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First published online August 28, 2003; 10.1104/pp.103.026252 Plant Physiology 133:748-760 (2003) © 2003 American Society of Plant Biologists Zeaxanthin Deficiency Enhances the High Light Sensitivity of an Ascorbate-Deficient Mutant of Arabidopsis1Department of Plant and Microbial Biology, University of California, Berkeley, California 94720–3102 (P.M.M., K.K.N.); and Commissariat à l'Energie Atomique/Cadarache, Département d'Écophysiologie Végétale et de Microbiologie, Laboratoire d'Ecophysiologie de la Photosynthèse, Unité Mixte de Recherche Centre National de la Recherche Scientifique/Commissariat à l'Energie Atomique 163, Université Méditerranée/Commissariat à l'Energie Atomique 1000, F–13108 Saint-Paul-lez-Durance, France (M.H.)
The ascorbate content of plants is usually increased in high light (HL), implying a function for ascorbate in the acclimation of plants to HL. Nevertheless, the importance of ascorbate in HL acclimation has not yet been tested directly. Here, we report on the acclimation process of an ascorbate-deficient Arabidopsis mutant to HL. The mutant vtc2 has only 10% to 30% of wild-type levels of ascorbate, and it is also slightly deficient in feedback de-excitation (qE), a photoprotective mechanism that causes the dissipation of excess light as heat. The vtc2 mutant was unable to acclimate to HL, when transferred from low light to HL. Its mature leaves bleached, and it showed an increased degree of lipid peroxidation and photoinhibition. In parallel, we tested the photosensitivity of an ascorbate-deficient xanthophyll cycle mutant, vtc2npq1, which also lacks zeaxanthin and nearly all qE. The double mutant bleached sooner and had higher degrees of lipid peroxidation and photoinhibition than the vtc2 mutant. This was in contrast to the npq1 single mutant that showed only slight deviations from the wild-type phenotype under the conditions used. These results demonstrate the antioxidant role of ascorbate in the acclimation process to HL and point to the relative importance of ascorbate in comparison with other photoprotective processes, such as specific xanthophylls or feedback de-excitation. The results also provide further support for the proposed role of zeaxanthin as an antioxidant and lipid stabilizer.
Plants have evolved several mechanisms to protect themselves from excess absorbed light energy. One important set of mechanisms against excess light are antioxidants and antioxidant enzymes, which scavenge or quench dangerous reactive oxygen species (ROS) that are produced as a result of excess light. Antioxidant enzymes include ascorbate peroxidase (APX), superoxide dismutase (SOD), and 2-Cys peroxiredoxin (2-CP). In the chloroplast, SOD and APX work together in the so-called water-water cycle to reduce superoxide, which is produced by reduction of oxygen to water by photosystem (PS) I. The water-water cycle is considered to be an important protective mechanism that also increases the electron transport rate (ETR) and dissipates excess energy (Asada, 1999  ). Chloroplast 2-CP belongs to a ubiquitous group of enzymes, alkyl hydroperoxide reductases, which reduce hydrogen peroxide and alkyl hydroperoxides (Baier and Dietz, 1999; Dietz et al., 2002). It is a stromal enzyme that is localized at thylakoid membranes (König et al., 2002), and it can reduce lipid hydroperoxides that are generated as a result of lipid peroxidation chain reactions (Dietz et al., 2002). Major antioxidants in the plant are the water-soluble molecules ascorbate and glutathione, which are both found in millimolar concentrations (Noctor and Foyer, 1998), and the lipid-soluble tocopherols and carotenoids.
Ascorbate is required for the production of zeaxanthin (Z) in the xanthophyll cycle as a cofactor for violaxanthin (V) de-epoxidase (VDE) and APX and plays an important role in modulating the cell cycle by stimulating cell division and by accelerating cell expansion and cell elongation (Horemans et al., 2000
Another important mechanism that protects against excess light is the harmless dissipation of light energy as heat, known as feedback de-excitation or the qE component of non-photochemical quenching of chlorophyll (Chl) fluorescence. qE requires the build-up of a proton gradient across the thylakoid membrane (Müller et al., 2001
Ascorbate has previously been found at higher levels in plants grown under full sunlight (Grace and Logan, 1996
Ascorbate-Deficient Mutants Bleached within 2 d after Transfer to HL
Plants were transferred to HL after 6 weeks of growth in LL. At this age, wild-type and the npq1 mutant plants were of the same size, whereas vtc2 and vtc2npq1, which had only about 20% of wild-type ascorbate levels (Müller-Moulé et al., 2002
The vtc2 and vtc2npq1 mutants also showed a dramatic increase in lipid peroxidation. The vtc2npq1 mutant showed higher degrees of lipid peroxidation with the thiobarbituric acid reactive substances (TBARS) assay after only 1 d, whereas vtc2 plants had higher levels after 2 d (Fig. 2). The double mutant exhibited twice as much lipid peroxidation as vtc2. There was no clear increase in lipid peroxidation for the wild type and the npq1 mutant during the course of the experiment.
