Photoinhibition and photoprotection in senescent leaves of field-grown wheat plants

Photosynthesis, photosystem II (PSII) photochemistry, photoinhibition and the xanthophyll cycle in the senescent flag leaves of wheat (Triticum aestivum L.) plants grown in the field were investigated. Compared to the non-senescent leaves, photosynthetic capacity was significantly reduced in senescent flag leaves. The light intensity at which photosynthesis was saturated also declined significantly. The light response curves of PSII photochemistry indicate that a down-regulation of PSII photochemistry occurred in senescent leaves in particular at high light. The maximal efficiency of PSII photochemistry in senescent flag leaves decreased slightly when measured at predawn but substantially at midday, suggesting that PSII function was largely maintained and photoinhibition occurred in senescent leaves when exposed to high light. At midday, PSII efficiency, photochemical quenching and the efficiency of excitation capture by open PSII centers decreased considerably, while non-photochemical quenching increased significantly. Moreover, compared with the values at early morning, a greater decrease in CO₂ assimilation rate was observed at midday in senescent leaves than in control leaves. The levels of antheraxanthin and zeaxanthin via the de-epoxidation of violaxanthin increased in senescent flag leaves from predawn to midday. An increase in the xanthophyll cycle pigments relative to chlorophyll was observed in senescent flag leaves. The results suggest that the xanthophyll cycle was activated in senescent leaves due to the decrease in CO₂ assimilation capacity and the light intensity for saturation of photosynthesis and that the enhanced formation of antheraxanthin and zeaxanthin at high light may play an important role in the dissipation of excess light energy and help to protect photosynthetic apparatus from photodamage. Our results suggest that the well-known function of the xanthophyll cycle to safely dissipate excess excitation energy is also important for maintaining photosynthetic function during leaf senescence.     

The xanthophyll cycle and sustained thermal energy dissipation activity in Vinca minor and Euonymus kiautschovicus in winter

The influence of low temperature on the operation of the xanthophyll cycle and energy dissipation activity, as ascertained through measurements of chlorophyll fluorescence, was examined in two broad-leaved evergreen species, Vinca minor L. and Euonymus kiautschovicus Loessner. In leaves examined under laboratory conditions, energy dissipation activity developed more slowly at lower leaf temperatures, but the final, steady-state level of such activity was greater at lower temperatures where the rate of energy utilization (through photosynthetic electron transport) was much lower. The rate at which energy dissipation activity increased was similar to that of the de-epoxidation of violaxanthin to antheraxanthin and zea-xanthin at different temperatures. However, leaves in the field examined prior to sunrise on mornings following cold days and nights exhibited a retention of antheraxanthin and zeaxanthin that was associated with sustained decreases in photosystem II efficiency. We therefore suggest that this phenomenon of ‘photoinhibition’ in response to light and cold temperatures during the winter results from sustained photoprotective thermal energy dissipation associated with the xanthophyll cycle. Such retention of the de-epoxidized components of the xanthophyll cycle responded to day-to-day changes in temperature, being greatest on the coldest mornings (when photoprotective energy dissipation might be most required) and less on warmer mornings when photosynthesis could presumably proceed at higher rates.     

Analysis of xanthophyll cycle carotenoids and chlorophyll fluorescence in light intensity-dependent chlorophyll-deficient mutants of wheat and barley

