The Xanthophyll Cycle Modulates the Kinetics of Nonphotochemical Energy Dissipation in Isolated Light-Harvesting Complexes, Intact Chloroplasts, and Leaves of Spinach1

We analyzed the kinetics of nonphotochemical quenching of chlorophyll fluorescence (qN) in spinach (Spinacia oleracea) leaves, chloroplasts, and purified light-harvesting complexes. The characteristic biphasic pattern of fluorescence quenching in dark-adapted leaves, which was removed by preillumination, was evidence of light activation of qN, a process correlated with the de-epoxidation state of the xanthophyll cycle carotenoids. Chloroplasts isolated from dark-adapted and light-activated leaves confirmed the nature of light activation: faster and greater quenching at a subsaturating transthylakoid pH gradient. The light-harvesting chlorophyll a/b-binding complexes of photosystem II were isolated from dark-adapted and light-activated leaves. When isolated from light-activated leaves, these complexes showed an increase in the rate of quenching in vitro compared with samples prepared from dark-adapted leaves. In all cases, the quenching kinetics were fitted to a single component hyperbolic function. For leaves, chloroplasts, and light-harvesting complexes, the presence of zeaxanthin was associated with an increased rate constant for the induction of quenching. We discuss the significance of these observations in terms of the mechanism and control of qN.

Surface reflectance properties of Antarctic moss and their relationship to plant species, pigment composition and photosynthetic function

In this study the variations in surface reflectance properties and pigment concentrations of Antarctic moss over species, sites, microtopography and with water content were investigated. It was found that species had significantly different surface reflectance properties, particularly in the region of the red edge (approximately 700 nm), but this did not correlate strongly with pigment concentrations. Surface reflectance of moss also varied in the visible region and in the characteristics of the red edge over different sites. Reflectance parameters, such as the photochemical reflectance index (PRI) and cold hard band were useful discriminators of site, microtopographic position and water content. The PRI was correlated both with the concentrations of active xanthophyll-cycle pigments and the photosynthetic light use efficiency, F-v/F-m, measured using chlorophyll fluorescence. Water content of moss strongly influenced the amplitude and position of the red-edge as well as the PRI, and may be responsible for observed differences in reflectance properties for different species and sites. All moss showed sustained high levels of photoprotective xanthophyll pigments, especially at exposed sites, indicating moss is experiencing continual high levels of photochemical stress.

Beziehungen zwischen den Xanthophyllzyklen und der Biosynthese von Lichtsammelxanthophyllen in Chlorophyll a/c-haltigen Algen

Zusammenfassung:In Chlorophyll(Chl) a/c-haltigen Algen leisten Xanthophylle einen wesentlichen Beitrag zur Lichtsammlung. Daneben finden sich weitere Xanthophylle, die an einem Schutzmechanismus bei überoptimalem Lichtangebot beteiligt sind, dem sog. Xanthophyllzyklus. Ein Teil der Chl a/c-haltigen Algen besitzt den auch bei Höheren Pflanzen anzutreffenden Violaxanthin/Antheraxanthin/Zeaxanthin-(Vx/Ax/Zx-)Zyklus. In anderen Gruppen wie den Dinophyta, Haptophyta und den Kieselalgen (Bacillariophyceae) ist statt dessen der Diadinoxanthin/Diatoxanthin-(Ddx/Dtx-)Zyklus zu finden. Die vorliegende Arbeit zeigt, daß schwachlichtadaptierte Turbidostatkulturen der Kieselalge Phaeodactylum tricornutum unter mehrstündiger Starklichtinkubation neben den Pigmenten des Ddx/Dtx-Zyklus auch die des Vx/Ax/Zx-Zyklus akkumulieren. Außerdem läßt sich ein dritter Xanthophyllzyklus zwischen beta-Cryptoxanthin (Cx) und beta-Cryptoxanthin-Epoxid (CxE) nachweisen, doch liegen diese beiden Pigmente nur in sehr geringen Konzentrationen vor. Für die Starklichtakkumulation von Zx ist eine hohe Deepoxidase-Aktivität und die de-novo-Synthese von Carotinoiden erforderlich. Aus Zx wird im anschließenden Schwachlicht über die Intermediate Vx und Ddx das Lichtsammelxanthophyll Fucoxanthin (Fx) synthetisiert. Dies bestätigt auch ein Vergleich der Kinetiken der einzelnen Umwandlungsschritte mit den anhand eines Modells der Xanthophyllbiosynthesewege ermittelten theoretischen Ratenkonstanten. Dieser Vergleich legt jedoch nahe, daß bei der Vx-Synthese aus beta-Carotin CxE anstelle von Zx involviert sein könnte. Eine Untersuchung weiterer Chl a/c-haltiger Algen mit Ddx/Dt-Zyklus ergab, daß sie unter Starklicht ebenfalls den Vx/Ax/Zx-Zyklus akkumulieren. Weiterhin sind, mit Einschränkungen bei den Dinophyten und Xanthophyceen, alle untersuchten Algen in der Lage, die unter Starklicht akkumulierten Xanthophyllzykluspigmente im nachfolgenden Schwachlicht zur Synthese des jeweiligen Lichtsammelxanthophylls zu nutzen. Unter energetischen Gesichtspunkten stellt dieses Pigment-Recycling insbesondere für die Fx-haltigen Algen einen Vorteil dar, da ihre Lichtsammelkomplexe im Vergleich zu denen der Höheren Pflanzen etwa die doppelte Anzahl an Xanthophyllen binden.

