Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion.

A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy.     

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.     

The protective functions of carotenoid and flavonoid pigments against excess visible radiation at chilling temperature investigated in Arabidopsis npq and tt mutants

The npq1 mutant of Arabidopsis thaliana (L.) Heynh. has no xanthophyll cycle due to a lack of functional violaxanthin de-epoxidase. Short-term exposure (<2 days) of detached leaves or whole plants to the combination of high photon flux density (1,000 micromol m(-2) s(-1)) and low temperature (10 degrees C) resulted in PSII photoinhibition which was more acute in npq1 than in the wild type. This increased photosensitivity of npql at chilling temperature was attributable to the inhibition of nonphotochemical energy quenching (NPQ) and not to the absence of zeaxanthin itself. In contrast to PSII, PSI was found to be phototolerant to chilling stress in the light in both genotypes. In the long term (10-12 days), PSII activity recovered in both npql and wild type, indicating that A. thaliana is able to acclimate to chilling stress in the light independently of the xanthophyll cycle. In npql, photoacclimation involved a substantial reduction of the light-harvesting pigment antenna of PSII and an improvement of photosynthetic electron transport. Chilling stress also induced synthesis of early light-inducedproteins (ELIPs) which, in the long term, disappeared in npql and remained stable in the wild type. In both genotypes, photoacclimation at low temperature induced the accumulation of various antioxidants including carotenoids (except beta-carotene), vitamin E (alpha- and -gamma-tocopherol) and non-photosynthetic pigments (anthocyanins and other flavonoids). Analysis of flavonoid-deficient tt mutants revealed that UV/blue-light-absorbing flavonols have a strong protective function against excess visible radiations. In contrast to the defect in npq1, the absence of flavonoids could not be overcome in the long term by compensatory mechanisms, leading to extensive photooxidative and photoinhibitory damage to the chloroplasts. Depth profiling of the leaf pigments by phase-resolved photoacoustic spectroscopy showed that the flavonoid-related photoprotection was due to light trapping, which decreased chlorophyll excitation by blue light. In contrast to flavonoids, the xanthophyll cycle and the associated NPQ seem to be mainly relevant to the protection of photosynthesis against sudden increases in light intensity.     

Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana

Vitamin E is considered a major antioxidant in biomembranes, but little evidence exists for this function in plants under photooxidative stress. Leaf discs of two vitamin E mutants, a tocopherol cyclase mutant (vte1) and a homogentisate phytyl transferase mutant (vte2), were exposed to high light stress at low temperature, which resulted in bleaching and lipid photodestruction. However, this was not observed in whole plants exposed to long-term high light stress, unless the stress conditions were extreme (very low temperature and very high light), suggesting compensatory mechanisms for vitamin E deficiency under physiological conditions. We identified two such mechanisms: nonphotochemical energy dissipation (NPQ) in photosystem II (PSII) and synthesis of zeaxanthin. Inhibition of NPQ in the double mutant vte1 npq4 led to a marked photoinhibition of PSII, suggesting protection of PSII by tocopherols. vte1 plants accumulated more zeaxanthin in high light than the wild type, and inhibiting zeaxanthin synthesis in the vte1 npq1 double mutant resulted in PSII photoinhibition accompanied by extensive oxidation of lipids and pigments. The single mutants npq1, npq4, vte2, and vte1 showed little sensitivity to the stress treatments. We conclude that, in cooperation with the xanthophyll cycle, vitamin E fulfills at least two different functions in chloroplasts at the two major sites of singlet oxygen production: preserving PSII from photoinactivation and protecting membrane lipids from photooxidation.     

The roles of specific xanthophylls in light utilization

To evaluate the role of specific xanthophylls in light utilization, wild-type and xanthophyll mutant plants (npq1, npq2, lut2, lut2npq1 and lut2npq2) from Arabidopsis thaliana were grown under three different light regimes: 30 (low light, LL), 150 (medium light, ML) and 450 (high light, HL) μmol photons m⁻² s⁻¹. We studied the pigment content, growth rate, xanthophyll cycle activity, chlorophyll fluorescence parameters and the response to photoinhibition. All genotypes differed strongly in the growth rates and the resistance against photoinhibition. In particular, replacement of lutein (Lut) by violaxanthin (Vx) in the lut2npq1 mutant did not affect the growth at non-saturating light intensities (LL and ML), but led to a pronounced reduction of growth under HL conditions, indicating an important photoprotective role of Lut. This was further supported by a much higher sensitivity of all Lut-deficient plants to photoinhibition in comparison with the wild type. In contrast, replacement of Lut by zeaxanthin (Zx) in lut2npq2 led to a pronounced reduction of growth under all light regimes, most likely related to the permanent non-photochemical dissipation of excitation energy by Zx at Vx-binding sites and the destabilization of antenna proteins by binding of Zx to Lut-binding sites. The high susceptibility of lut2npq2 to photoinhibition in comparison with npq2 further indicated that the photoprotective function of Zx is abolished in the absence of Lut. Thus, it can be concluded from our work that neither Vx nor Zx is able to fulfil the essential photoprotective function at Lut-binding sites under in vivo conditions.     

