Elevated ΔpH restores rapidly reversible photoprotective energy dissipation in <Emphasis Type="Italic">Arabidopsis</Emphasis> chloroplasts deficient in lutein and xanthophyll cycle activity

The xanthophylls of the light-harvesting complexes of photosystem II (LHCII), zeaxanthin, and lutein are thought to be essential for non-photochemical quenching (NPQ). NPQ is a process of photoprotective energy dissipation in photosystem II (PSII). The major rapidly reversible component of NPQ, qE, is activated by the transmembrane proton gradient, and involves the quenching of antenna chlorophyll excited states by the xanthophylls lutein and zeaxanthin. Using diaminodurene (DAD), a mediator of cyclic electron flow around photosystem I, to enhance ΔpH we demonstrate that qE can still be formed in the absence of lutein and light-induced formation of zeaxanthin in chloroplasts derived from the normally qE-deficient lut2npq1 mutant of Arabidopsis. The qE induced by high ΔpH in lut2npq1 chloroplasts quenched the level of fluorescence when all PSII reaction centers were in the open state (F _o state), protected PSII reaction centers from photoinhibition, was sensitive to the uncoupler nigericin, and was accompanied by absorption changes in the 410–565 nm region. Titrations show the ΔpH threshold for activation of qE in lut2npq1 chloroplasts lies outside the normal physiological range and is highly cooperative. Comparison of quenching in isolated trimeric (LHCII) and monomeric (CP26) light-harvesting complexes from lut2npq1 plants revealed a similarly shifted pH dependency compared with wild-type LHCII. The implications for the roles of lutein and zeaxanthin as direct quenchers of excitation energy are discussed. Furthermore, we argue that the control over the proton-antenna association constant, pK, occurs via influence of xanthophyll structure on the interconnected phenomena of light-harvesting antenna reorganization/aggregation and hydrophobicity.     

Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in Photosystem II antenna size and stability

Xanthophylls (oxygen derivatives of carotenes) are essential components of the plant photosynthetic apparatus. Lutein, the most abundant xanthophyll, is attached primarily to the bulk antenna complex, light-harvesting complex (LHC) II. We have used mutations in Arabidopsis thaliana that selectively eliminate (and substitute) specific xanthophylls in order to study their function(s) in vivo. These include two lutein-deficient mutants, lut1 and lut2, the epoxy xanthophyll-deficient aba1 mutant and the lut2aba1 double mutant. Photosystem stoichiometry, antenna sizes and xanthophyll cycle activity have been related to alterations in nonphotochemical quenching of chlorophyll fluorescence (NPQ). Nondenaturing polyacrylamide gel electrophoresis indicates reduced stability of trimeric LHC II in the absence of lutein (and/or epoxy xanthophylls). Photosystem (antenna) size and stoichiometry is altered in all mutants relative to wild type (WT). Maximal DeltapH-dependent NPQ (qE) is reduced in the following order: WT>aba1>lut1 approximately lut2>lut2aba1, paralleling reduction in Photosystem (PS) II antenna size. Finally, light-activation of NPQ shows that zeaxanthin and antheraxanthin present constitutively in lut mutants are not qE active, and hence, the same can be inferred of the lutein they replace. Thus, a direct involvement of lutein in the mechanism of qE is unlikely. Rather, altered NPQ in xanthophyll biosynthetic mutants is explained by disturbed macro-organization of LHC II and reduced PS II-antenna size in the absence of the optimal, wild-type xanthophyll composition. These data suggest the evolutionary conservation of lutein content in plants was selected for due to its unique ability to optimize antenna structure, stability and macro-organization for efficient regulation of light-harvesting under natural environmental conditions.     

Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in Photosystem II antenna size and stability