Sample characteristics and pigment concentration of arctic snow and permafrost algal strains

Ten algal strains from snow and permafrost substrates were tested for their ability to produce secondary carotenoids and ?-tocopherol in response to high light and decreased nitrogen levels. The Culture Collection of Cryophilic Algae at Fraunhofer IBMT in Potsdam served as the bioresource for this study. Eight of the strains belong to the Chlorophyceae and two strains are affiliated to the Trebouxiophyceae. While under low light, all 10 strains produced the normal spectrum of primary pigments known to be present in Chlorophyta, only the eight chlorophyceaen strains were able to synthesize secondary carotenoids under stress conditions, namely canthaxanthin, echinenone and astaxanthin; seven of them were also able to synthesize minor amounts of adonixanthin and an unidentified hydroxyechinenone. The two trebouxiophyceaen species of Raphidonema exhibited an unusually high pool of primary xanthophyll cycle pigments, possibly serving as a buffering reservoir against excessive irradiation. They also proved to be good alpha-tocopherol producers, which might also support the deactivation of reactive oxygen species. This study showed that some strains might be interesting novel candidates for biotechnological applications. Cold-adapted, snow and permafrost algae might serve as valuable production strains still exhibiting acceptable growth rates during the cold season in temperate regions.

The roles of specific xanthophylls in photoprotection

Xanthophyll pigments have critical structural and functional roles in the photosynthetic light-harvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga Chlamydomonas reinhardtii revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll fluorescence. Mutants with a defect in either the α- or β-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (α-carotene branch), zeaxanthin, and antheraxanthin (β-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), α-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.

Differential Control of Xanthophylls and Light-Induced Stress Proteins, as Opposed to Light-Harvesting Chlorophyll a/b Proteins, during Photosynthetic Acclimation of Barley Leaves to Light Irradiance

Barley (Hordeum vulgare L.) plants were grown at different photon flux densities ranging from 100 to 1800 μmol m−2 s−1 in air and/or in atmospheres with reduced levels of O2 and CO2. Low O2 and CO2 partial pressures allowed plants to grow under high photosystem II (PSII) excitation pressure, estimated in vivo by chlorophyll fluorescence measurements, at moderate photon flux densities. The xanthophyll-cycle pigments, the early light-inducible proteins, and their mRNA accumulated with increasing PSII excitation pressure irrespective of the way high excitation pressure was obtained (high-light irradiance or decreased CO2 and O2 availability). These findings indicate that the reduction state of electron transport chain components could be involved in light sensing for the regulation of nuclear-encoded chloroplast gene expression. In contrast, no correlation was found between the reduction state of PSII and various indicators of the PSII light-harvesting system, such as the chlorophyll a-to-b ratio, the abundance of the major pigment-protein complex of PSII (LHCII), the mRNA level of LHCII, the light-saturation curve of O2 evolution, and the induced chlorophyll-fluorescence rise. We conclude that the chlorophyll antenna size of PSII is not governed by the redox state of PSII in higher plants and, consequently, regulation of early light-inducible protein synthesis is different from that of LHCII.

