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.     

Time-resolved fluorescence analysis of the photosystem II antenna proteins in detergent micelles and liposomes

We have studied the time-resolved fluorescence properties of the light-harvesting complexes (Lhc) of photosystem II (Lhcb) in order to obtain information on the mechanism of energy dissipation (non-photochemical quenching) which is correlated to the conversion of violaxanthin to zeaxanthin in excess light conditions. The chlorophyll fluorescence decay of Lhcb proteins LHCII, CP29, CP26, and CP24 in detergent solution is mostly determined by two lifetime components of 1.2-1.5 and 3.6-4 ns while the contribution of the faster component is higher in CP29, CP26, and CP24 with respect to LHCII. The xanthophyll composition of Lhc proteins affects the ratio of the lifetime components: when zeaxanthin is bound into the site L2 of LHCII, the relative amplitude of the faster component is increased and, consequently, the chlorophyll fluorescence quenching is enhanced. Analysis of quenching in mutants of Arabidopsis thaliana, which incorporate either violaxanthin or zeaxanthin in their Lhc proteins, shows that the extent of quenching is enhanced in the presence of zeaxanthin. The origin of the two fluorescence lifetimes was analyzed by their temperature dependence: since lifetime heterogeneity was not affected by cooling to 77 K, it is concluded that each lifetime component corresponds to a distinct conformation of the Lhc proteins. Upon incorporation of Lhc proteins into liposomes, a quenching of chlorophyll fluorescence was observed due to shortening of all their lifetime components: this indicates that the equilibrium between the two conformations of Lhcb proteins is displaced toward the quenched conformation in lipid membranes or thylakoids with respect to detergent solution. By increasing the protein density in the liposomes, and therefore the probability of protein-protein interactions, a further decrease of fluorescence lifetimes takes place down to values typical of quenched leaves. We conclude that at least two major factors determine the quenching of chlorophyll fluorescence in Lhcb proteins, i.e., intrasubunit conformational change and intersubunit interactions within the lipid membranes, and that these processes are both important in the photoprotection mechanism of nonphotochemical quenching in vivo.     

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.     

Chlorophyll a/b-binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b-less barley mutant.

Monospecific polyclonal antibodies have been raised against synthetic peptides derived from the primary sequences from different plant light-harvesting Chl a/b-binding (LHC) proteins. Together with other monospecific antibodies, these were used to quantify the levels of the 10 different LHC proteins in wild-type and chlorina f2 barley (Hordeum vulgare L.), grown under normal and intermittent light (ImL). Chlorina f2, grown under normal light, lacked Lhcb1 (type I LHC II) and Lhcb6 (CP24) and had reduced amounts of Lhcb2, Lhcb3 (types II and III LHC II), and Lhcb4 (CP 29). Chlorina f2 grown under ImL lacked all LHC proteins, whereas wild-type ImL plants contained Lhcb5 (CP 26) and a small amount of Lhcb2. The chlorina f2 ImL thylakoids were organized in large parallel arrays, but wild-type ImL thylakoids had appressed regions, indicating a possible role for Lhcb5 in grana stacking. Chlorina f2 grown under ImL contained considerable amounts of violaxanthin (2-3/reaction center), representing a pool of phototransformable xanthophyll cycle pigments not associated with LHC proteins. Chlorina f2 and the plants grown under ImL also contained early light-induced proteins (ELIPs) as monitored by western blotting. The levels of both ELIPs and xanthophyll cycle pigments increased during a 1 h of high light treatment, without accumulation of LHC proteins. These data are consistent with the hypothesis that ELIPs are pigment-binding proteins, and we suggest that ELIPs bind photoconvertible xanthophylls and replace "normal" LHC proteins under conditions of light stress.     