Xanthophylls (oxygen derivatives of carotenes) are essential components of the plant photosynthetic apparatus. Lutein, the most abundant xanthophyll, is attached primarily to the bulk antenna complex, light-harvesting complex (LHC) II. We have used mutations in Arabidopsis thaliana that selectively eliminate (and substitute) specific xanthophylls in order to study their function(s) in vivo. These include two lutein-deficient mutants, lut1 and lut2, the epoxy xanthophyll-deficient aba1 mutant and the lut2aba1 double mutant. Photosystem stoichiometry, antenna sizes and xanthophyll cycle activity have been related to alterations in nonphotochemical quenching of chlorophyll fluorescence (NPQ). Nondenaturing polyacrylamide gel electrophoresis indicates reduced stability of trimeric LHC II in the absence of lutein (and/or epoxy xanthophylls). Photosystem (antenna) size and stoichiometry is altered in all mutants relative to wild type (WT). Maximal ΔpH-dependent NPQ (qE) is reduced in the following order: WT>aba1>lut1≈lut2>lut2aba1, paralleling reduction in Photosystem (PS) II antenna size. Finally, light-activation of NPQ shows that zeaxanthin and antheraxanthin present constitutively in lut mutants are not qE active, and hence, the same can be inferred of the lutein they replace. Thus, a direct involvement of lutein in the mechanism of qE is unlikely. Rather, altered NPQ in xanthophyll biosynthetic mutants is explained by disturbed macro-organization of LHC II and reduced PS II-antenna size in the absence of the optimal, wild-type xanthophyll composition. These data suggest the evolutionary conservation of lutein content in plants was selected for due to its unique ability to optimize antenna structure, stability and macro-organization for efficient regulation of light-harvesting under natural environmental conditions.     

Interactions between the photosystem II subunit PsbS and xanthophylls studied in vivo and in vitro

The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type.     

Carotenoid binding sites in LHCIIb. Relative affinities towards major xanthophylls of higher plants.

The major light-harvesting complex of photosystem II can be reconstituted in vitro from its bacterially expressed apoprotein with chlorophylls a and b and neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyll. Reconstitution of these one-carotenoid complexes requires low-stringency conditions during complex formation and isolation. Neoxanthin complexes (containing 30-50% of the all-trans isomer) disintegrate during electrophoresis, exhibit a largely reduced resistance against proteolytic attack; in addition, energy transfer from Chl b to Chl a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin or lutein contain nearly two xanthophylls per 12 chlorophylls and are more resistant against trypsin. Lutein-LHCIIb also exhibits an intermediate maintenance of energy transfer at higher temperature. Violaxanthin complexes approach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in recombinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin-LHCIIb exhibits a low thermal stability like neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein-LHCIIb and zeaxanthin-LHCIIb. Binary competition experiments were performed with two xanthophylls at varying ratios in the reconstitution. Analysis of the xanthophyll contents in the reconstitution products yielded information about relative carotenoid affinities of three assumed binding sites. In lutein/neoxanthin competition experiments, two binding sites showed a strong preference (> 200-fold) for lutein, whereas the third binding site had a higher affinity (25-fold) to neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36-fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: two binding sites had a fivefold higher affinity for lutein and one has a threefold higher affinity to zeaxanthin.     

Photoprotection mutants of Arabidopsis thaliana acclimate to high light by increasing photosynthesis and specific antioxidants

Biochemical and physiological acclimation to different light environments is crucial for plant growth and survival. In high light (HL), feedback de-excitation (qE) is a well-known photoprotective mechanism that dissipates excess excitation energy in the light-harvesting antenna of photosystem II (PSII) and relieves excitation pressure in the photosynthetic electron transport chain. The xanthophylls zeaxanthin (Z) and lutein (L) function in qE, but also have roles as antioxidants. Although several studies have shown that qE is important during short-term fluctuations in light intensity, here we show that it is not required for the growth of Arabidopsis thaliana in prolonged HL conditions in the laboratory. Mutants that are deficient in qE alone, qE and Z synthesis, or in qE, Z synthesis and also L synthesis were able to grow at 1800 micromol photons m(-2) s(-1) and exhibited no major symptoms of photooxidative stress. The mutants (and wild type) acclimated to HL by increasing photosynthetic capacity and decreasing light harvesting, which together rendered qE less important for photoprotection. At a metabolite level, the HL-grown mutants appeared to compensate for their remaining qE deficit with increased alpha-tocopherol and ascorbate levels compared to the wild type. The specificity of this response provides insight into the relationship between qE and the antioxidant network in plants.     

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.     

The role of lutein in the acclimation of higher plant chloroplast membranes to suboptimal conditions

Two mutants of Arabidopsis thaliana deficient in lutein have been investigated with respect to their responses to growth under a range of suboptimal conditions. The first mutant, lut1 , was enriched in violaxanthin, antheraxanthin, zeaxanthin and zeinoxanthin compared with the wild-type (WT). In the second mutant, lut2 , the lack of lutein was compensated for only by an increase in xanthophyll cycle (XC) carotenoids. Upon transfer of plants grown under optimal conditions to high light (HL), drought or HL + drought, both mutants acclimated during several days to the new conditions to the same extent as the WT. In contrast, transfer to chilling conditions (6°C) for 6 days induced responses that were different between WT and mutants and between the mutants themselves. In contrast to the WT, the lut2 mutant in particular exhibited a large increase in the Chl a/b ratio and the XC pool size, extensive de-epoxidation and an enhanced extent of non-photochemical quenching. It is suggested that although the role of lutein in the structure and organisation of the light-harvesting complexes can be fulfilled by other xanthophylls under excess light conditions at optimal temperatures, this is not the case at low temperature.     

