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Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that exceeds a plant's capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding that has implications for the functional evolution of pigment-binding proteins.

Photon yields of oxygen evolution at saturating CO2 were determined for 44 species of vascular plants, representing widely diverse taxa, habitats, life forms and growth conditions. The photonyield values on the basis of absorbed light (φ (a)) were remarkably constant among plants possessing the same pathway of photosynthetic CO2 fixation, provided the plants had not been subjected to environmental stress. The mean φ (a) value ±SE for 37 C3 species was 0.106±0.001 O2·photon(-1). The five C4 species exhibited lower photon yields and greater variation than the C3 species (φ (a)=0.0692±0.004). The φ (a) values for the two Crassulaceanacid-metabolism species were similar to those of C3 species. Leaf chlorophyll content had little influence on φ (a) over the range found in normal, healthy leaves. Chlorophyll fluorescence characteristics at 77 K were determined for the same leaves as used for the photon-yield measurements. Considerable variation in fluorescence emission both at 692 nm and at 734 nm, was found 1) among the different species; 2) between the upper and lower surfaces of the same leaves; and 3) between sun and shade leaves of the same species. By contrast, the ratio of variable to maximum fluorescence emission at 692 nm (Fv/FM, 692) remained remarkably constant (The mean value for the C3 species was 0.832±0.004). High-light treatments of shade leaves resulted in a reduction in both φ (a) and the Fv/FM, 692 ratio. The extent of the reductions increased with time of exposure to bright light. A linear relationship was obtained when φ (a) was plotted against Fv/FM, 692. The results show that determinations of the photon yield of O2 evolution and the Fv/FM, 692 ratio can serve as excellent quantitative measures of photoinhibition of overall photosynthetic energy-conversion system and of photochemistry of photosystem II, respectively. This is especially valuable in field work where it is often impossible to obtain appropriate controls.

No abstract

The role of the xanthophyll cycle in regulating the energy flow to the PS II reaction centers and therefore in photoprotection was studied by measurements of light-induced absorbance changes, Chl fluorescence, and photosynthetic O2 evolution in sun and shade leaves of Hedera canariensis. The light-induced absorbance change at 510 nm (ΔA510) was used for continuous monitoring of zeaxanthin formation by de-epoxidation of violaxanthin. Non-radiative energy dissipation (NRD) was estimated from non-photochemical fluorescence quenching (NPQ).High capacity for zeaxanthin formation in sun leaves was accompanied by large NRD in the pigment bed at high PFDs as indicated by a very strong NPQ both when all PS II centers are closed (F'm) and when all centers are open (F'o). Such Fo quenching, although present, was less pronounced in shade leaves which have a much smaller xanthophyll cycle pool.Dithiothreitol (DTT) provided through the cut petiole completely blocked zeaxanthin formation. DTT had no detectable effect on photosynthetic O2 evolution or the photochemical yield of PS II in the short term but fully inhibited the quenching of Fo and 75% of the quenching of Fm, indicating that NRD in the antenna was largely blocked. This inhibition of quenching was accompanied by an increased closure of the PS II reaction centers.In the presence of DTT a photoinhibitory treatment at a PFD of 200 μmol m(-2) s(-1), followed by a 45 min recovery period at a low PFD, caused a 35% decrease in the photon yield of O2 evolution, compared to a decrease of less than 5% in the absence of DTT. The Fv/Fm ratio, measured in darkness showed a much greater decrease in the presence than in the absence of DTT. In the presence of DTT Fo rose by 15-20% whereas no change was detected in control leaves.The results support the conclusion that the xanthophyll cycle has a central role in regulating the energy flow to the PS II reaction centers and also provide direct evidence that zeaxanthin protects against photoinhibitory injury to the photosynthetic system.

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. INTRODUCTIONPlants in nature experience variations in incident light quantity over several orders of magnitude on a daily basis. Although the reactions that convert solar energy into chemical energy are remarkably efficient, the capacity of these reactions is limited; therefore, plants often absorb more light energy than they are able to use for photosynthesis. In addition, many environmental stresses, including drought, extremes of temperature, or nutrient deprivation (DemmigAdams and Adams, 1992), can further limit the ability of a plant to utilize light energy. Absorption of excessive light energy can lead to sustained depressions in photosynthetic efficiency (photoinhibition), often due to oxidative damage to the photosynthetic apparatus.Photosynthetic organisms have evolved multiple mechanisms to cope with the absorption of excessive light and its consequences. Interception of incident light can be decreased by reorientation and/or movement of chloroplasts within cells (Brugnoli and Björkman, 1992) and, in some plants, by leaf movements (Björkman and Demmig-Adams, 1994). Carotenoids, including the xanthophylls, have essential photoprotective roles as quenchers of triplet chlorophyll ( 3 Chl) and singlet oxygen ( 1 O 2 ) and as inhibitors of lipid peroxidation (Cogdell and Frank, 1987; Frank and Cogdell, 1993;Demmig-Adams et al., 1996). Another important lipidsoluble antioxidant is ␣ -tocopherol (vita...

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

As a part of our investigations to test the hypothesis that zeaxanthin formed by reversible de-epoxidation of violaxanthin serves to dissipate any excessive and potentially harmful excitation energy we determined the influence of light climate on the size of the xanthophyll cycle pool (violaxanthin + antheraxanthin + zeaxanthin) in leaves of a number of species of higher plants. The maximum amount of zeaxanthin that can be formed by de-epoxidation of violaxanthin and antheraxanthin is determined by the pool size of the xanthophyll cycle. To quantitate the individual leaf carotenoids a rapid, sensitive and accurate HPLC method was developed using a non-endcapped Zorbax ODS column, giving baseline separation of lutein and zeaxanthin as well as of other carotenoids and Chl a and b.The size of the xanthophyll cycle pool, both on a basis of light-intercepting leaf area and of light-harvesting chlorophyll, was ca. four times greater in sun-grown leaves of a group of ten sun tolerant species than in shade-grown leaves in a group of nine shade tolerant species. In contrast there were no marked or consistent differences between the two groups in the content of the other major leaf xanthophylls, lutein and neoxanthin. Also, in each of four species examined the xanthophyll pool size increased with an increase in the amount of light available during leaf development whereas there was little change in the content of the other xanthophylls. However, the α-carotene/β-carotene ratio decreased and little or no α-carotene was detected in sun-grown leaves. Among shade-grown leaves the α-carotene/β-carotene ratio was considerably higher in species deemed to be umbrophilic than in species deemed to be heliophilic.The percentage of the xanthophyll cycle pool present as violaxanthin (di-epoxy-zeaxanthin) at solar noon was 96-100% for shade-grown plants and 4-53% for sun-grown plants with zeaxanthin accounting for most of the balance. The percentage of zeaxanthin in leaves exposed to midday solar radiation was higher in those with low than in those with high photosynthetic capacity.The results are consistent with the hypothesis that the xanthophyll cycle is involved in the regulation of energy dissipation in the pigment bed, thereby preventing a buildup of excessive excitation energy at the reaction centers.