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.     

Absence of lutein, violaxanthin and neoxanthin affects the functional chlorophyll antenna size of photosystem-II but not that of photosystem-I in the green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii double mutant npq2 lor1 lacks the beta, epsilon-carotenoids lutein and loroxanthin as well as all beta,beta-epoxycarotenoids derived from zeaxanthin (e.g. violaxanthin and neoxanthin). Thus, the only carotenoids present in the thylakoid membranes of the npq2 lor1 cells are beta-carotene and zeaxanthin. The effect of these mutations on the photochemical apparatus assembly and function was investigated. In cells of the mutant strain, the content of photosystem-II (PSII) and photosystem-I (PSI) was similar to that of the wild type, but npq2 lor1 had a significantly smaller PSII light-harvesting Chl antenna size. In contrast, the Chl antenna size of PSI was not truncated in the mutant. SDS-PAGE and Western blot analysis qualitatively revealed the presence of all LHCII and LHCI apoproteins in the thylakoid membrane of the mutant. The results showed that some of the LHCII and most of the LHCI were assembled and functionally connected with PSII and PSI, respectively. Photon conversion efficiency measurements, based on the initial slope of the light-saturation curve of photosynthesis and on the yield of Chl a fluorescence in vivo, showed similar efficiencies. However, a significantly greater light intensity was required for the saturation of photosynthesis in the mutant than in the wild type. It is concluded that zeaxanthin can successfully replace lutein and violaxanthin in most of the functional light-harvesting antenna of the npq2 lor1 mutant.     

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.     

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.     

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.     

Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress

Acclimation to changing environments, such as increases in light intensity, is necessary, especially for the survival of sedentary organisms like plants. To learn more about the importance of ascorbate in the acclimation of plants to high light (HL), vtc2, an ascorbate-deficient mutant of Arabidopsis, and the double mutants vtc2npq4 and vtc2npq1 were tested for growth in low light and HL and compared with the wild type. The vtc2 mutant has only 10% to 30% of wild-type levels of ascorbate, vtc2npq4 has lower ascorbate levels and lacks non-photochemical quenching of chlorophyll fluorescence (NPQ) because of the absence of the photosystem II protein PsbS, and vtc2npq1 is NPQ deficient and also lacks zeaxanthin in HL but has PsbS. All three genotypes were able to grow in HL and had wild-type levels of Lhcb1, cytochrome f, PsaF, and 2-cysteine peroxiredoxin. However, the mutants had lower electron transport and oxygen evolution rates and lower quantum efficiency of PSII compared with the wild type, implying that they experienced chronic photooxidative stress. The mutants lacking NPQ in addition to ascorbate were only slightly more affected than vtc2. All three mutants had higher glutathione levels than the wild type in HL, suggesting a possible compensation for the lower ascorbate content. These results demonstrate the importance of ascorbate for the long-term acclimation of plants to HL.     

The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major route for stress-induced ABA accumulation is via neoxanthin isomers

A novel abscisic acid (ABA)-deficient mutant, aba4, was identified in a screen for paclobutrazol-resistant germination. Compared with wild-type, the mutant showed reduced endogenous ABA levels in both dehydrated rosettes and seeds. Carotenoid composition analysis demonstrated that the defective locus affects neoxanthin synthesis. The ABA4 gene was identified by map-based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted protein has four putative helical transmembrane domains and shows significant similarity to predicted proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis transgenic plants led to increased accumulation of trans-neoxanthin, indicating that the ABA4 protein has a direct role in neoxanthin synthesis. aba4 mutant phenotypes were mild compared with previously identified ABA-deficient mutants that exhibit vegetative tissue phenotypes. Indeed, ABA levels in seeds of aba4 mutants were higher than those of aba1 mutants. As aba1 mutants are also affected in a unique gene, this suggests that ABA can be produced in the aba4 mutant by an alternative pathway using violaxanthin as a substrate. It appears, therefore, that in Arabidopsis both violaxanthin and neoxanthin are in vivo substrates for 9-cis-epoxycarotenoid dioxygenases. Furthermore, significantly reduced levels of ABA were synthesized in the aba4 mutant on dehydration, demonstrating that ABA biosynthesis in response to stress must occur mainly via neoxanthin isomer precursors.     

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.     