The Arabidopsis aba4-1 mutant reveals a specific function for neoxanthin in protection against photooxidative stress

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions.     

Lutein Accumulation in the Absence of Zeaxanthin Restores Nonphotochemical Quenching in the Arabidopsis thaliana npq1 Mutant

Plants protect themselves from excess absorbed light energy through thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). The major component of NPQ, qE, is induced by high transthylakoid ΔpH in excess light and depends on the xanthophyll cycle, in which violaxanthin and antheraxanthin are deepoxidized to form zeaxanthin. To investigate the xanthophyll dependence of qE, we identified suppressor of zeaxanthin-less1 (szl1) as a suppressor of the Arabidopsis thaliana npq1 mutant, which lacks zeaxanthin. szl1 npq1 plants have a partially restored qE but lack zeaxanthin and have low levels of violaxanthin, antheraxanthin, and neoxanthin. However, they accumulate more lutein and α-carotene than the wild type. szl1 contains a point mutation in the lycopene β-cyclase (LCYB) gene. Based on the pigment analysis, LCYB appears to be the major lycopene β-cyclase and is not involved in neoxanthin synthesis. The Lhcb4 (CP29) and Lhcb5 (CP26) protein levels are reduced by 50% in szl1 npq1 relative to the wild type, whereas other Lhcb proteins are present at wild-type levels. Analysis of carotenoid radical cation formation and leaf absorbance changes strongly suggest that the higher amount of lutein substitutes for zeaxanthin in qE, implying a direct role in qE, as well as a mechanism that is weakly sensitive to carotenoid structural properties.     

Photoprotection in a zeaxanthin- and lutein-deficient double mutant of Arabidopsis

When light absorption by a plant exceeds its capacity for light utilization, photosynthetic light harvesting is rapidly downregulated by photoprotective thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). To address the involvement of specific xanthophyll pigments in NPQ, we have analyzed mutants affecting xanthophyll metabolism in Arabidopsis thaliana. An npq1 lut2 double mutant was constructed, which lacks both zeaxanthin and lutein due to defects in the violaxanthin de-epoxidase and lycopene -cyclase genes. The npq1 lut2 strain had normal Photosystem II efficiency and nearly wild-type concentrations of functional Photosystem II reaction centers, but the rapidly reversible component of NPQ was completely inhibited. Despite the defects in xanthophyll composition and NPQ, the npq1 lut2 mutant exhibited a remarkable ability to tolerate high light.This revised version was published online in October 2005 with corrections to the Cover Date.     

The role of the PsbS protein in the protection of photosystems I and II against high light in Arabidopsis thaliana

The PsbS protein is recognised in higher plants as an important component in dissipating excess light energy via its regulation of non-photochemical quenching. We investigated photosynthetic responses in the arabidopsis npq4 mutant, which lacks PsbS, and in a mutant over-expressing PsbS (oePsbS). Growth under low light led to npq4 and wild-type plants being visibly indistinguishable, but induced a phenotype in oePsbS plants, which were smaller and had shorter flowering spikes. Here we report that chloroplasts from npq4 generated more singlet oxygen (¹O₂) than those from oePsbS. This accompanied a higher extent of photosystem II photoinhibition of leaves from npq4 plants. In contrast, oePsbS was more damaged by high light than npq4 and the wild-type at the level of photosystem I. The plastoquinone pool, as measured by thermoluminescence, was more oxidised in the oePsbS than in npq4, whilst the amount of photo-oxidisable P₇₀₀, as probed with actinic light or saturating flashes, was higher in oePsbS compared to wild-type and npq4. Taken together, this indicates that the level of PsbS has a regulatory role in cyclic electron flow. Overall, we show that under high light oePsbS plants were more protected from ¹O₂ at the level of photosystem II, whereas lack of cyclic electron flow rendered them susceptible to damage at photosystem I. Cyclic electron flow is concluded to be essential for protecting photosystem I from high light stress.     