Lipid peroxidation was also measured using a thermoluminescence technique. In plant leaves, lipid peroxidative damage is associated with two thermoluminescence bands peaking at 80°C to 90°C and 135°C to 140°C (Hideg and Vass, 1993 As can be seen in Figure 3A, the four genotypes showed the same differences in degree of lipid peroxidation as were observed in the previous experiment (Fig. 2). The wild type and the npq1 mutant showed a slight increase in lipid peroxidation only after 25 h, whereas both ascorbate-deficient mutants showed much stronger lipid peroxidation already at early time points. The double mutant in particular showed a doubling of lipid peroxidation after only 2.5 h. To ensure that the TBARS and thermoluminescence measurements correlated, TBARS were measured simultaneously with thermoluminescence in this experiment. Figure 3B shows that the TBARS and thermoluminescence measurements for vtc2npq1 were correlated, and that TBARS therefore can be used as an estimate of lipid peroxidation in Arabidopsis. It should be mentioned though that this strong correlation was not found for samples that were extensively bleached, possibly because the thermoluminescence measurements depend on Chl content (Vavilin et al., 1998).
In the first 4 h, all genotypes showed a rapid decrease in Fv/Fm, a Chl fluorescence parameter that is commonly used to measure photoinhibition (Fig. 4A). Fv/Fm continued to decrease drastically in the vtc2npq1 mutant during the 1st d of HL, and also substantially in the vtc2 mutant. After 1 d of HL, the vtc2npq1 mutant had an Fv/Fm value of less than 0.1, whereas the vtc2 mutant still showed a value of 0.35. After 2 d in HL, both ascorbate-deficient mutants had the same low level of Fv/Fm. In contrast, the decline in Fv/Fm was stabilized in the npq1 mutant and the wild type, which did not show any further decrease in Fv/Fm after the 1st d of HL, but rather showed recovery during the next few days of the experiment. No increase in the Fo value were apparent in any genotypes showing that the effects on PSII activity were likely not due to heat stress.
The ETR was estimated from Chl fluorescence parameters (Genty et al., 1989 As expected, the npq1 and the vtc2npq1 mutant exhibited only a very low level of qE (approximately 0.2) when transferred to HL (Fig. 4C). The wild type, on the other hand, showed a maximum qE of 1.0 after 1 d in HL, after which qE started to decrease again. The vtc2 mutant had less qE than the wild type, with the highest values after 1 h in HL. The vtc2 mutant did not show any further increases in qE during the next 2 d in HL.
The ascorbate content of wild type and npq1 started to increase within 1 d in HL, from 2.8 µmol g–1 fresh weight in LL to 9 µmol –1 fresh weight after 5 d in HL (Fig. 5A). In LL, the vtc2 and vtc2npq1 mutants had only about 20% of the ascorbate found in wild type and npq1, and these two mutants showed no change in ascorbate content during the first 2 d of HL. There was no major change in the reduction state of ascorbate in the wild type and the npq1 mutant, but there was a small decrease after 4 h and 1 d in HL, which showed recovery after 2 d in HL (Fig. 5B). The ascorbate pool in the vtc2 mutant was as reduced as that of the wild type in LL, but it was more oxidized after only 1 h in HL and never recovered to LL levels. In LL, the vtc2npq1 double mutant had already a more oxidized pool, which became even more oxidized after only 1 h in HL.No recovery occurred during the first 2 d of the treatment.