De-epoxidation of violaxanthin in the minor antenna proteins of photosystem II, LHCB4, LHCB5, and LHCB6

The conversion of violaxanthin to zeaxanthin is essentially required for the pH-regulated dissipation of excess light energy in the antenna of photosystem II. Violaxanthin is bound to each of the antenna proteins of both photosystems. Former studies with recombinant Lhcb1 and different Lhca proteins implied that each antenna protein contributes specifically to violaxanthin conversion related to protein-specific affinities of the different violaxanthin binding sites. We investigated the violaxanthin de-epoxidation in the minor antenna proteins of photosystem II, Lhcb4-6. Recombinant proteins were reconstituted with different xanthophyll mixtures to study the conversion of violaxanthin at different xanthophyll binding sites in these proteins. The extent and kinetics of violaxanthin de-epoxidation were found to be dependent on the respective protein and, for each protein, also on the binding site of violaxanthin. In particular, violaxanthin bound to Lhcb4 was nearly inconvertible for de-epoxidation, whereas violaxanthin bound to Lhcb5 was fully convertible but with slow kinetics. Lhcb6 exhibited heterogeneous violaxanthin conversion characteristics, which could be assigned to different populations of reconstituted Lhcb6 complexes with respect to violaxanthin binding sites. The results support the proposed different binding affinities of violaxanthin to the three putative violaxanthin binding sites (V1, N1, and L2) in antenna proteins. Under consideration of former studies with Lhcb1 and Lhca proteins, the data imply that violaxanthin bound to the V1 and N1 binding site of antenna proteins is easily accessible for de-epoxidation in all antenna proteins, whereas violaxanthin bound to L2 is either only slowly or not convertible to zeaxanthin, depending on the respective protein.     

Xanthophyll cycle and energy-dependent fluorescence quenching in leaves from pea plants grown under intermittent light

The possible role of zeaxanthin formation and antenna proteins in energy-dependent chlorophyll fluorescence quenching (qE) has been investigated. Intermittent-light-grown pea (Pisum sativum L.) plants that lack most of the chlorophyll a/b antenna proteins exhibited a significantly reduced qE upon illumination with respect to control plants. On the other hand, the violaxanthin content related to the number of reaction centers and to xanthophyll cycle activity, i.e. the conversion of violaxanthin into zeaxanthin, was found to be increased in the antenna-protein-depleted plants. Western blot analyses indicated that, with the exception of CP 26, the content of all chlorophyll a/b-binding proteins in these plants is reduced to less than 10% of control values. The results indicate that chlorophyll a/b-binding antenna proteins are involved in the energy-dependent fluorescence quenching but that only a part of qE can be attributed to quenching by chlorophyll a/b-binding proteins. It seems very unlikely that xanthophylls are exclusively responsible for the qE mechanism.Abbreviations CAB chlorophyll a/b-binding - Chl chlorophyll - F_V variable fluorescence - IML intermittent light - LHC light harvesting complex - PFD photon flux density - qP photochemical quenching of chlorophyll fluoresence - qN non-photochemical quenching - qE energy-dependent quenching - qI photoinhibitory quenching - qT quenching by state transition     

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.     

Dynamics of zeaxanthin binding to the photosystem II monomeric antenna protein Lhcb6 (CP24) and modulation of its photoprotection properties

Lhcb6 (CP24) is a monomeric antenna protein of photosystem II, which has been shown to play special roles in photoprotective mechanisms, such as the Non-Photochemical Quenching and reorganization of grana membranes in excess light conditions. In this work we analyzed Lhcb6 in vivo and in vitro: we show this protein, upon activation of the xanthophyll cycle, accumulates zeaxanthin into inner binding sites faster and to a larger extent than any other pigment-protein complex. By comparative analysis of Lhcb6 complexes violaxanthin or zeaxanthin binding, we demonstrate that zeaxanthin not only down-regulates chlorophyll singlet excited states, but also increases the efficiency of chlorophyll triplet quenching, with consequent reduction of singlet oxygen production and significant enhancement of photo-stability. On these bases we propose that Lhcb6, the most recent addition to the Lhcb protein family which evolved concomitantly to the adaptation of photosynthesis to land environment, has a crucial role in zeaxanthin-dependent photoprotection.     