Xanthophyll cycle mutants from Chlamydomonas reinhardtii indicate a role for zeaxanthin in the D1 protein turnover

Photosynthetic activity, pigment conversion and D1 protein degradation under high light stress has been investigated in a wild type strain and two xanthophyll cycle mutants (npq1 and npq2) of Chlamydomonas reinhardtii. Wild type cells exhibited the well-known inactivation of photosystem II in high light, which was accompanied by the loss of β-carotene and a concomitant increase of zeaxanthin. Complete degradation of D1 protein was found after 2 h of illumination in the presence of chloramphenicol, an inhibitor of chloroplast protein synthesis. The npq1 mutant, which is unable to convert violaxanthin to zeaxanthin, showed a very similar behaviour. For the npq2 mutant, however, which is unable to form violaxanthin from zeaxanthin and thus contains high amounts of zeaxanthin even in low light, photosystem II inactivation was less pronounced. This was paralleled by a much slower D1 protein degradation in chloramphenicol treated cells. Our results support a protective role for zeaxanthin against high light-induced photosystem II inactivation resulting in a slowed-down D1 protein turnover.     

Xanthophyll cycle pool size and composition in relation to the nitrogen content of apple leaves

The objective of this study was to determine xanthophyll cycle pool size and composition in response to N status and their relationships to non-photochemical quenching in apple leaves. Bench-grafted Fuji/M.26 trees were fertilized with different N concentrations (0-20 mM) in a modified Hoagland's solution for 6 weeks to create a wide range of leaf N status (1-4.4 g m(-2)). Chlorophyll content, xanthophyll cycle pool size, lutein, total carotene, and neoxanthin on a leaf area basis all increased linearly with increasing leaf N. However, only the ratios of the xanthophyll cycle pool and of lutein to chlorophyll were higher in low N leaves than in high N leaves. Under high light at midday, both zeaxanthin (Z), expressed on a chlorophyll basis, and the percentage of the xanthophyll cycle pool present as Z, increased as leaf N decreased. Thermal dissipation of excitation energy, measured as non-photochemical quenching of chlorophyll fluorescence, was positively related to, whereas efficiency of excitation transfer and photosystem II quantum efficiency were negatively related to, Z, expressed on a chlorophyll basis or on a xanthophyll cycle pool basis. It is concluded that both xanthophyll cycle pool size (on a chlorophyll basis) and conversion of violaxanthin to zeaxanthin are enhanced in response to N limitation to dissipate excessive absorbed light under high irradiance.     

Zeaxanthin epoxidation - an in vitro approach

Zeaxanthin epoxidase (ZE) is an enzyme operating in the violaxanthin cycle, which is involved in photoprotective mechanisms. In this work model systems to study zeaxanthin (Zx) epoxidation were developed. Two assay systems are presented in which epoxidation of Zx was observed. In these assays two mutants of Arabidopsis thaliana which have active only one of the two xanthophyll cycle enzymes were used. The npq1 mutant possesses an active ZE and is thus able to convert Zx to violaxanthin (Vx) but the violaxanthin de-epoxidase (VDE) is inactive, so that Vx cannot be converted to Zx. The other mutant, npq2, possesses an active VDE and can convert exogenous Vx to Zx under strong light conditions but reverse reaction is not possible. The first assay containing thylakoids from npq1 and npq2 mutants of A. thaliana gave positive results and high efficiency of epoxidation reaction was observed. The amount of Zx was reduced by 25%. To optimize high efficiency of epoxidation reaction additional factors facilitating both fusion of the two types of thylakoids and incorporation of Zx to their membranes were also studied. The second kind of assay contained npq1 mutant thylakoids of A. thaliana supplemented with exogenous Zx and monogalactosyldiacylglycerol (MGDG). Experiments with different proportions of Zx and MGDG showed that their optimal ratio is 1:60. In such system, due to epoxidation, the amount of Zx was reduced by 38% of its initial level. The in vitro systems of Zx epoxidation described in this paper enable analysis some properties of the ZE without necessity of its isolation.     