Carotenoid-binding sites of the major light-harvesting complex II of higher plants.

Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron crystallography in the helix A/helix B cross, have the highest affinity for lutein, but also bind violaxanthin and zeaxanthin with lower affinity. The latter xanthophyll causes disruption of excitation energy transfer. The occupancy of at least one of these sites, probably L1, is essential for protein folding. Neoxanthin is bound to a distinct site (N1) that is highly selective for this species and whose occupancy is not essential for protein folding. Whereas xanthophylls in the L1 and L2 sites interact mainly with chlorophyll a, neoxanthin shows strong interaction with chlorophyll b, inducing the hyperchromic effect of the 652 nm absorption band. This observation explains the recent results of energy transfer from carotenoids to chlorophyll b obtained by femtosecond absorption spectroscopy. Whereas xanthophylls in the L1 and L2 sites are active in photoprotection through chlorophyll-triplet quenching, neoxanthin seems to act mainly in (1)O(2)(*) scavenging.     

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.     

Analysis of the pigment stoichiometry of pigment-protein complexes from barley (Hordeum vulgare). The xanthophyll cycle intermediates occur mainly in the light-harvesting complexes of photosystem I and photosystem II.

The carotenoid zeaxanthin has been implicated in a nonradiative dissipation of excess excitation energy. To determine its site of action, we have examined the location of zeaxanthin within the thylakoid membrane components. Five pigment-protein complexes were isolated with little loss of pigments: photosystem I (PSI); core complex (CC) I, the core of PSI; CC II, the core of photosystem II (PSII); light-harvesting complex (LHC) IIb, a trimer of the major light-harvesting protein of PSII; and LHC IIa, c, and d, a complex of the monomeric minor light-harvesting proteins of PSII. Zeaxanthin was found predominantly in the LHC complexes. Lesser amounts were present in the CCs possibly because these contained some extraneous LHC polypeptides. The LHC IIb trimer and the monomeric LHC II a, c, and d pigment-proteins from dark-adapted plants each contained, in addition to lutein and neoxanthin, one violaxanthin molecule but little antheraxanthin and no zeaxanthin. Following illumination, each complex had a reduced violaxanthin content, but now more antheraxanthin and zeaxanthin were present. PSI had little or no neoxanthin. The pigment content of LHC I was deduced by subtracting the pigment content of CC I from that of PSI. Our best estimate for the carotenoid content of a LHC IIb trimer from dark-adapted plants is one violaxanthin, two neoxanthins, six luteins, and 0.03 mol of antheraxanthin per mol trimer. The xanthophyll cycle occurs mainly or exclusively within the light-harvesting antennae of both photosystems.     

The Arabidopsis szl1 Mutant Reveals a Critical Role of β-Carotene in Photosystem I Photoprotection

Carotenes and their oxygenated derivatives, the xanthophylls, are structural determinants in both photosystems (PS) I and II. They bind and stabilize photosynthetic complexes, increase the light-harvesting capacity of chlorophyll-binding proteins, and have a major role in chloroplast photoprotection. Localization of carotenoid species within each PS is highly conserved: Core complexes bind carotenes, whereas peripheral light-harvesting systems bind xanthophylls. The specific functional role of each xanthophyll species has been recently described by genetic dissection, however the in vivo role of carotenes has not been similarly defined. Here, we have analyzed the function of carotenes in photosynthesis and photoprotection, distinct from that of xanthophylls, by characterizing the suppressor of zeaxanthin-less (szl) mutant of Arabidopsis (Arabidopsis thaliana) which, due to the decreased activity of the lycopene-β-cyclase, shows a lower carotene content than wild-type plants. When grown at room temperature, mutant plants showed a lower content in PSI light-harvesting complex I complex than the wild type, and a reduced capacity for chlorophyll fluorescence quenching, the rapidly reversible component of nonphotochemical quenching. When exposed to high light at chilling temperature, szl1 plants showed stronger photoxidation than wild-type plants. Both PSI and PSII from szl1 were similarly depleted in carotenes and yet PSI activity was more sensitive to light stress than PSII as shown by the stronger photoinhibition of PSI and increased rate of singlet oxygen release from isolated PSI light-harvesting complex I complexes of szl1 compared with the wild type. We conclude that carotene depletion in the core complexes impairs photoprotection of both PS under high light at chilling temperature, with PSI being far more affected than PSII.     