Antisense inhibition of the photosynthetic antenna proteins CP29 and CP26: implications for the mechanism of protective energy dissipation

The specific roles of the chlorophyll a/b binding proteins CP29 and CP26 in light harvesting and energy dissipation within the photosynthetic apparatus have been investigated. Arabidopsis was transformed with antisense constructs against the genes encoding the CP29 or CP26 apoprotein, which gave rise to several transgenic lines with remarkably low amounts of the antisense target proteins. The decrease in the level of CP24 protein in the CP29 antisense lines indicates a physical interaction between these complexes. Analysis of chlorophyll fluorescence showed that removal of the proteins affected photosystem II function, probably as a result of changes in the organization of the light-harvesting antenna. However, whole plant measurements showed that overall photosynthetic rates were similar to those in the wild type. Both antisense lines were capable of the qE type of nonphotochemical fluorescence quenching, although there were minor changes in the capacity for quenching and in its induction kinetics. High-light-induced violaxanthin deepoxidation to zeaxanthin was not affected, although the pool size of these pigments was decreased slightly. We conclude that CP29 and CP26 are unlikely to be sites for nonphotochemical quenching.     

Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29. Effects of zeaxanthin, pH and phosphorylation.

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.     

Zeaxanthin has enhanced antioxidant capacity with respect to all other xanthophylls in Arabidopsis leaves and functions independent of binding to PSII antennae

The ch1 mutant of Arabidopsis (Arabidopsis thaliana) lacks chlorophyll (Chl) b. Leaves of this mutant are devoid of photosystem II (PSII) Chl-protein antenna complexes and have a very low capacity of nonphotochemical quenching (NPQ) of Chl fluorescence. Lhcb5 was the only PSII antenna protein that accumulated to a significant level in ch1 mutant leaves, but the apoprotein did not assemble in vivo with Chls to form a functional antenna. The abundance of Lhca proteins was also reduced to approximately 20% of the wild-type level. ch1 was crossed with various xanthophyll mutants to analyze the antioxidant activity of carotenoids unbound to PSII antenna. Suppression of zeaxanthin by crossing ch1 with npq1 resulted in oxidative stress in high light, while removing other xanthophylls or the PSII protein PsbS had no such effect. The tocopherol-deficient ch1 vte1 double mutant was as sensitive to high light as ch1 npq1, and the triple mutant ch1 npq1 vte1 exhibited an extreme sensitivity to photooxidative stress, indicating that zeaxanthin and tocopherols have cumulative effects. Conversely, constitutive accumulation of zeaxanthin in the ch1 npq2 double mutant led to an increased phototolerance relative to ch1. Comparison of ch1 npq2 with another zeaxanthin-accumulating mutant (ch1 lut2) that lacks lutein suggests that protection of polyunsaturated lipids by zeaxanthin is enhanced when lutein is also present. During photooxidative stress, alpha-tocopherol noticeably decreased in ch1 npq1 and increased in ch1 npq2 relative to ch1, suggesting protection of vitamin E by high zeaxanthin levels. Our results indicate that the antioxidant activity of zeaxanthin, distinct from NPQ, can occur in the absence of PSII light-harvesting complexes. The capacity of zeaxanthin to protect thylakoid membrane lipids is comparable to that of vitamin E but noticeably higher than that of all other xanthophylls of Arabidopsis leaves.     

Dynamics of chromophore binding to Lhc proteins in vivo and in vitro during operation of the xanthophyll cycle

Three plant xanthophylls are components of the xanthophyll cycle in which, upon exposure of leaves to high light, the enzyme violaxanthin de-epoxidase (VDE) transforms violaxanthin into zeaxanthin via the intermediate antheraxanthin. Previous work () showed that xanthophylls are bound to Lhc proteins and that substitution of violaxanthin with zeaxanthin induces conformational changes and fluorescence quenching by thermal dissipation. We have analyzed the efficiency of different Lhc proteins to exchange violaxanthin with zeaxanthin both in vivo and in vitro. Light stress of Zea mays leaves activates VDE, and the newly formed zeaxanthin is found primarily in CP26 and CP24, whereas other Lhc proteins show a lower exchange capacity. The de-epoxidation system has been reconstituted in vitro by using recombinant Lhc proteins, recombinant VDE, and monogalactosyl diacylglycerol (MGDG) to determine the intrinsic capacity for violaxanthin-to-zeaxanthin exchange of individual Lhc gene products. Again, CP26 was the most efficient in xanthophyll exchange. Biochemical and spectroscopic analysis of individual Lhc proteins after de-epoxidation in vitro showed that xanthophyll exchange occurs at the L2-binding site. Xanthophyll exchange depends on low pH, implying that access to the binding site is controlled by a conformational change via lumenal pH. These findings suggest that the xanthophyll cycle participates in a signal transduction system acting in the modulation of light harvesting versus thermal dissipation in the antenna system of higher plants.     

Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complexes

Xanthophylls have a crucial role in the structure and function of the light harvesting complexes of photosystem II (LHCII) in plants. The binding of xanthophylls to LHCII has been investigated, particularly with respect to the xanthophyll cycle carotenoids violaxanthin and zeaxanthin. It was found that most of the violaxanthin pool was loosely bound to the major complex and could be removed by mild detergent treatment. Gentle solubilization of photosystem II particles and thylakoids allowed the isolation of complexes, including a newly described oligomeric preparation, enriched in trimers, that retained all of the in vivo violaxanthin pool. It was estimated that each LHCII monomer can bind at least one violaxanthin. The extent to which different pigments can be removed from LHCII indicated that the relative strength of binding was chlorophyll b > neoxanthin > chlorophyll a > lutein > zeaxanthin > violaxanthin. The xanthophyll binding sites are of two types: internal sites binding lutein and peripheral sites binding neoxanthin and violaxanthin. In CP29, a minor LHCII, both a lutein site and the neoxanthin site can be occupied by violaxanthin. Upon activation of the violaxanthin de-epoxidase, the highest de-epoxidation state was found for the main LHCII component and the lowest for CP29, suggesting that only violaxanthin loosely bound to LHCII is available for de-epoxidation.     

The effect of zeaxanthin as the only xanthophyll on the structure and function of the photosynthetic apparatus in Arabidopsis thaliana

In green plants, the xanthophyll carotenoid zeaxanthin is synthesized transiently under conditions of excess light energy and participates in photoprotection. In the Arabidopsis lut2 npq2 double mutant, all xanthophylls were replaced constitutively by zeaxanthin, the only xanthophyll whose synthesis was not impaired. The relative proportions of the different chlorophyll antenna proteins were strongly affected with respect to the wild-type strain. The major antenna, LHCII, did not form trimers, and its abundance was strongly reduced as was CP26, albeit to a lesser extent. In contrast, CP29, CP24, LHCI proteins, and the PSI and PSII core complexes did not undergo major changes. PSII-LHCII supercomplexes were not detectable while the PSI-LHCI supercomplex remained unaffected. The effect of zeaxanthin accumulation on the stability of the different Lhc proteins was uneven: the LHCII proteins from lut2 npq2 had a lower melting temperature as compared with the wild-type complex while LHCI showed increased resistance to heat denaturation. Consistent with the loss of LHCII, light-state 1 to state 2 transitions were suppressed, the photochemical efficiency in limiting light was reduced and photosynthesis was saturated at higher light intensities in lut2 npq2 leaves, resulting in a photosynthetic phenotype resembling that of high light-acclimated leaves. Zeaxanthin functioned in vivo as a light-harvesting accessory pigment in lut2 npq2 chlorophyll antennae. As a whole, the in vivo data are consistent with the results obtained by using recombinant Lhc proteins reconstituted in vitro with purified zeaxanthin. While PSII photoinhibition was similar in wild type and lut2 npq2 exposed to high light at low temperature, the double mutant was much more resistant to photooxidative stress and lipid peroxidation than the wild type. The latter observation is consistent with an antioxidant and lipid protective role of zeaxanthin in vivo.     

Genomic analysis of mutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

When the absorption of light energy exceeds the capacity for its utilization in photosynthesis, regulation of light harvesting is critical in order for photosynthetic organisms to minimize photo-oxidative damage. Thermal dissipation of excess absorbed light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is induced rapidly in response to excess light conditions, and it is known that xanthophylls such as zeaxanthin and lutein, the transthylakoid pH gradient, and the PsbS protein are involved in this mechanism. Although mutants affecting NPQ and the biosynthesis of zeaxanthin and lutein were originally isolated and characterized at the physiological level in the unicellular green alga Chlamydomonas reinhardtii, the molecular basis of several of these mutants, such as npq1 and lor1, has not been determined previously. The recent sequencing of the C. reinhardtii nuclear genome has facilitated the search for C. reinhardtii homologs of plant genes involved in xanthophyll biosynthesis and regulation of light harvesting. Here we report the identification of C. reinhardtii genes encoding PsbS and lycopene ɛ-cyclase, and we show that the lor1 mutation, which affects lutein synthesis, is located within the lycopene ɛ-cyclase gene. In contrast, no homolog of the plant violaxanthin de-epoxidase (VDE) gene was found. Molecular markers were used to map the npq1 mutation, which affects VDE activity, as a first step toward the map-based cloning of the NPQ1 gene.     