Three light intensity-dependent Chl b-deficient mutants, two in wheat and one in barley, were analyzed for their xanthophyll cycle carotenoids and Chl fluorescence characteristics under two different growth PFDs (30 versus 600 mol photons·m^–2 s^–1 incident light). Mutants grown under low light possessed lower levels of total Chls and carotenoids per unit leaf area compared to wild type plants, but the relative proportions of the two did not vary markedly between strains. In contrast, mutants grown under high light had much lower levels of Chl, leading to markedly greater carotenoid to Chl ratios in the mutants when compared to wild type. Under low light conditions the carotenoids of the xanthophyll cycle comprised approximately 15% of the total carotenoids in all strains; under high light the xanthophyll cycle pool increased to over 30% of the total carotenoids in wild type plants and to over 50% of the total carotenoids in the three mutant strains. Whereas the xanthophyll cycle remained fairly epoxidized in all plants grown under low light, plants grown under high light exhibited a considerable degree of conversion of the xanthophyll cycle into antheraxanthin and zeaxanthin during the diurnal cycle, with almost complete conversion (over 90%) occurring only in the mutants. 50 to 95% of the xanthophyll cycle was retained as antheraxanthin and zeaxanthin overnight in these mutants which also exhibited sustained depressions in PS II photochemical efficiency (F_v/F_m), which may have resulted from a sustained high level of photoprotective energy dissipation activity. The relatively larger xanthophyll cycle pool in the Chl b-deficient mutant could result in part from the reported concentration of the xanthophyll cycle in the inner antenna complexes, given that the Chl b-deficient mutants are deficient in the peripheral LHC-II complexes.Abbreviations A antheraxanthin - Chl chlorophyll - F_o and F_m minimal yield (at open PS II reaction centers) and maximal yield (at closed centers) of chlorophyll fluorescence in darkness - F level of fluorescence during illumination with photosynthetically active radiation - F_m maximal yield (at closed centers) of chlorophyll fluorescence during illumination with photosynthetically active radiation - (F_m–F)/F_m actual efficiency of PS II during illumination with photosynthetically active radiation - F_v/F_m+(F_m–F_o)/F_m intrinsic efficiency of PS II in darkness - LHC_II light-harvesting chlorophyll-protein complex of Photosystem II - PFD photon flux density (between 400 and 700 nm) - PS I Photosystem I - PS II Photosystem II - V violaxanthin - Z zeaxanthin     

Leaf Xanthophyll content and composition in sun and shade determined by HPLC

As a part of our investigations to test the hypothesis that zeaxanthin formed by reversible de-epoxidation of violaxanthin serves to dissipate any excessive and potentially harmful excitation energy we determined the influence of light climate on the size of the xanthophyll cycle pool (violaxanthin + antheraxanthin + zeaxanthin) in leaves of a number of species of higher plants. The maximum amount of zeaxanthin that can be formed by de-epoxidation of violaxanthin and antheraxanthin is determined by the pool size of the xanthophyll cycle. To quantitate the individual leaf carotenoids a rapid, sensitive and accurate HPLC method was developed using a non-endcapped Zorbax ODS column, giving baseline separation of lutein and zeaxanthin as well as of other carotenoids and Chl a and b.The size of the xanthophyll cycle pool, both on a basis of light-intercepting leaf area and of light-harvesting chlorophyll, was ca. four times greater in sun-grown leaves of a group of ten sun tolerant species than in shade-grown leaves in a group of nine shade tolerant species. In contrast there were no marked or consistent differences between the two groups in the content of the other major leaf xanthophylls, lutein and neoxanthin. Also, in each of four species examined the xanthophyll pool size increased with an increase in the amount of light available during leaf development whereas there was little change in the content of the other xanthophylls. However, the -carotene/-carotene ratio decreased and little or no -carotene was detected in sun-grown leaves. Among shade-grown leaves the -carotene/-carotene ratio was considerably higher in species deemed to be umbrophilic than in species deemed to be heliophilic.The percentage of the xanthophyll cycle pool present as violaxanthin (di-epoxy-zeaxanthin) at solar noon was 96–100% for shade-grown plants and 4–53% for sun-grown plants with zeaxanthin accounting for most of the balance. The percentage of zeaxanthin in leaves exposed to midday solar radiation was higher in those with low than in those with high photosynthetic capacity.The results are consistent with the hypothesis that the xanthophyll cycle is involved in the regulation of energy dissipation in the pigment bed, thereby preventing a buildup of excessive excitation energy at the reaction centers.Abbreviations A antheraxanthin - C -carotene - C -carotene - EPS epoxidation state (V+0.5A)/(V+A+Z) - L lutein - N neoxanthin - PFD photon flux density - V violaxanthin - Z zeaxanthin C.I.W.-D.P.B. Publiation No. 1035     

Characterization of the Xanthophyll Cycle and Other Photosynthetic Pigment Changes Induced by Iron Deficiency in Sugar Beet (Beta vulgaris L.)