Arabidopsis carotenoid mutants demonstrate that lutein is not essential for photosynthesis in higher plants.

Lutein, a dihydroxy beta, epsilon-carotenoid, is the predominant carotenoid in photosynthetic plant tissue and plays a critical role in light-harvesting complex assembly and function. To further understand lutein synthesis and function, we isolated four lutein-deficient mutants of Arabidopsis that define two loci, lut1 and lut2 (for lutein deficient). These loci are required for lutein biosynthesis but not for the biosynthesis of beta, beta-carotenoids. The lut1 mutations are recessive, accumulate high levels of zeinoxanthin, which is the immediate precursor of lutein, and define lut1 as a disruption in epsilon ring hydroxylation. The lut2 mutations are semidominant, and their biochemical phenotype is consistent with a disruption of epsilon ring cyclization. The lut2 locus cosegregates with the recently isolated epsilon cyclase gene, thus, providing additional evidence that the lut2 alleles are mutations in the epsilon cyclase gene. It appears likely that the epsilon cyclase is a key step in regulating lutein levels and the ratio of lutein to beta,beta-carotenoids. Surprisingly, despite the absence of lutein, neither the lut1 nor lut2 mutation causes a visible deleterious phenotype or altered chlorophyll content, but both mutants have significantly higher levels of beta, beta-carotenoids. In particular, there is a stable increase in the xanthophyll cycle pigments (violaxanthin, antheraxanthin, and zeaxanthin) in both lut1 and lut2 mutants as well as an increase in zeinoxanthin in lut1 and beta-carotene in lut2. The accumulation of specific carotenoids is discussed as it pertains to the regulation of carotenoid biosynthesis and incorporation into the photosynthetic apparatus. Presumably, particular beta, beta-carotenoids are able to compensate functionally and structurally for lutein in the photosystems of Arabidopsis.     

Mechanistic aspects of the xanthophyll dynamics in higher plant thylakoids

Plant thylakoids have a highly conserved xanthophyll composition, consisting of β-carotene, lutein, neoxanthin and a pool of violaxanthin that can be converted to antheraxanthin and zeaxanthin in excess light conditions. Recent work has shown that xanthophylls undergo dynamic changes, not only in their composition but also in their distribution among Lhc proteins. Xanthophylls are released from specific binding site in the major trimeric LHCII complex of photosystem II and are subsequently bound to different sites into monomeric Lhcb proteins and dimeric Lhca proteins. In this work we review available evidence from in vivo and in vitro studies on the structural determinants that control xanthophyll exchange in Lhc proteins. We conclude that the xanthophyll exchange rate is determined by the structure of individual Lhc gene products and it is specifically controlled by the lumenal pH independently from the activation state of the violaxanthin de-epoxidase enzyme. The xanthophyll exchange induces important modifications in the organization of the antenna system of Photosystem II and, possibly of Photosystem I. Major changes consist into a modulation of the light harvesting efficiency and an increase of the protection from lipid peroxidation. The xanthophyll cycle thus appears to be a signal transduction system for co-ordinated regulation of the photoprotection mechanisms under persistent stress from excess light.     

Genetic manipulation of carotenoid biosynthesis and photoprotection

There are multiple complementary and redundant mechanisms to provide protection against photo-oxidative damage, including non-photochemical quenching (NPQ). NPQ dissipates excess excitation energy as heat by using xanthophylls in combination with changes to the light-harvesting complex (LHC) antenna. The xanthophylls are oxygenated carotenoids that in addition to contributing to NPQ can quench singlet or triplet chlorophyll and are necessary for the assembly and stability of the antenna. We have genetically manipulated the expression of the epsilon-cyclase and beta-carotene hydroxylase carotenoid biosynthetic enzymes in Arabidopsis thaliana. The epsilon-cyclase overexpression confirmed that lut2 (lutein deficient) is a mutation in the epsilon-cyclase gene and demonstrated that lutein content can be altered at the level of mRNA abundance with levels ranging from 0 to 180% of wild-type. Also, it is clear that lutein affects the induction and extent of NPQ. The deleterious effects of lutein deficiency on NPQ in Arabidopsis and Chlamydomonas are additive, no matter what the genetic background, whether npq1 (zeaxanthin deficient), aba1 or antisense beta-hydroxylase (xanthophyll cycle pool decreased). Additionally, increasing lutein content causes a marginal, but significant, increase in the rate of induction of NPQ despite a reduction in the xanthophyll cycle pool size.     