Impact of changes in natural ultraviolet radiation on pigment composition, physiological and morphological characteristics of the Antarctic moss, Grimmia antarctici

The impact of ambient ultraviolet (UV)-B radiation on the endemic bryophyte, Grimmia antarctici, was studied over 14 months in East Antarctica. Over recent decades, Antarctic plants have been exposed to the largest relative increase in UV-B exposure as a result of ozone depletion. We investigated the effect of reduced UV and visible radiation on the pigment concentrations, surface reflectance and physiological and morphological parameters of this moss. Plexiglass screens were used to provide both reduced UV levels (77%) and a 50% decrease in total radiation. The screen combinations were used to separate UV photoprotective from visible photoprotective strategies, because these bryophytes are growing in relatively high light environments compared with many mosses. G. antarctici was affected negatively by ambient levels of UV radiation. Chlorophyll content was significantly lower in plants grown under near-ambient UV, while the relative proportions of photoprotective carotenoids, especially beta-carotene and zeaxanthin, increased. However, no evidence for the accumulation of UV-B-absorbing pigments in response to UV radiation was observed. Although photosynthetic rates were not affected, there was evidence of UV effects on morphology. Plants that were shaded showed fewer treatment responses and these were similar to the natural variation observed between moss growing on exposed microtopographical ridges and in more sheltered valleys within the turf. Given that other Antarctic bryophytes possess UV-B-absorbing pigments which should offer better protection under ambient UV-B radiation, these findings suggest that G. antarctici may be disadvantaged in some settings under a climate with continuing high levels of springtime UV-B radiation.

Effects of N supply on the accumulation of photosynthetic pigments and photoprotectors in Gracilaria tenuistipitata (Rhodophyta) cultured under UV radiation

We have studied the effects of nitrate supply under photosynthetic active radiation (PAR) plus ultraviolet radiation (UVR) exposure on photosynthetic pigments (chlorophyll a and carotenoids), photoprotective UV screen mycosporine-like amino acids (MAAs), and photosynthetic parameters, including the maximum quantum yield (F(v)/F(m)) and electron transport rate (ETR) on the red agarophyte Gracilaria tenuistipitata. Apical tips of G. tenuistipitata were cultivated under ten different concentrations of NO(3)(-) for 7 days. It has been shown that G. tenuistipitata cultured under laboratory conditions has the ability to accumulate high amounts of MAAs following a nitrate concentration-dependent manner under PAR+UVR. Two MAAs were identified, shinorine and porphyra-334. The relative concentration of the first increased under high concentrations of nitrate, while the second one decreased. The presence of antheraxanthin is reported for the first time in this macro-algae, which also contains zeaxanthin, lutein, and beta-carotene. The accumulation of pigments, photoprotective compounds, and photosynthetic parameters of G. tenuistipitata is directly related to N availability. All variables decreased under low N supplies and reached constant maximum values with supplements higher than 0.5 mM NO(3)(-). Our results suggest a high potential to acclimation and photoprotection against stress factors (including high PAR and UVR) directly related to N availability for G. tenuistipitata.

Response of holosymbiont pigments from the scleractinian coral Montipora monasteriata to short-term heat stress

