and saltwater flooding in the evergreen coastal shrub, Myrica cerifera, under controlled conditions. M. cerifera forms large monospecific thickets that facilitate scaling up from leaf-level measurements to the landscape. Based on physiological responses, stress began by day 3 in flooded plants treated with 5, 10, and 15 g L −1 salinity, as seen by significant decreases in stomatal conductance and net photosynthesis relative to control plants. Decreases in physiological measurements occurred by day 9 in freshwater flooded plants. Visible signs of stress occurred by day 5 for plants treated with 15 g L −1, day 8 for flooded plants exposed to 10 g L −1, and day 10 for those treated with 5 g L −1 salinity. Significant differences in light-adapted fluorescence yield ( Δ F / F ′ m ) were observed by day 3 in plants flooded with 5, 10, and 15 g L −1 salinity and day 6 in freshwater flooded plants. Non-photochemical quenching ( Φ NPQ) increased with decreasing Δ F / F ′ m . In comparison, statistical differences in dark-adapted fluorescence yield ( F v/ F m) were observed by day 12 in plants flooded with 5, 10, and 15 g L −1 salinity, well after visible signs of stress were apparent. Fluorescence parameters were successful at detecting and distinguishing both freshwater and saltwater flooding stress. A positive, linear correlation ( r 2 = 0.80) was observed between Δ F / F ′ m and the physiological reflectance index (PRI). Xanthophyll-cycle dependent energy dissipation appears to be the underlying mechanism in protecting photosystem II from excess energy in saltwater flooded plants. Δ F / F ′ m was useful in detecting stress-induced changes in the photosystem before any visible signs of damage were evident at the leaf-level. This parameter may be linked to hyperspectral reflectance data for rapid detection of stress at the canopy-level.

Alternate energy dissipation? Phenolic metabolites and the xanthophyll cycle

The dynamics of phenolic galloylglucoses (di-, tri-, tetra- and penta-galloylglucose), flavonoids (quercitin and quercitin glycosides) and sideroxylonal were compared with that of xanthophyll cycle-dependent energy dissipation during rapid induction of chilling-dependent photo-inhibition. Pre-dawn xanthophyll cycle engagement of seedlings of Eucalyptus nitens transferred from mild nursery conditions to a low temperature controlled environment increased logarithmically during eight days of treatment. Photochemical efficiency and flavonoids decreased after four days of treatment and non-photochemical quenching after two days of treatment. Galloylglucoses and sideroxylonal decreased linearly during treatment. These results demonstrate that rapid changes in foliar phenolic levels are associated with abrupt changes in the plant environment. It is argued that under these growth-chamber conditions, the xanthophyll cycle facilitated dissipation of excess light energy, lessening the requirement for the dissipation of energy or antioxidant activity through phenolic metabolites.

Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation.

High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly 'photoinhibited', leaves. Functional implications of these observations are discussed.

Transgenic tobacco with suppressed zeaxanthin formation is susceptible to stress-induced photoinhibition.

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with the antisense construct of tobacco violaxanthin de-epoxidase was analyzed for responses in growth chambers to both short and long-term stress treatments. Following a short-term (2 or 3 h) high-light treatment, antisense plants had a greater reduction in F(v)/F(m) relative to wild-type, indicating a greater susceptibility to photoinhibition. The responses of antisense plants to long-term stress were examined in two separate experiments, one with high light alone and the other wherein high light and water stress were combined. In the light-stress experiment, plants were grown at 1300 mumol photons m(-2) s(-1) under a 12 h photoperiod. In the light and water-stress experiment, plants were grown under moderately high light of 900 mumol photons m(-2) s(-1), under a 16 h photoperiod, in combination with water stress. Both conditions caused formation of high antheraxanthin and zeaxanthin levels in wild-type plants but not in antisense plants. In both cases, antisense plants showed significant reductions in F(v)/F(m) and total leaf-pigment content relative to wild-type. The data demonstrate a critical photoprotective function of the xanthophyll cycle-dependent energy dissipation in tobacco exposed suddenly to high amounts of excess light over extended times.