Interestingly, glutathione levels in the wild type and the npq1 mutant increased only during the first 4 h in HL (Fig. 5C), whereas the redox state was not significantly affected (Fig. 5D). The ascorbate-deficient mutants also showed a significant increase in glutathione during the first 4 h of HL, after which it remained stable (vtc2) or decreased (vtc2npq1). The redox state of glutathione was similar to that of the wild type except in the vtc2npq1 mutant, which showed a decrease in reduction state after 2 d in HL.
The wild type and the npq1 mutant also exhibited an increase in
All genotypes showed the same pigment phenotype in LL (Figs. 6 and 7), with the exception of vtc2npq1, which had slightly lower Chl a/b ratios. After 1 h in HL, the wild type, which has a functional xanthophyll cycle, showed the typical decrease in V (Fig. 6B) and increase in A, Z, and the de-epoxidation state (Fig. 6, B–E). The vtc2 mutant also showed these changes, but to a lesser extent due to its lower ascorbate content (Müller-Moulé et al., 2002
APX activity increased for all genotypes after transfer to HL (Fig. 8). There was no difference between the genotypes with regard to APX activity in LL or during the first 4 h of the treatment. After 1 d in HL, APX activity of vtc2npq1 was significantly lower than the activities of the wild type, vtc2, and npq1. After 2 d in HL, the APX activity of vtc2 was as low as that of vtc2npq1. Total SOD activity did not change during the course of the experiment, although the data were somewhat variable (data not shown). Plants possess several isozymes of SOD. Arabidopsis has iron (Fe) SOD isozymes and one copper/zinc (CuZn) SOD isozyme in the chloroplast, another CuZnSOD in the cytosol, and a manganese (Mn) SOD in the mitochondria (Kliebenstein et al., 1998
Wild-Type Mutant Plants Acclimated Well to HL
Wild-type Arabidopsis plants grown in LL were able to acclimate to HL conditions. They showed a transient decrease in Fv/Fm (Fig. 4A) and a small increase in lipid peroxidation after 5 d (Fig. 2), but otherwise no visible damage (Fig. 1). Plants also acclimated by decreasing their antenna (total Chl decreased, Chl a/b ratio increased; Fig. 7, D and E) and increasing their ETR (Fig. 4B), ascorbate (Fig. 5A), glutathione (Fig. 5C),
Plants increased their antioxidant capacity also by increasing their antioxidant enzyme content. Total APX activity levels increased already after 1 h and by more than 2-fold after 5 d in HL (Fig. 8), and a small increase in FeSOD protein levels was also seen (Fig. 9A). An increase in FeSOD protein level in response to HL has been reported previously (Kliebenstein et al., 1998
The npq1 mutant behaved similarly to the wild type, but was somewhat more photoinhibited and showed lipid peroxidation slightly earlier than the wild type. No differences were found in regard to ETR, antioxidant, or pigment levels (Figs. 4,5,6,7), except for the lack of V de-epoxidation (Fig. 6). Compared with the wild type, the npq1 mutant showed a faster increase in 2-CP levels (Fig. 9B). This increase in 2-CP might be necessary to compensate for the lack of Z, which functions as an antioxidant (Havaux, 1998
The small difference in photosensitivity between the wild type and the npq1 mutant is somewhat different from a previous result, where the npq1 mutant showed more pronounced lipid peroxidation than the wild type when transferred to HL (Havaux and Niyogi, 1999
In contrast to the npq1 mutant, the ascorbate-deficient mutants were much more sensitive to the HL. The vtc2 and vtc2npq1 mutants showed extensive bleaching (Figs. 1, 6, and 7), and higher degrees of lipid peroxidation (Figs. 2 and 3A) and photoinhibition (Fig. 4A) than the wild type or the npq1 mutant. All (vtc2npq1) or most (vtc2) mature leaves were fully bleached and dried up by the 3rd d of HL, making it impossible to collect tissue at later time points. The vtc2 and vtc2npq1 mutants also had lowered ETR in HL instead of the increase seen in the other two genotypes (Fig. 4B). This could be due to a low operation of the Calvin-Benson cycle due to inactivation of several H2O2-sensitive enzymes, such as Rubisco, glyceraldehyde-3-dehydrogenase, and Fru bisphosphatase (Asada, 1994
Which of the many functions of ascorbate might explain the HL sensitivity of the ascorbate-deficient mutants? It most likely was not the lowered amount of Z (Fig. 6D), due to substrate limitation of VDE (Müller-Moulé et al., 2002
Plants generally respond to an increase in light intensity and the subsequent rise in ROS by increasing the ascorbate and the xanthophyll cycle pool sizes (Grace and Logan, 1996
A mutant totally devoid of all tocopherols, vte1, on the other hand shows no apparent growth defect under optimal conditions (Porfirova et al., 2002 The ascorbate-deficient mutants showed a rapid oxidation of the ascorbate pool within 1 h of HL (Fig. 5B) but showed a difference in regard to the redox state of the glutathione pool only after 2 d in HL (Fig. 5D). It is conceivable that the ascorbate pool was oxidized more rapidly than in the wild type because of the smaller ascorbate pool size. Interestingly, an oxidation of the pool was also observed during aging (see legend to Fig. 5). APX levels increased in the two ascorbate-deficient mutants, but this increase was smaller than in the wild type or the npq1 mutant after 1 d (vtc2npq1) or 2 d (vtc2) in HL. This is probably due to the fact that some regions of the leaf were already bleached, whereas other regions still remained functional and were able to increase APX activity. The ascorbate-deficient mutants also showed an increase in 2-CP levels after 1 d in HL, similar to the npq1 mutant (Fig. 9).