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.     

The P450-type carotene hydroxylase PuCHY1 from Porphyra suggests the evolution of carotenoid metabolism in red algae

Carotene hydroxylases catalyze the hydroxylation of a-and b-carotene hydrocarbons into xanthophylls. In red algae, b-carotene is a ubiquitously distributed carotenoid, and hydroxylated carotenoids such...     

Stark effect measurements on monomers and trimers of reconstituted light-harvesting complex II of plants

The electric-field induced absorption changes (Stark effect) of reconstituted light-harvesting complex II (LHCII) in different oligomerisation states-monomers and trimers-with different xanthophyll content have been probed at 77 K. The Stark spectra of the reconstituted control samples, containing the xanthophylls lutein and neoxanthin, are very similar to previously reported spectra of native LHCII. Reconstituted LHCII, containing lutein but no neoxanthin, shows a similar electrooptical response in the Chl a region, but the Stark signal of Chl b around 650 nm amounts to at most approximately 25% of that of the control samples. We conclude that neoxanthin strongly modifies the electronic states of the nearby Chl b molecules causing a large electrooptical response at 650 nm stemming from one or more Chls b in the control samples. Ambiguities about the assignment of several bands in the Soret region [Biochim. Biophys. Acta 1605 (2003) 83] are resolved and the striking difference in electric field response between the two lutein molecules is confirmed. The Stark effect in the carotenoid spectral region in both control and neoxanthin-deficient samples is almost identical, showing that the neoxanthin Stark signal is small and much less intense than the lutein Stark signal.     

The photoprotective molecular switch in the photosystem II antenna

We have reviewed the current state of multidisciplinary knowledge of the photoprotective mechanism in the photosystem II antenna underlying non-photochemical chlorophyll fluorescence quenching (NPQ). The physiological need for photoprotection of photosystem II and the concept of feed-back control of excess light energy are described. The outline of the major component of nonphotochemical quenching, qE, is suggested to comprise four key elements: trigger (ΔpH), site (antenna), mechanics (antenna dynamics) and quencher(s). The current understanding of the identity and role of these qE components is presented. Existing opinions on the involvement of protons, different LHCII antenna complexes, the PsbS protein and different xanthophylls are reviewed. The evidence for LHCII aggregation and macrostructural reorganization of photosystem II and their role in qE are also discussed. The models describing the qE locus in LHCII complexes, the pigments involved and the evidence for structural dynamics within single monomeric antenna complexes are reviewed. We suggest how PsbS and xanthophylls may exert control over qE by controlling the affinity of LHCII complexes for protons with reference to the concepts of hydrophobicity, allostery and hysteresis. Finally, the physics of the proposed chlorophyll-chlorophyll and chlorophyll-xanthophyll mechanisms of energy quenching is explained and discussed. This article is part of a Special Issue entitled: Photosystem II.     

Seasonal changes of violaxanthin cycle pigment de-epoxidation in wintergreen and evergreen plants

We studied carotenoids composition and the activities of the xanthophylls pigments in evergreen conifers (Abies sibirica, Juniperus communis, Picea obovata) and dwarf-shrub (Vaccinium vitis-idaea), and in wintergreen herbaceous plants (Ajuga reptans, Pyrola rotundifolia) growing near Syktyvkar (61°67(/) N 50°77(/) E). The carotenoid pool consisted mainly of following xanthophylls: lutein (70%), neoxanthin (7-10%) and a xanthophylls cycle component - violaxanthin (3-15%). Zeaxanthin and antheraxanthin were found in conifer samples collected in December-March while in other species - during all year. A direct connection between xanthophyll pigment de-epoxidation level and light energy thermal dissipation was shown only for boreal conifer species. It is proposed that zeaxanthin plays a central role in the dissipation of excess excitation energy (nonphotochemical quenching) in the antenna of photosystem II (PSII). We conclude that the increase in the extent of de-epoxidation is beneficial for the retention of PSII activity for conifers in early spring and for herbs in summer.