In this work we characterize the changes induced by iron deficiency in the pigment composition of sugar beet (Beta vulgaris L.) leaves. When sugar beet plants were grown hydroponically under limited iron supply, neoxanthin and β-carotene decreased concomitantly with chlorophyll a, whereas lutein and the carotenoids within the xanthophyll cycle were less affected. Iron deficiency caused major increases in the lutein/chlorophyll a and xanthophyll cycle pigments/chlorophyll a molar ratios. Xanthophyll cycle carotenoids in Fe-deficient plants underwent epoxidations and de-epoxidations in response to ambient light conditions. In dark adapted Fe-deficient plants most of the xanthophyll cycle pigment pool was in the epoxidated form violaxanthin. We show, both by HPLC and by in vivo 505 nanometers absorbance changes, that in Fe deficient plants and in response to light, the de-epoxidated forms antheraxanthin and zeaxanthin were rapidly formed at the expense of violaxanthin. Several hours after returning to dark, the xanthophyll cycle was shifted again toward violaxanthin. The ratio of variable to maximum chlorophyll fluorescence from intact leaves was decreased by iron deficiency. However, in iron deficient leaves this ratio was little affected by light conditions which displace the xanthophyll cycle toward epoxidation or de-epoxidation. This suggests that the functioning of the xanthophyll cycle is not necessarily linked to protection against excess light input.     

Xanthophyll cycle and light stress in nature: uniform response to excess direct sunlight among higher plant species

Photosystem II (PS II) efficiency, nonphotochemical fluorescence quenching, and xanthophyll cycle composition were determined in situ in the natural environment at midday in (i) a range of differently angled sun leaves ofEuonymus kiautschovicus Loesener and (ii) in sun leaves of a wide range of different plant species, including trees, shrubs, and herbs. Very different degrees of light stress were experienced by these leaves (i) in response to different levels of incident photon flux densities at similar photosynthetic capacities amongEuonymus leaves and (ii) as a result of very different photosynthetic capacities among species at similar incident photon flux densities (that were equivalent to full sunlight). ForEuonymus as well as the interspecific comparison all data fell on one single, close relationship for changes in intrinsic PSII efficiency, nonphotochemical fluorescence quenching, or the levels of zeaxanthin + antheraxanthin in leaves, respectively, as a function of the actual level of light stress. Thus, the same conversion state of the xanthophyll cycle and the same level of energy dissipation were observed for a given degree of light stress independent of species or conditions causing the light stress. Since all increases in thermal energy dissipation were associated with increases in the levels of zeaxanthin + antheraxanthin in these leaves, there was thus no indication of any form of xanthophyll cycle-independent energy dissipation in any of the twenty-four species or varieties of plants examined in their natural environment. It is also concluded that transient diurnal changes in intrinsic PSII efficiency in nature are caused by changes in the efficiency with which excitation energy is delivered from the antennae to PSII centers, and are thus likely to be purely photoprotective. Consequently, the possibility of quantifying the allocation of absorbed light into PSII photochemistry versus energy dissipation in the antennae from changes in intrinsic PSII efficiency is explored.Abbreviations A antheraxanthin - F actual level of fluorescence - F_a, F _o minimal fluorescence in the absence, presence of thylakoid energization - F_m, F _m maximal fluorescence in the absence, presence of thylakoid energization - F_m, - F)/F _m actual PSII efficiency ( = percent of absorbed light utilized in PSII photochemistry) - F_v/F_m, F _v /F_m/ PSII efficiency of open centers in the absence, presence of thylakoid energization - NPQ nonphotochemical fluorescence quenching - F_m/F _m - 1; q_p quenching coefficient for photochemical quenching - V violaxanthin - Z zeaxanthin     

Relationship between photosynthetic radiation-use efficiency of barley canopies and the photochemical reflectance index (PRI)