Heating the scleractinian coral, Montipora monasteriata (Forskal 1775) to 32 degrees C under < 650 mu mol quanta m(-2) s(-1) led to bleaching in the form of a reduction in Peridinin, xanthophyll pool, chlorophyll c(2) and chlorophyll a, but areal dinoflagellates densities did not decline. Associated with this bleaching, chlorophyll (Chl) allomerization and dinoflagellate xanthophyll cycling increased. Chl allomerization is believed to result from the interaction of Chl with singlet oxygen (O-1(2)) or other reactive oxygen species. Thermally induced increases in Chl allomerization are consistent with other studies that have demonstrated that thermal stress generates reactive oxygen species in symbiotic dinoflagellates. Xanthophyll cycling requires the establishment of a pH gradient across the thylakoid membrane. Our results indicate that, during the early stages of thermal stress, thylakoid membranes are intact. Different morphs of M. monasteriata responded differently to the heat stress applied: heavily pigmented coral hosts taken from a high-light environment showed significant reductions in green fluorescent protein (GFP)-like homologues, whereas nonhost pigmented high-light morphs experienced a significant reduction in water-soluble protein content. Paradoxically, the more shade acclimated cave morph were, based on Chl fluorescence data, less thermally stressed than either of the high-light morphs. These results Support the importance of coral pigments for the regulation of the light environment within the host tissue.

Role of xanthophyll and water-water cycles in the protection of photosynthetic apparatus in Arbutus unedo and Arabidopsis thaliana

Photosynthetic organisms have sought out the delicate balance between efficient light harvesting under limited irradiance and regulated energy dissipation under excess irradiance. One of the protective mechanisms is the thermal energy dissipation through the xanthophyll cycle that may transform harmlessly the excitation energy into heat and thereby prevent the formation of damaging active oxygen species (AOS). Violaxanthin deepoxidase (VDE) converts violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) defending the photosynthetic apparatus from excess of light. Another important biological pathway is the chloroplast water-water cycle, which is referred to the electrons from water generated in PSII reducing atmospheric O2 to water in PSI. This mechanism is active in the scavenging of AOS, when electron transport is slowed down by the over-reduction of NADPH pool. The control of the VDE gene and the variations of a set of physiological parameters, such as chlorophyll florescence and AOS content, have been investigated in response to excess of light and drought condition using Arabidopsis thaliana and Arbutus unedo.. Pigment analysis showed an unambiguous relationship between xanthophyll de-epoxidation state ((A+Z)/(V+A+Z)) and VDE mRNA amount in not-irrigated plants. Unexpectedly, gene expression is higher during the night when xanthophylls are mostly epoxidated and VDE activity is supposed to be very low than during the day. The importance of the water-water cycle in protecting the chloroplasts from light stress has been examined through Arabidopsis plant with a suppressed expression of the key enzyme of the cycle: the thylakoid-attached copper/zinc superoxide dismutase. The analysis revealed changes in transcript expression during leaf development consistent with a signalling role of AOS in plant defence responses but no difference was found any in photosynthesis efficiency or in AOS concentration after short-term exposure to excess of light. Environmental stresses such as drought may render previously optimal light levels excessive. In these circumstances the intrinsic regulations of photosynthetic electron transport like xanthophyll and water-water cycles might modify metabolism and gene expression in order to deal with increasing AOS.

Antioxidative Defense System, Pigment Composition, and Photosynthetic Efficiency in Two Wheat Cultivars Subjected to Drought1

We analyzed antioxidative defenses, photosynthesis, and pigments (especially xanthophyll-cycle components) in two wheat (Triticum durum Desf.) cultivars, Adamello and Ofanto, during dehydration and rehydration to determine the difference in their sensitivities to drought and to elucidate the role of different protective mechanisms against oxidative stress. Drought caused a more pronounced inhibition in growth and photosynthetic rates in the more sensitive cv Adamello compared with the relatively tolerant cv Ofanto. During dehydration the glutathione content decreased in both wheat cultivars, but only cv Adamello showed a significant increase in glutathione reductase and hydrogen peroxide-glutathione peroxidase activities. The activation states of two sulfhydryl-containing chloroplast enzymes, NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphatase, were maintained at control levels during dehydration and rehydration in both cultivars. This indicates that the defense systems involved are efficient in the protection of sulfhydryl groups against oxidation. Drought did not cause significant effects on lipid peroxidation. Upon dehydration, a decline in chlorophyll a, lutein, neoxanthin, and β-carotene contents, and an increase in the pool of de-epoxidized xanthophyll-cycle components (i.e. zeaxanthin and antheraxanthin), were evident only in cv Adamello. Accordingly, after exposure to drought, cv Adamello showed a larger reduction in the actual photosystem II photochemical efficiency and a higher increase in nonradiative energy dissipation than cv Ofanto. Although differences in zeaxanthin content were not sufficient to explain the difference in drought tolerance between the two cultivars, zeaxanthin formation may be relevant in avoiding irreversible damage to photosystem II in the more sensitive cultivar.