Molecular and global time-resolved analysis of a psbS gene dosage effect on pH- and xanthophyll cycle-dependent nonphotochemical quenching in photosystem II

Photosynthetic light harvesting in plants is regulated by a pH- and xanthophyll-dependent nonphotochemical quenching process (qE) that dissipates excess absorbed light energy and requires the psbS gene product. An Arabidopsis thaliana mutant, npq4-1, lacks qE because of a deletion of the psbS gene, yet it exhibits a semidominant phenotype. Here it is shown that the semidominance is due to a psbS gene dosage effect. Diploid Arabidopsis plants containing two psbS gene copies (wild-type), one psbS gene (npq4-1/NPQ4 heterozygote), and no psbS gene (npq4-1/npq4-1 homozygote) were compared. Heterozygous plants had 56% of the wild-type psbS mRNA level, 58% of the wild-type PsbS protein level, and 60% of the wild-type level of qE. Global analysis of the chlorophyll a fluorescence lifetime distributions revealed three components in wild-type and heterozygous plants, but only a single long lifetime component in npq4-1. The short lifetime distribution associated with qE was inhibited by more than 40% in heterozygous plants compared with the wild type. Thus, the extent of qE measured as either the fractional intensities of the PSII chlorophyll a fluorescence lifetime distributions or steady state intensities was stoichiometrically related to the amount of PsbS protein.     

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.     

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.     

Acclimation of the Photosynthetic Apparatus to Growth Irradiance in a Mutant Strain of Synechococcus Lacking Iron Superoxide Dismutase

The acclimation of the photosynthetic apparatus to growth irradiance in a mutant strain of Synechococcus sp. PCC 7942 lacking detectable iron superoxide dismutase activity was studied. The growth of the mutant was inhibited at concentrations of methyl viologen 4 orders of magnitude smaller than those required to inhibit the growth of the wild-type strain. An increased sensitivity of photosynthetic electron transport near photosystem I (PSI) toward photooxidative stress was also observed in the mutant strain. In the absence of methyl viologen, the mutant exhibited similar growth rates compared with those of the wild type, even at high growth irradiance (350 [mu]E m-2 s-1) where chronic inhibition of photosystem II (PSII) was observed in both strains. Under high growth irradiance, the ratios of PSII to PSI and of [alpha]-phycocyanin to chlorophyll a were less than one-third of the values for the wild type. In both strains, cellular contents of chlorophyll a, [alpha]-phycocyanin, and [beta]-carotene, as well as the length of the phycobilisome rods, declined with increasing growth irradiance. Only the cellular content of the carotenoid zeaxanthin seemed to be independent of growth irradiance. These results suggest an altered acclimation to growth irradiance in the sodB mutant in which the stoichiometry between PSI and PSII is adjusted to compensate for the loss of PSI efficiency occurring under high growth irradiance. Similar shortening of the phycobilisome rods in the sodB mutant and wild-type strain suggest that phycobilisome rod length is regulated independently of photosystem stoichiometry.     

Photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas

When there is an imbalance between the light energy absorbed by a photosynthetic organism and that which can be utilized in photosynthesis, photo-oxidative stress can damage pigments, proteins, lipids, and nucleic acids. In this work we compared the wild type and a xanthophyll-deficient mutant of Chlamydomonas reinhardtii in their response to high amounts of light. Wild-type Chlamydomonas cells were able to acclimate to high amounts of light following transfer from low light conditions. In contrast, the npq1 lor1 double mutant, which lacks protective xanthophylls (zeaxanthin and lutein) in the chloroplast, progressively lost viability and photosynthetic capacity along with destruction of thylakoid membrane protein-pigment complexes and accumulation of reactive oxygen species and membrane lipid peroxides. Loss of viability was partially rescued by lowered oxygen tension, suggesting that the high sensitivity of the mutant to light stress is caused by the production of reactive oxygen species in the chloroplast. Cell death was not prevented by the addition of an organic carbon source to the growth medium, demonstrating that the photo-oxidative damage can target other essential chloroplast processes besides photosynthesis. From the differential sensitivity of the mutant to exogenously added pro-oxidants, we infer that the reactive oxygen species produced during light stress in npq1 lor1 may be singlet oxygen and/or superoxide but not hydrogen peroxide. The bleaching phenotype of npq1 lor1 was not due to enhanced photodamage to photosystem II but rather to a less localized phenomenon of accumulation of photo-oxidation products in chloroplast membranes.     

Photodamage of the photosynthetic apparatus and its dependence on the leaf developmental stage in the npq1 Arabidopsis mutant deficient in the xanthophyll cycle enzyme violaxanthin de-epoxidase

The npq1 Arabidopsis mutant is deficient in the violaxanthin de-epoxidase enzyme that converts violaxanthin to zeaxanthin in excess light (xanthophyll cycle). We have compared the behavior of mature leaves (ML) and developing leaves of the mutant and the wild type in various light environments. Thermoluminescence measurements indicated that high photon flux densities (>500 micromol m(-2) s(-1)) promoted oxidative stress in the chloroplasts of npq1 ML, which was associated with a loss of chlorophyll and an inhibition of the photochemical activity. Illuminating leaf discs in the presence of eosin, a generator of singlet oxygen, brought about pronounced lipid peroxidation in npq1 ML but not in wild-type leaves. No such effects were seen in young leaves (YL) of npq1, which were quite tolerant to strong light and eosin-induced singlet oxygen. Non-photochemical energy quenching was strongly inhibited in npq1 YL and ML and was not improved with high-light acclimation. Our results confirm that the xanthophyll cycle protects chloroplasts from photooxidation by a mechanism distinct from non-photochemical energy quenching and they reveal that the absence of xanthophyll cycle can be compensated by other protective mechanisms. npq1 YL were observed to accumulate considerable amounts of vitamin E during photoacclimation, suggesting that this lipophilic antioxidant could be involved in the high phototolerance of those leaves.     