Some processes of excess radiation dissipation have been associated with changes in leaf reflectance near 531 nm. We aimed to study the relations between the photochemical reflectance index (PRI) derived from this signal, and photosynthetic radiation-use efficiency (defined as net CO₂ assimilation rate/incident photon flux density) in a cereal canopy. Measurements of reflectance, fluorescence, gas exchange and xanthophyll cycle pigments were made in the morning, midday and afternoon in barley canopies with two levels of nitrogen fertilization. The photosynthetic radiation-use efficiency decreased at midday, mainly in the third leaf, in both treatments, with lower values for the nitrogen deficient leaves. The zeaxanthin content showed the inverse pattern, increasing at midday and in the nitrogen deficient treatment. The photosynthetic radiation-use efficiency was well correlated with the epoxidation state, EPS (violaxanthin + 0.5 antheraxanthin)/(violaxanthin + antheraxanthin + zeaxanthin). The PRI [here defined as (R₅₃₉ - R₅₇₀)/(R₅₃₉+ R₅₇₀)] was significantly correlated with epoxidation state and zeaxanthin and with photosynthetic radiation-use efficiency. These results validate the utility of PRI in the assessment of radiation-use efficiency at canopy level.     

Operation of the xanthophyll cycle in higher plants in response to diurnal changes in incident sunlight

Changes in the carotenoid composition of leaves in response to diurnal changes in sunlight were determined in the crop species Helianthus annuus L. (sunflower), Cucurbita pepo L. (pumpkin), and Cucumls sativus L. (cucumber), in the diaheliotropic mesophyte Malva neglecta Wallr., and in the perennial shrub Euonymus kiautschovicus Loesner. Large daily changes were observed in the relative proportions of the components of the xanthophyll cycle, violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) in plants grown in full sunlight. In all leaves large amounts of Z were formed at peak irradiance, with the changes in Z content closely following changes in incident photon flux density (PFD) over the course of the day. All leaves also contained large total pools of the three xanthophyll-cycle components. However, the extent to which the V pool present at dawn became de-epoxidized during the day varied widely among leaves, from a 27% decrease in M. neglecta to a 90% decrease in E. kiautschovicus. The largest amounts of Z and the lowest amounts of V at peak irradiance (full sunlight) were observed in the species with the lower rates of photosynthesis (particularly in E. kiautschovicus and pumpkin), and smaller amounts of Z and a lesser decrease in V content were found at peak irradiance in those species with the higher rates of photosynthesis (particularly in M. neglecta and sunflower). In all species some Z was present in the leaves prior to sunrise. Furthermore, in individuals of sunflower, pumpkin, and cucumber grown at 85% of full sunlight and transferred to full sunlight, a further increase in the already large pool of the xanthophyll-cycle pigments occurred over the course of 1 d.Abbreviations A antheraxanthin - -Car, -Car - and -carotene - EPS epoxidation state - PFD photon flux density, between 400 and 700 nm - V violaxanthin - Z zeaxanthin This work was supported by the U.S. Department of Agriculture, Competitive Research Grants Office, award No. 90-37130-5422, and a Faculty Development Award from the University of Colorado to W.W. Adams III.     

Photosynthesis and chlorophyllafluorescence during flag leaf senescence of field-grown wheat plants

The characteristics of photosynthetic gas exchange, chlorophyll a fluorescence, and xanthophyll cycle pigments during flag leaf senescence of field-grown wheat plants were investigated. With senescence progressing, the light-saturated net CO₂ assimilation rate expressed either on a basis of leaf area or chlorophyll decreased significantly. The apparent quantum yield of net photosynthesis decreased when expressed on a leaf area basis but increased when expressed on a chlorophyll basis. The maximal efficiency of PSII photochemistry decreased very little while actual PSII efficiency, photochemical quenching, and the efficiency of excitation capture by open PSII centers decreased considerably. At the same time, non-photochemical quenching increased significantly. A substantial decrease in the contents of violaxanthin and zeaxanthin, but a slight decrease in the content of antheraxanthin were observed. However, the de-epoxidation status of the xanthophyll cycle was positively correlated with progressive senescence. This increase was due mainly to a smaller decrease in zeaxanthin than in violaxanthin. Our results suggest that PSII apparatus remained functional, but a down-regulation of PSII occurred under the steady state of photosynthesis in senescent flag leaves. Such a down-regulation was associated with the closure of PSII centers and an enhanced xanthophyll cycle-related thermal dissipation in the PSII antennae.     