Interestingly, young, developing leaves of both ascorbate-deficient mutants did not show any signs of bleaching (Fig. 1), suggesting that young leaves have a higher capacity for photoprotection. Because leaf temperatures of young and mature leaves of the vtc2npq1 mutant were similar (data not shown), this phenomenon cannot be explained by differences in evaporative cooling between young and mature leaves. However, one likely cause for the increased protection is an increased antioxidant content. For example, young leaves of the npq1 mutant have been shown to be more resistant to HL or oxidative stress, and these leaves had higher levels of
The vtc2npq1 mutant showed more bleaching (Figs. 1, 6, and 7), earlier and higher degrees of lipid peroxidation (Figs. 2 and 3A), lower Fv/Fm values (Fig. 4A), lower ETRs (Fig. 4B), and a more oxidized ascorbate pool in LL than the vtc2 mutant (Fig. 5B). The lack of Z in the vtc2npq1 mutant could conceivably affect a Z-dependent sustained form of thermal energy dissipation (Demmig et al., 1987 Only a small negative impact on performance was noticed in the npq1 mutant relative to the wild type. On the other hand, the lack of Z clearly had a negative effect in the vtc2 mutant background, where the antioxidant content was already reduced compared with the wild type. This clearly points to a synergism between two different photoprotective mechanisms. It also underscores the complexity and multitude of photoprotective mechanisms that have evolved in plants to keep them alive. In conclusion, we have demonstrated the importance of ascorbate for the acclimation to HL in Arabidopsis, and an additional antioxidant role of Z that was especially evident in an ascorbate-deficient background.
Plant Material and Growth Conditions All genotypes used (wild type, npq1, vtc2, and vtc2npq1) were of the Arabidopsis ecotype Col-0. The double mutant (vtc2npq1) was constructed by crossing vtc2-1 with npq1-2. The plants were grown in 10 h of light, 22°C/14 h of dark, 18°C cycle with a photon flux density of 160 µmol photons m–2 s–1 (LL) for 5 weeks and then transferred to a high-intensity discharge lighting chamber (E15, Conviron, Winnipeg, Canada) with a photon flux density of 1,800 µmol photons m–2 s–1 and a day temperature of 20°C. Samples were taken after 1 h in the LL before transfer, and at 1, 4, 24, 48, 72, 96, and 120 h after transfer to the HL. In addition, control plants were measured that had remained in the LL for 144 h. Any significant differences between the control plants and the LL-grown plants before the experiments are noted in the figure legends. Only fully expanded leaves were sampled. Severely bleached leaves were taken only if they were not dried out yet. The experiment was repeated two to four times with independently grown sets of plants. As a precaution, leaf temperatures of young and mature leaves of the wild type and the vtc2npq1 mutant were measured. Leaf temperatures increased by approximately 5°C after transfer to HL and did not differ between young and mature leaves or between genotypes, either in LL or after transfer to HL (data not shown). Light stress was also imposed on detached leaves (Fig. 3). The leaves placed on wet filter paper were exposed to white light (1,000 µmol photon m–2 s–1) produced by 150-W metal halide lamps equipped with two infrared suppressor filters. Leaf temperature was maintained constant at 9°C ± 1°C.