Photoinhibition and photoprotection in symbiotic dinoflagellates from reef-building corals

Pulse-amplitude-modulation fluorometry and oxygen respirometry were used to investigate diel photosynthetic responses by symbiotic dinoflagellates to light levels in summer and winter on a high latitude coral reef. The symbiotic dinoflagellates from 2 species of reef-building coral (Porites cylindrica and Stylophora pistillata) showed photoinhibitory decreases in the ratio of variable (F-v) to maximal (F-m) fluorescence (F-v/F-m) as early as 09:00 h on both summer and winter days on the reefs associated with One Tree Island (23 degrees 30' S, 152 degrees 06' E; Great Barrier Reef, Australia). This was due to decreases in maximum, F-m, and to a smaller extent minimum, F-0, chlorophyll fluorescence. Complete recovery took 4 to 6 h and began to occur as soon as light levels fell each day. Chlorophyll fluorescence quenching analysis of corals measured during the early afternoon revealed classic regulation of photosystem II (PSII) efficiency through non-photochemical quenching (NPQ). These results appear to be similar to data collected for other algae and higher plants, suggesting involvement of the xanthophyll cycle of symbiotic dinoflagellates in regulating the quantum efficiency of PSII. The ability of symbiotic dinoflagellates to develop significant NPQ, however, depended strongly on when the symbiotic dinoflagellates were studied. Whereas symbiotic dinoflagellates from corals in the early afternoon showed a significant capacity to regulate the efficiency of PSII using NPQ, those sampled before sunrise had a slower and much reduced capacity, suggesting that elements of the xanthophyll cycle are suppressed prior to sunrise. A second major finding of this study is that the quantum efficiency of PSII in symbiotic dinoflagellates is strongly diurnal, and is as much as 50% lower just prior to sunrise than later in the day. When combined with oxygen flux data, these results indicate that a greater portion of the electron transport occurring later in the day is likely to be due to the increases in the rate of carbon fixation by Rubisco or to higher flutes through the Mehler-Ascorbate-Peroxidase (MAP) cycle.

Photoregulation and photodamage in Schefflera arboricola leaves adapted to different light environments

Leaves of the subtropical understorey shrub Schefflera arboricola Hayata growing in full sunlight had higher specific leaf weight, higher chlorophyll a/b ratios, lower total chlorophyll content and a threefold higher xanthophyll cycle pigment content than leaves growing in a naturally shaded, but sunfleck-punctuated, environment. A number of measurements, all made in situ and during natural day/night cycles, were taken as follows: current photochemical capacity (F-v/F-m after 10 min dark-adaptation), size and epoxidation state of the xanthophyll cycle, CO2 gas exchange and determination of the D1 synthesis rate. In sun leaves the lowest daily F-v/F-m was found to be approximately 0.6, the change from maximum correlating with an increase in zeaxanthin. Daily changes in zeaxanthin were partly due to de novo synthesis and turnover. We suggest that sun leaves can dissipate most of the excess light energy absorbed safely via the photoprotective xanthophyll cycle. D1 synthesis rates did not correlate with photosynthetic photon flux density or F-v/F-m. The shade leaves had high F-v/F-m values and constant photosynthetic rates throughout the day except during sunflecks, when photosynthetic rates increased and D1 synthesis accelerated, all without a substantial decrease in F-v/F-m. It seems that leaves of S. arboricola adapted to natural shade conditions can use sunflecks to contribute significantly to their productivity. The third leaf type investigated was from greenhouse-grown plants of S. arboricola after exposure to full sunlight. These leaves showed a rapid and large reduction in F-v/F-m (to 0.3), which neither correlated with zeaxanthin formation nor recovered within the same day. From long-term effects following full sunlight exposure of greenhouse-grown plants we suggest that this F-v/F-m reduction actually reflects photodestruction.