Photochemical efficiency of Photosystem II and xanthophyll cycle components in Zea mays leaves exposed to water stress and high light

The effects of two light treatments (photosynthetically active photon flux density of either 650 or 1950 µmol m^–2 s^–1) on the photochemical efficiency of Photosystem II (PS II) (measured as variable to maximum fluorescence ratio) and on the xanthophyll cycle components was studied in wilted Zea mays leaves. For comparison, these parameters were followed under the same light conditions in well-hydrated leaves maintained either in normal or CO₂-free air. The net CO₂ assimilation of dehydrated leaves declined rapidly as their relative water content (RWC) decreased from 100 to 60% while the PS II efficiency measured after a prolonged dark period of 16 h declined only when RWC leaves was lower than 60%. Furthermore, drought caused an increase in the pool size of the xanthophyll cycle pigments and the presence of a sustained elevated level of zeaxanthin and antheraxanthin at the end of the long dark period. The leaf water deficit enhanced the sensitivity of PS II efficiency to light exposure. During illumination, strong inhibition of PS II efficiency and large violaxanthin deepoxidation was observed in wilted leaves even under moderate photon flux density compared to control leaves in the same conditions. After 2 h of darkness following the light treatment, the PS II efficiency that is dependent on the previous PPFD, decreased with leaf water deficit. Moreover, zeaxanthin epoxidation led to an accumulation of antheraxanthin in dehydrated leaves. All these drought effects on PS II efficiency and xanthophyll cycle components were also obtained in well-hydrated leaves by short-term CO₂ deprivation during illumination. We conclude that the increased susceptibility of PS II efficiency to light in wilted maize leaves is mainly explained by the decrease of CO₂ availability and the resulting low net CO₂ assimilation.     

A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light‐harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes

The epoxy‐xanthophylls antheraxanthin and violaxanthin are key precursors of light‐harvesting carotenoids and participate in the photoprotective xanthophyll cycle. Thus, the invention of zeaxanthin epoxidase (ZEP) catalyzing their formation from zeaxanthin has been a fundamental step in the evolution of photosynthetic eukaryotes. ZEP genes have only been found in Viridiplantae and chromalveolate algae with secondary plastids of red algal ancestry, suggesting that ZEP evolved in the Viridiplantae and spread to chromalveolates by lateral gene transfer. By searching publicly available sequence data from 11 red algae covering all currently recognized red algal classes we identified ZEP candidates in three species. Phylogenetic analyses showed that the red algal ZEP is most closely related to ZEP proteins from photosynthetic chromalveolates possessing secondary plastids of red algal origin. Its enzymatic activity was assessed by high performance liquid chromatography (HPLC) analyses of red algal pigment extracts and by cloning and functional expression of the ZEP gene from Madagascaria erythrocladioides in leaves of the ZEP‐deficient aba2 mutant of Nicotiana plumbaginifolia. Unlike other ZEP enzymes examined so far, the red algal ZEP introduces only a single epoxy group into zeaxanthin, yielding antheraxanthin instead of violaxanthin. The results indicate that ZEP evolved before the split of Rhodophyta and Viridiplantae and that chromalveolates acquired ZEP from the red algal endosymbiont and not by lateral gene transfer. Moreover, the red algal ZEP enables engineering of transgenic plants incorporating antheraxanthin instead of violaxanthin in their photosynthetic machinery.     

Influence of solar radiation and leaf angle on leaf xanthophyll concentrations in mangroves