A total of nine samples (three sets with three samples each) were measured for each time point. Standard modulated Chl fluorescence parameters were measured with an FMS2 instrument (Hansatech, King's Lynn, UK). qE was calculated as (Fm – Fm')/Fm' and
A total of six samples (three sets with two samples each) were measured for each time point following the protocol described by Li et al. (2002
Total and reduced ascorbate were determined by a spectrophotometric method using the UV absorption of reduced ascorbate at 265 nm (Conklin et al., 1996
Total and reduced glutathione were determined by HPLC followed by electrochemical detection (CoulArray, ESA, Chelmsford, MA). A total of six to nine leaf-disc samples (three sets with two–three samples each) were taken for each time point and immediately frozen in liquid nitrogen. The frozen discs were ground to a fine powder and extracted with 300 µL of 2% (w/v) metaphosphoric acid/2 mM EDTA, followed by 30 s of vortexing. The extract was centrifuged at 15,300g for 3 min, and 20 µL of the filtered supernatant was subjected to HPLC and separated on a TSK-GEL ODS-80Tm column at 30°C with a flow rate of 1 mL min–1 for 20 min. The mobile phase consisted of 0.2 M potassium phosphate (pH 3), 100 mg L–1 1-pentanesulfonic acid, and 1% (v/v) acetonitrile. The eight channels of the electrochemical detector were set at –200, –100, 0, +450, +650, +750, +880, and +900 mV. GSH was detected at +650 and +750 mV with +650 mV being the major channel. Oxidized glutathione was detected at +650, +750, and +880 mV with +750 mV being the major channel.
Lipid peroxidation was determined as the level of TBARS (Hodges et al., 1999
Leaves were harvested and immediately frozen in liquid nitrogen. For total protein extraction, leaves were ground in extraction buffer (4% [w/v] SDS, 25 mM Tris-HCl [pH 8.8], and 2.5% [v/v] glycerol). Samples were then boiled in solubilization buffer (2x buffer: 16% [w/v] SDS, 100 mM Tris-HCl, pH 8.8, and 10% [v/v] glycerol) followed by a 30-min incubation after the addition of 10% [v/v]
Two leaf discs were taken and immediately frozen in liquid nitrogen. The leaf discs were ground to a fine powder in 1.5 mL of extraction buffer using mortar and pestle. A previously described, APX assay was used (Grace and Logan, 1996 For SOD, the reduction of cytochrome c by superoxide was determined after treatment of the extracts with ascorbate oxidase. The same extraction buffer as for APX was used. Superoxide was generated by a phenazine methosulfate/NADH system using 100 µM NADH, 50 nM phenazine methosulfate, and 10 µM cytochrome c. SOD activity was also measured as a plate assay. One control rate and seven samples were measured simultaneously with nonspecific rates measured in neighboring wells. One unit of SOD activity causes a half-maximal inhibition of cytochrome c reduction at a rate of 0.025 A550 units min–1 in a volume of 300 µL.
We thank Margarete Baier, Patricia Conklin, Karl-Josef Dietz, and Anna Haldrup for providing us with antibodies. We also thank Heidi Ledford for critical reading of the manuscript and Barry Logan for assistance with the APX activity measurements and critical reading of the manuscript. Received May 1, 2003; returned for revision May 25, 2003; accepted June 18, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.026252.
1 This work was supported by the U.S. Department of Agriculture National Research Initiative (grant no. 98–35306–6600), by the Director, Office of Science, Office of Basic Energy Sciences, Chemical Science Division, U.S. Department of Energy (contract no. DE–AC03–765F00098), by the Searle Scholars Program/The Chicago Community Trust, and by the France-Berkeley Fund. * Corresponding author; e-mail niyogi{at}nature.berkeley.edu; fax 510–642–4995.
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ABSTRACT
RESULTS
-Tocopherol. All LL controls measured after the experiment were within the SE of the initial LL mean.
-carotene, and total Chl (
PSII as (Fm' – Fs)/Fm', with Fm' being measured in the light and Fm being defined as the value measured after 15 min dark-adaptation. ETR was calculated as photosynthetically active radiation * 0.5 * 0.84 * 