Manipulation of the Xanthophyll Cycle Increases Plant Susceptibility to Sclerotinia sclerotiorum

The xanthophyll cycle is involved in dissipating excess light energy to protect the photosynthetic apparatus in a process commonly assessed from non-photochemical quenching (NPQ) of chlorophyll fluorescence. Here, it is shown that the xanthophyll cycle is modulated by the necrotrophic pathogen Sclerotinia sclerotiorum at the early stage of infection. Incubation of Sclerotinia led to a localized increase in NPQ even at low light intensity. Further studies showed that this abnormal change in NPQ was closely correlated with a decreased pH caused by Sclerotinia-secreted oxalate, which might decrease the ATP synthase activity and lead to a deepening of thylakoid lumen acidification under continuous illumination. Furthermore, suppression (with dithiothreitol) or a defect (in the npq1-2 mutant) of violaxanthin de-epoxidase (VDE) abolished the Sclerotinia-induced NPQ increase. HPLC analysis showed that the Sclerotinia-inoculated tissue accumulated substantial quantities of zeaxanthin at the expense of violaxanthin, with a corresponding decrease in neoxanthin content. Immunoassays revealed that the decrease in these xanthophyll precursors reduced de novo abscisic acid (ABA) biosynthesis and apparently weakened tissue defense responses, including ROS induction and callose deposition, resulting in enhanced plant susceptibility to Sclerotinia. We thus propose that Sclerotinia antagonizes ABA biosynthesis to suppress host defense by manipulating the xanthophyll cycle in early pathogenesis. These findings provide a model of how photoprotective metabolites integrate into the defense responses, and expand the current knowledge of early plant-Sclerotinia interactions at infection sites.     

Leaf chloroplast ultrastructure and photosynthetic properties of a chlorophyll-deficient mutant of rice

Leaf chloroplast ultrastructure and photosynthetic properties of a natural, yellow-green leaf mutant (ygl1) of rice were characterized. Our results showed that chloroplast development was significantly delayed in the mutant leaves compared with the wild-type rice (WT). As leaves matured, more grana stacks formed concurrently with increasing leaf chlorophyll (Chl) content. Except for the lower intercellular CO₂ concentration, the ygl1 plants had a higher leaf net photosynthetic rate, stomatal conductance, and transpiration rate than those of the WT plants. Under equal amounts of Chl, the excitation energy of PSI and PSII was much stronger in the mutant than that in the WT. The ygl1 plants showed higher nonphotochemical quenching and lower photochemical quenching. They also exhibited higher actual photochemical efficiency of PSII with a higher electron transport rate. Under the light of 200 μmol(photon) m^?2 s^?1, the ygl1 mutant showed lesser deepoxidation of violaxanthin in the xanthophyll cycle than WT, but it increased substantially under strong light conditions. In conclusion, the photosynthetic machinery of the ygl1 remained stable during leaf development. The plants were less sensitive to photoinhibition compared with WT due to the active xanthophyll cycle. The ygl1 plants were efficient in both light harvesting and conversion of solar energy.     

The Lhcb protein and xanthophyll composition of the light harvesting antenna controls the DeltapH-dependency of non-photochemical quenching in Arabidopsis thaliana

Nonphotochemical quenching (NPQ) is the photoprotective dissipation of energy in photosynthetic membranes. The hypothesis that the DeltapH-dependent component of NPQ (qE) component of non-photochemical quenching is controlled allosterically by the xanthophyll cycle has been tested using Arabidopsis mutants with different xanthophyll content and composition of Lhcb proteins. The titration curves of qE against DeltapH were different in chloroplasts containing zeaxanthin or violaxanthin, proving their roles as allosteric activator and inhibitor, respectively. The curves differed in mutants deficient in lutein and specific Lhcb proteins. The results show that qE is determined by xanthophyll occupancy and the structural interactions within the antenna that govern allostericity.     

A mechanism of nonphotochemical energy dissipation, independent from PsbS, revealed by a conformational change in the antenna protein CP26