Summary Mangroves have similar xanthophyll cycle components/chlorophyll ratios [i.e. (V+A+Z)/chl] to other plant species. (V+A+Z)/chl ratios were sensitive to the light environment in which leaves grew, decreasing as light levels decreased over a vertical transect through a forest canopy. The (V+A+Z)/chl ratio also varied among species. However, in sun leaves over all species, the (V+A+Z)/chl ratios correlate with the proportion of leaf area displayed on a horizontal plane, which is determined by leaf angle. Thus, leaf angle and the xanthophyll cycle may both be important in providing protection from high light levels in mangrove species. A canopy survey assessed whether (V+A+Z)/chl ratios could be correlated with species dominance of exposed positions in forest canopies.Rhizophora mangroves, with near-vertical leaf angles, andBruguiera parviflora, with small, horizontal, xanthophyllrich leaves, dominated the canopy, whileB. gymnorrhiza, a species with large, horizontally arranged leaves, was less abundant at the top of the canopy. Thus, two different strategies for adapting to high solar radiation levels may exist in these species. The first strategy is avoidance through near vertical leaf angles, and the second is a large capacity to dissipate energy through zeaxanthin. The (V+A+Z)/chl ratio was also negatively correlated with the epoxidation state of the xanthophyll cycle pool (the proportion present as violaxanthin and half that present as antheraxanthin) at midday. This suggested that the requirement for dissipation of excess light (represented by the midday epoxidation state) may influence the (V+A+Z)/chl ratio.     

The transiently pubescent young leaves of plane (Platanus orientalis) are deficient in photodissipative capacity

We have analyzed reflectance changes and carotenoid composition of young and mature leaves of Platanus orientalis L. in order to test the hypothesis that the transient occurrence of highly absorptive and reflective leaf hairs of young leaves (M. Ntefidou and Y. Manetas 1996, Aust. J. Plant Physiol. 23: 535–538) may be correlated to a weakly developed photodissipative capacity in the chloroplast. Compared to mature leaves, young leaves showed negligible reflectance changes at 530 nm upon sudden illumination, possibly indicating a limited production of zeaxanthin. In addition, actual pigment analysis confirmed lower pools of xanthophyll cycle components and reduced capacity for violaxanthin photoconversion in young leaves. Accordingly, the epoxidation state at saturating photon fluence rates was particularly high. A notable feature of xanthophyll cycle interconversions in young leaves was the inability to drive the system to complete de-epoxidation, as antheraxanthin in the light was always higher than zeaxanthin. Among the rest of the carotenoids, the levels of β-carotene were particularly low. Moreover, most of the photosynthetic pigments were considerably bleached when young leaves were exposed to high light. The above results strongly suggest that young leaves possess a limited photodissipative capacity and therefore, the presence of leaf hairs affords protection against excess light. When the leaf has matured and presumably the concentrations of photoprotective compounds are adequate, the loss of hairs is not of consequence. In fact, their presence on mature leaves may reduce the photosynthetically active radiation to non-saturating levels for photosynthesis.     

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

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

Antisense suppression of violaxanthin de-epoxidase in tobacco does not affect plant performance in controlled growth conditions

Violaxanthin de-epoxidase (VDE) catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Tobacco was transformed with an antisense VDE construct under control of the cauliflower mosaic virus 35S promoter to determine the effect of reduced levels of VDE on plant growth. Screening of 40 independent transformants revealed 18 antisense lines with reduced levels of VDE activity with two in particular (TAS32 and TAS39) having greater than 95% reduction in VDE activity. Northern analysis demonstrated that these transformants had greatly suppressed levels of VDE mRNA. De-epoxidation of violaxanthin was inhibited to such an extent that no zeaxanthin and only very low levels of antheraxanthin could be detected after exposure of leaves to high light (2000 μmol m⁻² s⁻¹ for 20 min) with no observable effect on levels of other carotenoids and chlorophyll. Non-photochemical quenching was greatly reduced in the antisense VDE tobacco, demonstrating that a significant level of the non-photochemical quenching in tobacco requires de-epoxidation of violaxanthin. Although the antisense plants demonstrated a greatly impaired de-epoxidation of violaxanthin, no effect on plant growth or photosynthetic rate was found when plants were grown at a photon flux density of 500 or 1000 μmol m⁻² s⁻¹ under controlled growth conditions as compared to wild-type tobacco. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chlamydomonas Xanthophyll Cycle Mutants Identified by Video Imaging of Chlorophyll Fluorescence Quenching