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 cycledependent 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 photo respiration and/or mitochondrial respiration. Leaf α-tocopherol did not exhibit growth PPFD-dependent trends.

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

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

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 predawn F v /F m . 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.

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

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

Seasonal differences in xanthophyll cycle characteristics and antioxidants in Mahonia repens growing in different light environments.

We investigated differences between summer and winter in photosynthesis, xanthophyll cycle-dependent energy dissipation, and antioxidant systems in populations of Mahonia repens (Lindley) Don growing in the eastern foothills of the Colorado Rocky Mountains in deep shade, full exposure, and under a single-layered canopy of Pinus ponderosa (partially shaded). In summer, increasing growth irradiance (from deep shade to partial shade to full exposure) was associated with increased xanthophyll cycle-dependent energy dissipation in PSII and an increased capacity to detoxify reactive reduced oxygen species, as measured by increases in the activities of ascorbate peroxidase, superoxide scavenging, glutathione reductase, and monodehydroascorbate reductase, as well as increases in leaf ascorbate and glutathione content. Leaves of exposed and partially shaded plants exhibited decreased capacities for photosynthetic O2 evolution in winter compared to summer, while in the deeply shaded plants this parameter did not differ seasonally. Seasonal differences in the levels of antioxidants generally exhibited an inverse response to photosynthesis, being higher in winter compared to summer in the exposed and partially shaded populations, but remaining unchanged in the deeply shaded population. In addition, total pool size and conversion state of the xanthophyll cycle were higher in winter than in summer in all populations. These trends suggest that both xanthophyll cycle-dependent energy dissipation in PSII and the capacity to detoxify reactive reduced oxygen species responded to the level of excess light absorption.

9-Aminoacridine and dibucaine exhibit competitive interactions and complicated inhibitory effects that interfere with measurements of ΔpH and xanthophyll cycle-dependent Photosystem II energy dissipation

This study concerns measurements and interpretations of the trans-thylakoid membrane pH gradient, ΔpH, and xanthophyll cycle-dependent energy dissipation in Photosystem II (PS II). Compared and contrasted are the concentration-dependent inhibitory effects and interactions between two lipophilic tertiary amines, namely, 9-aminoacridine the ΔpH indicator and dibucaine a local anesthetic reported to inhibit both the ΔpH and xanthophyll cycle deepoxidation. Chlorophyll a fluorescence monitored both electron transport efficiency and xanthophyll cycle-dependent energy dissipation, high-performance liquid chromatography monitored deepoxidase and chloroplast ATPase activities and steady-state fluorescence monitored various activities of the amines in solution. Low concentrations (up to 2 μM) of both 9-aminoacridine and dibucaine showed similar fluorescence properties and ΔpH-dependent uptake into thylakoids. Importantly both amines exhibited mutually competitive inhibitory effects with respect to this ΔpH-dependent uptake and fluorescence quenching. The fluorescence yields of both compounds in aqueous solution were strongly quenched by sodium ascorbate, a necessary cofactor for in vitro deepoxidation. Both compounds similarly inhibited several light induced activities including deepoxidation, photosynthetic electron transport and PS II energy dissipation. However, for all these activities 9-aminoacridine was 2 to 5 times more potent. Importantly, 9-aminoacridine inhibited deepoxidation with an I50≈1 μM, a concentration far below that which inhibits the ΔpH, ATP synthesis/hydrolysis or electron transport. The inhibitory effects of both compounds on PS II energy dissipation were exerted at 3 to 5 times lower concentration if added before as opposed to after a saturating level of deepoxidation. This result confirms the important role for deepoxidation in mediating PS II energy dissipation. Compared to 9-aminoacridine and in contrast to similar effects on the light-induced activities, dibucaine exhibited significantly different inhibitory effects on ATPase activity and ATPase mediated PS II energy dissipation. However, we conclude from the more potent inhibition by 9-aminoacridine and the similar inhibitory patterns of all the light-induced activities that neither 9-aminoacridine nor dibucaine possess unique capacities to neutralize the light-mediated ΔpH. DCMU–3-(3,4-dichlorophenyl)-1,1-dimethylurea; DTT–dithiothreitol; fx–fractional intensity of fluorescence lifetime component x; F(′)m–maximal PS II Chl a fluorescence intensity with all QA reduced in the absence (presence) of thylakoid membrane energization; Fo–minimal PS II Chl a fluorescence intensity with all QA oxi dized; Fs–steady state PS II Chl a fluorescence; HPLC–high performance liquid chromatography; I(o)–intensity of fluorescence in the presence (absence) of quencher; Ka–association constant between Z (and A) and protonated PS II units; LA–local anesthetic; NaAsc–sodium ascorbate; NR–neutral red; PAM–pulse-amplitude modulation fluorometer; PFD–photon-flux density, μmols photons m-2 s-1; PS I–Photosystem I; PS II–Photosystem II; [PS II-+]–concentration of PS II units with inactive/deprotonated (active/protonated ) xanthophyll binding sites; [PS IItot]–total concentration of PS II units; [PS II+-Z]–concentration of PS II units with Z or A bound; Q–fraction of fluorescence intensity that is quenched; Qmax–fraction of fluorescence intensity that is quenched under control conditions; QA–primary quinone electron acceptor of PS II; V–violaxanthin; Z–zeaxanthin; 9AA–9-aminoacridine; ΔpH–trans-thylakoid membrane proton gradient; τf–lifetime of Chl a fluorescence