Simple replacement of violaxanthin by zeaxanthin in LHC-II does not cause chlorophyll fluorescence quenching

Recently, a mechanism for the energy-dependent component (qE) of non-photochemical quenching (NPQ), the fundamental photo-protection mechanism in green plants, has been suggested. Replacement of violaxanthin by zeaxanthin in the binding pocket of the major light harvesting complex LHC-II may be sufficient to invoke efficient chlorophyll fluorescence quenching. Our quantum chemical calculations, however, show that the excited state energies of violaxanthin and zeaxanthin are practically identical when their geometry is constrained to the naturally observed structure of violaxanthin in LHC-II. Therefore, since violaxanthin does not quench LHC-II, zeaxanthin should not either. This theoretical finding is nicely in agreement with experimental results obtained by femtosecond spectroscopy on LHC-II complexes containing violaxanthin or zeaxanthin.     

Absence of the Lhcb1 and Lhcb2 proteins of the light-harvesting complex of photosystem II - effects on photosynthesis,grana stacking and fitness

We have constructed Arabidopsis thaliana plants that are virtually devoid of the major light-harvesting complex, LHC II. This was accomplished by introducing the Lhcb2.1 coding region in the antisense orientation into the genome by Agrobacterium-mediated transformation. Lhcb1 and Lhcb2 were absent, while Lhcb3, a protein present in LHC II associated with photosystem (PS) II, was retained. Plants had a pale green appearance and showed reduced chlorophyll content and an elevated chlorophyll a/b ratio. The content of PS II reaction centres was unchanged on a leaf area basis, but there was evidence for increases in the relative levels of other light harvesting proteins, notably CP26, associated with PS II, and Lhca4, associated with PS I. Electron microscopy showed the presence of grana. Photosynthetic rates at saturating irradiance were the same in wild-type and antisense plants, but there was a 10-15% reduction in quantum yield that reflected the decrease in light absorption by the leaf. The antisense plants were not able to perform state transitions, and their capacity for non-photochemical quenching was reduced. There was no difference in growth between wild-type and antisense plants under controlled climate conditions, but the antisense plants performed worse compared to the wild type in the field, with decreases in seed production of up to 70%.     

Acclimation- and mutation-induced enhancement of PsbS levels affects the kinetics of non-photochemical quenching in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The efficiency of photosystem II antenna complexes (LHCs) in higher plants must be regulated to avoid potentially damaging overexcitation of the reaction centre in excess light. Regulation is achieved via a feedback mechanism known as non-photochemical quenching (NPQ), triggered the proton gradient (ΔpH) causing heat dissipation within the LHC antenna. ΔpH causes protonation of the LHCs, the PsbS protein and triggers the enzymatic de-epoxidation of the xanthophyll, violaxanthin, to zeaxanthin. A key step in understanding the mechanism is to decipher whether PsbS and zeaxanthin cooperate to promote NPQ. To obtain clues about their respective functions we studied the effects of PsbS and zeaxanthin on the rates of NPQ formation and relaxation in wild-type Arabidopsis leaves and those overexpressing PsbS (L17) or lacking zeaxanthin (npq1). Overexpression of PsbS was found to increase the rate of NPQ formation, as previously reported for zeaxanthin. However, PsbS overexpression also increased the rate of NPQ relaxation, unlike zeaxanthin, which is known decrease the rate. The enhancement of PsbS levels in plants lacking zeaxanthin (npq1) by either acclimation to high light or crossing with L17 plants showed that the effect of PsbS was independent of zeaxanthin. PsbS levels also affected the kinetics of the 535 nm absorption change (ΔA535), which monitors the formation of the conformational state of the LHC antenna associated with NPQ, in an identical way. The antagonistic action of PsbS and zeaxanthin with respect to NPQ and ΔA535 relaxation kinetics suggests that the two molecules have distinct regulatory functions.