The biochemical, biophysical, and physiological properties of the PsbS protein were studied in relation to mutations of two symmetry-related, lumen-exposed glutamate residues, Glu-122 and Glu-226. These two glutamates are targets for protonation during lumen acidification in excess light. Mutation of PsbS did not affect xanthophyll cycle pigment conversion or pool size. In conditions of excess light, photosynthetic light harvesting is regulated by a feedback de-excitation mechanism termed energy-dependent quenching (qE), 1 which increases thermal dissipation of excess absorbed light energy in photosystem II (PSII). The qE mechanism is triggered by conditions that limit photosynthetic carbon fixation and result in increased acidification of the chloroplast thylakoid lumen (1-4). The thermal dissipation of excess excitation energy is most commonly measured and referred to as nonphotochemical quenching (NPQ) of PSII chlorophyll (Chl) a fluorescence. Although there are several components of NPQ, in higher plants qE can account for the major part of NPQ and is characterized by its relatively fast induction and relaxation kinetics, on a physiological time scale of seconds to minutes. The decrease in the intensity of Chl fluorescence is the result of the decrease in the electronic excited state lifetime of Chl caused by an increased thermal dissipation rate constant (5). The rapid response of the qE process is chemically associated with changes in the trans-thylakoid membrane pH gradient (⌬pH). The ⌬pH change has at least two functions in qE. First, it activates the violaxanthin de-epoxidase that converts violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) (6). A and/or Z are essential elements of qE (7-9). Second, the lower pH in the lumen results in protonation of PSII proteins, including the 22-kDa PSII subunit, PsbS, which plays a key role in qE (10). When both pH-induced changes occur together it is believed that Chls in PSII can transfer their excess energy to Z, which can return to the ground state via thermal decay (7,11,12). Plants containing PsbS mutations of both glutamatesThe pH-sensing mechanism of the PsbS protein is influenced by two pairs of symmetrically arranged glutamate residues, each located within or close to the two lumen-exposed loops of the protein (13). Dicyclohexylcarbodiimide (DCCD), a well known inhibitor of qE (14 -16) is a carboxylate-modifying agent (17) that binds to PsbS (18). Although it was suggested that the DCCD binding site is in the lumenal loops of PsbS, the exact binding site has not been determined. Importantly, site-directed mutagenesis experiments indicated that two of the PsbS glutamates, Glu-122 and Glu-226, are necessary for the function of PsbS (13).In this article we used single and double mutations of PsbS (E122Q/E226Q) to make a detailed biochemical and biophysical analysis of the role of these two glutamates in pH sensing and DCCD binding. We probed the role of the Glu-122 and Glu-226 residues by monitoring the changes in the PSII Chl a fluores-

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. During the course of their life cycle, plants are exposed to a varying light environment, such as slow seasonal changes and a sudden increase in light intensity because of an opening in the leaf canopy. Plants have been evolving to cope with this changing light environment in a way that not only enables them to harvest light optimally but also to protect themselves from excess light. Excess absorbed light is dangerous to plants because it can lead to the enhanced production of reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), superoxide, hydroxyl radicals, and singlet oxygen (Niyogi, 1999), which can damage many cellular components (Foyer, 1997), including PSII and PSI.Much is already known about how plants acclimate to high light (HL). Important responses include a reduction in the size of the light-harvesting complex and an increase in the rate of photosynthesis, which correlates with increases in ATP synthase, electron transport components, and Calvin-Benson cycle enzymes (Anderson and Osmond, 1987;Walters and Horton, 1994). Other acclimation responses include changes in morphology. Leaves that have developed in HL (sun leaves) are generally thicker because of more cell layers with a higher leaf mass per unit area than leaves that have developed in low light (LL; shade leaves; Bjö rkman, 1981; Pearcy, 1998).One very fast response to an increased light intensity is non-photochemical quenching of chlorophyll (Chl) fluorescence (NPQ), which dissipates excess energy as heat (Mü ller et al., 2001). NPQ is composed of three parts: qT, qI, and qE. qT is caused by state transitions, and in vascular plants, it is gen...

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 wellknown 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 m mol 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 a -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 participation of the amino acid ␤83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by sitedirected mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid ␤83 was changed from Glu to Asp (␤E83D) and to Lys (␤E83K), and the highly conserved tetrapeptide ␤T82-E83-G84-L85 (⌬TEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15⌬atpB and FUD50 with the mutated atpB genes. The transformants containing the ␤E83D and ␤E83K mutant genes grew well photoautotrophically. The ⌬TEGL transformant did not grow photoautotrophically, and no CF 1 subunits were detected by immunostaining of Western blots using CF 1 specific antibodies. The rates of ATP synthesis at clamped ⌬pH with thylakoids isolated from cw15 and the two mutants, ␤E83D and ␤E83K, were similar. However, only the phosphorylation activity of the mutant ␤E83D was inhibited by tentoxin with 50% inhibition attained at 4 M. These results confirm that amino acid ␤83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF 1 s isolated from cw15, ␤E83D, and ␤E83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF 1 from the ␤E83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the ␤E83K-CF 1 than that of CF 1 from the other two strains. Moreover, the optimal activity of the ␤E83K-CF 1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the "crown" domain affects the active conformation of the CF 1 -ATPase.The eukaryotic unicellular green algae Chlamydomonas reinhardtii constitutes a powerful experimental model system for the study of the photosynthetic machinery. It is accessible to genetic analysis and grows photoautotrophically on minimal medium or heterotrophically with acetate as the sole carbon source. These properties have been used to isolate numerous photosynthetic mutants which have helped to examine the function of the photosynthetic apparatus (1).The chloroplast of C. reinhardtii contains approximately 80 copies of its 196-kb 1 circular genome (2). Due to recent progress in the molecular genetics of C. reinhardtii, chloroplast proteins can be altered by site-directed mutagenesis of the corresponding genes followed by transformation into the chloroplast (3-7). Chloroplast transformation was first demonstrated in 1988 by Boynton and co-workers (3), by complementation of an atpB deletion mutant with the cloned wild type gene. The transforming DNA integrates into the recipient chloroplast DNA by homologous recombination. Goldschmidt-Clermont (5) has constructed a chimeric selectable marker using ...

No abstract