Photochemistry and xanthophyll cycle-dependent energy dissipation in differently oriented cladodes of Opuntia stricta during the winter

The photosynthetic and energy dissipation responses of four differently oriented photosynthetic surfaces (cladodes) from the cactus Opuntia stricta (Haw.) Haw. were studied in the field during the winter in Australia. Even under very low PFD (i.e. -2 s-1) all surfaces experienced a dramatic decline in photosystem II (PSII) efficiency during the morning period when temperatures were below freezing. However, light energy absorbed during the warmer afternoon period was more efficiently utilised for photochemistry with less diversion through the thermal energy dissipation pathway. Low temperature presumably reduced the proportion of excitation energy that could be utilised photosynthetically, resulting in a high rate of energy dissipation with a concomitant decline in PSII efficiency. A lag in the diurnal de-acidification of malic acid, and therefore the availability of endogenous CO2, may have also contributed to the low rate of photochemistry during the morning period. We interpret the increase in energy dissipation and decline in PSII efficiency as a controlled response of PSII that is dependent upon the de-epoxidised components of the xanthophyll cycle under conditions when the absorption of light exceeds the capacity of the photosynthetic apparatus to process the excitation energy through photochemistry.

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.

Effects of Nitrogen on the Photosynthetic Apparatus of Clematis vitalba Grown at Several Irradiances

Effects of nitrogen supply (N-supply) on the photosynthetic apparatus of Clematis vitalba L. grown at several irradiances were determined by measuring soluble protein content, rubisco activity, photosynthetic pigment content and composition, and the photochemical efficiency of photosystem II (Fv/Fm). Compared to low irradiance (3 and 10% full sunlight), leaves grown at higher irradiance (up to full sunlight) had up to 5–6 times the soluble protein content and rubisco activity, and up to 2–4 times the total carotenoid content, on both a leaf area and a chlorophyll basis. On a leaf area basis, decreased N-supply reduced soluble protein concentration, rubisco activity and total carotenoid concentration to a greater extent at high compared to low irradiance. On a chlorophyll basis, in contrast, soluble protein and rubisco activity decreased by over 40% with increased N-supply (1.0–0.1 mol m-3) at high irradiance but N-supply did not influence the concentration of total carotenoids. Leaves grown at high compared to low irradiance had a greater concentration of xanthophyll cycle pigments (V+A+Z), β-carotene and lutein (but not neoxanthin) on a chlorophyll basis, and a slightly lower Fv/Fm. Nitrogen- supply did not influence the composition of the photosynthetic pigment pool, Fv/Fm, or the extent of de-epoxidation of the V+A+Z pool. The results suggest that irradiance-acclimation of C. vitalba can occur regardless of N-supply. Under N limitation at high irradiance, a balance between light capture and photosynthetic capacity is important rather than an increase in xanthophyll cycle-dependent energy dissipation. The importance of lutein as a light-harvesting pigment is questioned. A rapid method for the reversed phase-HPLC separation of carotenoids is described.