Accumulation of Zeaxanthin in Abscisic Acid-Deficient Mutants of Arabidopsis Does Not Affect Chlorophyll Fluorescence Quenching or Sensitivity to Photoinhibition in Vivo

Abscisic acid (ABA)-deficient mutants of Arabidopsis do not synthesize the epoxy-xanthophylls antheraxanthin, violaxanthin, or neoxanthin. However, thylakoid membranes from these mutants contain 3-fold more zeaxanthin than wild-type plants. This increase in zeaxanthin occurs as a stoichiometric replacement of the missing violaxanthin and neoxanthin within the pigment-protein complexes of both photosystem I and photosystem II (PSII). The retention of zeaxanthin in the dark by ABA-deficient mutants sensitizes the leaves to the development of nonphotochemical quenching (NPQ) during the first 2 to 4 min following a dark-light transition. However, the increase in pool size does not result in any increase in steady-state NPQ. When we exposed wild-type and ABA-deficient mutants leaves to twice growth irradiance, the mutants developed lower maximal NPQ but suffered similar photoinhibition to wildtype, measured both as a decline in the ratio of variable to maximal fluorescence and as a loss of functional PSII centers from oxygen flash yield measurements. These results suggest that only a few of the zeaxanthin molecules present within the light-harvesting antenna of PSII may be involved in NPQ and neither the accumulation of a large pool of zeaxanthin within the antenna of PSII nor an increase in conversion of violaxanthin to zeaxanthin will necessarily enhance photoprotective energy dissipation.     

Stomata from npq1, a zeaxanthin-less Arabidopsis mutant, lack a specific response to blue light

The Arabidopsis mutant npq1, which cannot accumulate zeaxanthin because of a defective violaxanthin deepoxidase, was used to investigate the role of zeaxanthin in the stomatal response to blue light. Neither dark-adapted nor light-treated guard cells or mesophyll cells of the npq1 mutant contained detectable zeaxanthin. In contrast, wild-type guard cells had a significant zeaxanthin content in the dark and accumulated large amounts of zeaxanthin when illuminated. The well-documented red light enhancement of blue light-stimulated stomatal opening, in which increasing fluence rates of background red light result in increased response to blue light, was used to probe the specific blue light response of Arabidopsis stomata. Stomata from the npq1 mutant did not have a specific blue light response under all fluence rates of background red light tested. On the other hand, stomata from leaves of hy4 (cry 1), an Arabidopsis mutant lacking blue light-dependent inhibition of hypocotyl elongation, had a typical enhancement of the blue light response by background red light. The lack of a specific blue light response in the zeaxanthinless npq1 mutant provides genetic evidence for the role of zeaxanthin as a blue light photoreceptor in guard cells.     

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.     

Abscisic-acid-deficient mutants at the aba gene locus of Arabidopsis thaliana are impaired in the epoxidation of zeaxanthin

Abstract. The xanthophyll content of wild type and abscisic acid (ABA) - deficient mutants of pea and Arabidopsis thaliana was determined. The wilty mutant of pea was indistinguishable from the non-mutant control. In contrast, plants homozygous for mutant alleles at the aba locus of Arabidopsis were very different from wild type. In these mutants, zeaxanthin accumulated to abnormally high levels. The major carotenoids, violaxanthin and 9'- cis -neoxanthin were virually absent from the mutant chromatograms. It was concluded that the aba genetic lesion impairs the epoxidation of zeaxanthin to violaxanthin and that this results in an inability to accumulate ABA. This provides clear evidence that zeaxanthin is a precursor of ABA.     

Light-induced stomatal movement of selected Arabidopsis thaliana mutants

Various Arabidopsis thaliana mutants with defects in phytohormone signal transduction or the reception of light were analysed with regard to their stomatal response in a red, red/blue light irradiation programme. Stomatal response to light was detected with a customized gas exchange measurement device, optimized for the small model plant. Small transpiration-kinetic variations of the two wild-type lines Columbia (Col) and Landsberg erecta (Ler) were observed. A comparison of the mutant lines to the respective wild type revealed significant differences for the phytochrome A (phyA-103), the abscisic acid insensitive (aba3-2) and the auxin resistant (axr1-3) mutant. Furthermore, the zeaxanthin-less mutant line npq1-2 showed no alterations in stomatal response to light.