The regulation of light harvesting in higher plant photosynthesis, defined as stress-dependent modulation of the ratio of energy transfer to the reaction centers versus heat dissipation, was studied by means of carotenoid biosynthesis mutants and recombinant light harvesting complexes (LHCs) with modified chromophore binding. The npq2 mutant of Arabidopsis thaliana, blocked in the biosynthesis of violaxanthin and thus accumulating zeaxanthin, was shown to have a lower fluorescence yield of chlorophyll in vivo and, correspondingly, a higher level of energy dissipation, with respect to the wild-type strain and npq1 mutant, the latter of which is incapable of zeaxanthin accumulation. Experiments on purified thylakoid membranes from all three mutants showed that the major source of the difference between the npq2 and wild-type preparations was a change in pigment to protein interactions, which can explain the lower chlorophyll fluorescence yield in the npq2 samples. Analysis of the xanthophyll binding LHC proteins showed that the Lhcb5 photosystem II subunit (also called CP26) undergoes a change in its pI upon binding of zeaxanthin. The same effect was observed in wild-type CP26 upon treatment that leads to the accumulation of zeaxanthin in the membrane and was interpreted as the consequence of a conformational change. This hypothesis was confirmed by the analysis of two recombinant proteins obtained by overexpression of the Lhcb5 apoprotein in Escherichia coli and reconstitution in vitro with either violaxanthin or zeaxanthin. The V and Z containing pigment-protein complexes obtained by this procedure showed different pIs and high and low fluorescence yields, respectively. These results confirm that LHC proteins exist in multiple conformations, an idea suggested by previous spectroscopic measurements (Moya et al., 2001), and imply that the switch between the different LHC protein conformations is activated by the binding of zeaxanthin to the allosteric site L2. The results suggest that the quenching process induced by the accumulation of zeaxanthin contributes to qI, a component of NPQ whose origin was previously poorly understood.     

Different roles of alpha- and beta-branch xanthophylls in photosystem assembly and photoprotection

Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively alpha-branch (chy1chy2lut5) or beta-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of beta-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that alpha-branch (lutein) and beta-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection.     

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.     

Comparative profiling of lipid-soluble antioxidants and transcripts reveals two phases of photo-oxidative stress in a xanthophyll-deficient mutant of<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Excess light can impose severe oxidative stress on photosynthetic organisms. We have characterized high-light responses in wild-type Chlamydomonas reinhardtii and in the npq1 lor1 double mutant. The npq1 lor1 strain lacks two photoprotective carotenoids, lutein and zeaxanthin, and experiences acute photo-oxidative stress upon exposure to excess light. To examine the ability of npq1 lor1 cells to respond to photo-oxidative stress, we measured changes in lipid-soluble antioxidants following a shift from low light to high light in the wild type and the double mutant. The size of the xanthophyll cycle pool increased in both the wild type and mutant during the first 6 h of exposure to high light levels, but then decreased in the mutant during photo-oxidative bleaching. The level of alpha-tocopherol (vitamin E) was constant in the wild type and mutant during the first 6 h; then it increased by three-fold in the wild type but declined in npq1 lor1 cells. We also used cDNA microarrays and RNA gel-blot analysis to monitor differences in gene expression. Both strains showed an initial light-stress response in the form of a transient increase in expression of (1) GPXH, a glutathione peroxidase gene that has been shown to respond specifically to singlet oxygen and lipid peroxidation; (2) SMT1, a gene for a putative sterol C-methyltransferase; and (3) LI818r, a stress-responsive member of the light-harvesting complex superfamily. These transient changes in gene expression in high light were followed by a second series of changes in npq1 lor1, coincident with declines in lipid-soluble antioxidants but preceding detectable photo-oxidative damage to proteins and lipids. Thus, the response of npq1 lor1 to high light is unexpectedly complex, with initial changes in lipid-soluble antioxidants and RNA levels that are associated with acclimation in the wild type and a second wave of changes that accompanies photo-oxidative bleaching.     

Mechanisms shaping the synergism of zeaxanthin and PsbS in photoprotective energy dissipation in the photosynthetic apparatus of plants

Safe operation of photosynthesis is vital to plants and is ensured by the activity of processes protecting chloroplasts against photo-damage. The harmless dissipation of excess excitation energy is considered to be the primary photoprotective mechanism and is most effective in the combined presence of PsbS protein and zeaxanthin, a xanthophyll accumulated in strong light as a result of the xanthophyll cycle. Here we address the problem of specific molecular mechanisms underlying the synergistic effect of zeaxanthin and PsbS. The experiments were conducted with Arabidopsis thaliana, using wild-type plants, mutants lacking PsbS (npq4), and mutants affected in the xanthophyll cycle (npq1), with the application of molecular spectroscopy and imaging techniques. The results lead to the conclusion that PsbS interferes with the formation of densely packed aggregates of thylakoid membrane proteins, thus allowing easy exchange and incorporation of xanthophyll cycle pigments into such structures. It was found that xanthophylls trapped within supramolecular structures, most likely in the interfacial protein region, determine their photophysical properties. The structures formed in the presence of violaxanthin are characterized by minimized dissipation of excitation energy. In contrast, the structures formed in the presence of zeaxanthin show enhanced excitation quenching, thus protecting the system against photo-damage.