The photosynthetic apparatus in plants is protected against oxidative damage by processes that dissipate excess absorbed light energy as heat within the light-harvesting complexes. This dissipation of excitation energy is measured as nonphotochemical quenching of chlorophyll fluorescence. Nonphotochemical quenching depends primarily on the [delta]pH that is generated by photosynthetic electron transport, and it is also correlated with the amounts of zeaxanthin and antheraxanthin that are formed from violaxanthin by the operation of the xanthophyll cycle. To perform a genetic dissection of nonphotochemical quenching, we have isolated npq mutants of Chlamydomonas by using a digital video-imaging system. In excessive light, the npq1 mutant is unable to convert violaxanthin to antheraxanthin and zeaxanthin; this reaction is catalyzed by violaxanthin de-epoxidase. The npq2 mutant appears to be defective in zeaxanthin epoxidase activity, because it accumulates zeaxanthin and completely lacks antheraxanthin and violaxanthin under all light conditions. Characterization of these mutants demonstrates that a component of nonphotochemical quenching that develops in vivo in Chlamydomonas depends on the accumulation of zeaxanthin and antheraxanthin via the xanthophyll cycle. However, observation of substantial, rapid, [delta]pH-dependent nonphotochemical quenching in the npq1 mutant demonstrates that the formation of zeaxanthin and antheraxanthin via violaxanthin de-epoxidase activity is not required for all [delta]pH-dependent nonphotochemical quenching in this alga. Furthermore, the xanthophyll cycle is not required for survival of Chlamydomonas in excessive light.     

Operation of the xanthophyll cycle in the seagrass Zostera marina in response to variable irradiance

Changes in the photobiology and photosynthetic pigments of the seagrass Zostera marina from Chesapeake Bay (USA) were examined under a range of natural and manipulated irradiance regimes. Photosynthetic activity was assessed using chlorophyll-a fluorescence, and photosynthetic pigments were measured by HPLC. Large changes in the violaxanthin, zeaxanthin, and antheraxanthin content were concomitant with the modulation of non-photochemical quenching (NPQ). Photokinetics (F_v/F_m, rapid light curves (RLC), and non-photochemical quenching) varied as a result of oscillating irradiance and were highly correlated to xanthophyll pigment content. Zeaxanthin and antheraxanthin concentrations increased under elevated light conditions, while violaxanthin increased in darkened conditions. Unusually high concentrations of antheraxanthin were found in Z. marina under a wide range of light conditions, and this was associated with the partial conversion of violaxanthin to zeaxanthin. These results support the idea that xanthophyll intermediate pigments induce a photoprotective response during exposure to high irradiances in this seagrass.     

Plant lipocalins: violaxanthin de-epoxidase and zeaxanthin epoxidase

Violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the interconversions between the carotenoids violaxanthin, antheraxanthin and zeaxanthin in plants. These interconversions form the violaxanthin or xanthophyll cycle that protects the photosynthetic system of plants against damage by excess light. These enzymes are the first reported lipocalin proteins identified from plants and are only the second examples of lipocalin proteins with enzymatic activity. This review summarizes the discovery and characterization of these two unique lipocalin enzymes and examines the possibility of other potential plant lipocalin proteins.     

Seasonal changes in xanthophyll composition and photosynthesis of cork oak (Quercus suber L.) leaves under mediterranean climate

Seasonal changes in pigment composition of sun and shade leavesof cork oak (Quercus suber) were studied under field conditionsin Portugal. Expanding leaves showed a high concentration ofxanthophyll cycle components, violaxanthin, antheraxanthin andzeaxanthin. The pool of violaxanthin plus antheraxanthin pluszeaxanthin (V+A+Z) varied greatly between the seasons, beinghigher at the end of summer and in winter when photosynthesiswas limited by water stress and cold, respectively. The sizeof V+A+Z pool was associated to synthesis of zeaxanthin in responseto an excess of light. In sun leaves, midday A+Z relative contentwas positively correlated with the V+A+Z pool, whereas in shadeleaves A+Z decreased with leaf ageing. In both leaf types A+Zwas positively correlated with the non-photochemical quenching(NPQ) of chlorophyll a fluorescence. However, in winter NPQdid not change significantly throughout the day, whereas the(A+Z)/(V+A+Z) increased following the typical daily trend observedin other seasons. Key words: Chlorophyll fluorescence, pigments, Quercus suber, thermal dissipation, xanthophylls