The Response of Xanthophyll Cycle-dependent Energy Dissipation in Alocasia brisbanensis to Sunflecks in a Subtropical Rainforest

The photosynthetic responses of leaves of Alocasia brisbanensis (F.M. Bailey) Domin (Araceae) to sunflecks were monitored via chlorophyll fluorescence beneath the canopy of a subtropical rainforest in Australia. Additionally, the size and conversion state of the xanthophyll cycle were determined. Acclimation to understory environments that regularly experienced sunflecks involved small increases in the size of the xanthophyll cycle pool in comparison to understory plants that never received sunflecks. In understory plants that regularly experienced sunflecks the rate of photochemistry and the level of xanthophyll cycle-dependent energy dissipation closely tracked changes in incident PFD. Subsequent to the first sunfleck plants tended to retain their pool of xanthophyll cycle carotenoids as the deepoxidised forms (antheraxanthin and zeaxanthin) throughout the day. Retention of these deepoxidised forms apparently allows the trans-thylakoid membrane proton gradient to engage and disengage dissipation rapidly in response to a sunfleck, thereby mitigating photooxidative damage and ensuring a rapid return to efficient light utilisation via photosynthesis in limiting light. Our results were also in agreement with previous studies that demonstrated a requirement for light activation of photosynthesis.

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.

Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants: Photochemical quenching and xanthophyll cycle-dependent nonphotochemical quenching of fluorescence

Photosystem II (PS II) chlorophyll (Chl) a fluorescence lifetimes were measured in thylakoids and leaves of barley wild-type and chlorina f104 and f2 mutants to determine the effects of the PS II Chl a+b antenna size on the deexcitation of absorbed light energy. These barley chlorina mutants have drastically reduced levels of PS II light-harvesting Chls and pigment-proteins when compared to wild-type plants. However, the mutant and wild-type PS II Chl a fluorescence lifetimes and intensity parameters were remarkably similar and thus independent of the PS II light-harvesting antenna size for both maximal (at minimum Chl fluorescence level, Fo) and minimal rates of PS II photochemistry (at maximum Chl fluorescence level, Fm). Further, the fluorescence lifetimes and intensity parameters, as affected by the trans-thylakoid membrane pH gradient (ΔpH) and the carotenoid pigments of the xanthophyll cycle, were also similar and independent of the antenna size differences. In the presence of a ΔpH, the xanthophyll cycle-dependent processes increased the fractional intensity of a Chl a fluorescence lifetime distribution centered around 0.4-0.5 ns, at the expense of a 1.6 ns lifetime distribution (see Gilmore et al. (1995) Proc Natl Acad Sci USA 92: 2273-2277). When the zeaxanthin and antheraxanthin concentrations were measured relative to the number of PS II reaction center units, the ratios of fluorescence quenching to [xanthophyll] were similar between the wild-type and chlorina f104. However, the chlorina f104, compared to the wild-type, required around 2.5 times higher concentrations of these xanthophylls relative to Chl a+b to obtain the same levels of xanthophyll cycle-dependent fluorescence quenching. We thus suggest that, at a constant ΔpH, the fraction of the short lifetime distribution is determined by the concentration and thus binding frequency of the xanthophylls in the PS II inner antenna. The ΔpH also affected both the widths and centers of the lifetime distributions independent of the xanthophyll cycle. We suggest that the combined effects of the xanthophyll cycle and ΔpH cause major conformational changes in the pigment-protein complexes of the PS II inner or core antennae that switch a normal PS II unit to an increased rate constant of heat dissipation. We discuss a model of the PS II photochemical apparatus where PS II photochemistry and xanthophyll cycle-dependent energy dissipation are independent of the Peripheral antenna size.