Resposta antioxidante, formação de fitoquelatinas e composição de pigmentos fotoprotetores em Brachiaria decumbens Stapf submetida à contaminação com Cd e Zn

In order to evaluate the response of Brachiaria decumbens Stapf to Cd and Zn, plants were hydroponically exposed to 50 and 100 mmol L-1 of Cd and 500 and 2000 mmol L-1 of Zn. Metal content of shoots and roots was determined, as well as alterations in photosynthetic and photoprotective pigments, antioxidant metabolites and phytochelatin synthesis. Plants concentrated elevated levels of Cd and Zn, especially in roots. Zinc exposure negatively affected chlorophyll and β-carotene content, whereas the highest dose of Cd reduced VAZ cycle pigments and tocopherol levels in plant shoots. Cadmium was the maximum inducer of the phytochelatin synthesis pathway.

Localization of the mechanism of pH-dependent non- photochemical fluorescence quenching in thylakoid membranes

Higher plants have evolved a well-conserved set of photoprotective mechanisms, collectively designated Non-Photochemical Quenching of chlorophyll fluorescence (qN), to deal with the inhibitory absorption of excess light energy by the photosystems. Their main contribution originates from safe thermal deactivation of excited states promoted by a highly-energized thylakoid membrane, detected via lumen acidification. The precise origins of this energy- or LlpH-dependent quenching (qE), arising from either decreased energy transfer efficiency in PSII antennae (~ Young & Frank, 1996; Gilmore & Yamamoto, 1992; Ruban et aI., 1992), from alternative electron transfer pathways in PSII reaction centres (~ Schreiber & Neubauer, 1990; Thompson &Brudvig, 1988; Klimov et aI., 1977), or from both (Wagner et aI., 1996; Walters & Horton, 1993), are a source of considerable controversy. In this study, the origins of qE were investigated in spinach thylakoids using a combination of fluorescence spectroscopic techniques: Pulse Amplitude Modulated (PAM) fluorimetry, pump-probe fluorimetry for the measurement of PSII absorption crosssections, and picosecond fluorescence decay curves fit to a kinetic model for PSII. Quenching by qE (,..,600/0 of maximal fluorescence, Fm) was light-induced in circulating samples and the resulting pH gradient maintained during a dark delay by the lumenacidifying capabilities of thylakoid membrane H+ ATPases. Results for qE were compared to those for the addition of a known antenna quencher, 5-hydroxy-1,4naphthoquinone (5-0H-NQ), titrated to achieve the same degree of Fm quenching as for qE. Quenching of the minimal fluorescence yield, F0' was clear (8 to 130/0) during formation of qE, indicative of classical antenna quenching (Butler, 1984), although the degree was significantly less than that achieved by addition of 5-0H-NQ. Although qE induction resulted in an overall increase in absorption cross-section, unlike the decrease expected for antenna quenchers like the quinone, a larger increase in crosssection was observed when qE induction was attempted in thylakoids with collapsed pH gradients (uncoupled by nigericin), in the absence of xanthophyll cycle operation (inhibited by DTT), or in the absence of quenching (LlpH not maintained in the dark due to omission of ATP). Fluorescence decay curves exhibited a similar disparity between qE-quenched and 5-0H-NQ-quenched thylakoids, although both sets showed accelerated kinetics in the fastest decay components at both F0 and Fm. In addition, the kinetics of dark-adapted thylakoids were nearly identical to those in qEquenched samples at F0' both accelerated in comparison with thylakoids in which the redox poise of the Oxygen-Evolving Complex was randomized by exposure to low levels of background light (which allowed appropriate comparison with F0 yields from quenched samples). When modelled with the Reversible Radical Pair model for PSII (Schatz et aI., 1988), quinone quenching could be sufficiently described by increasing only the rate constant for decay in the antenna (as in Vasil'ev et aI., 1998), whereas modelling of data from qE-quenched thylakoids required changes in both the antenna rate constant and in rate constants for the reaction centre. The clear differences between qE and 5-0H-NQ quenching demonstrated that qE could not have its origins in the antenna alone, but is rather accompanied by reaction centre quenching. Defined mechanisms of reaction centre quenching are discussed, also in relation to the observed post-quenching depression in Fm associated with photoinhibition.