High Light Acclimation Mechanisms Deficient in a PsbS-Knockout Arabidopsis Mutant.

The photosystem II PsbS protein of thylakoid membranes is responsible for regulating the energy-dependent, non-photochemical quenching of excess chlorophyll excited states as a short-term mechanism for protection against high light (HL) stress. However, the role of PsbS protein in long-term HL acclimation processes remains poorly understood. Here we investigate the role of PsbS protein during long-term HL acclimation processes in wild-type (WT) and npq4-1 mutants of Arabidopsis which lack the PsbS protein. During long-term HL illumination, photosystem II photochemical efficiency initially dropped, followed by a recovery of electron transport and photochemical quenching (qL) in WT, but not in npq4-1 mutants. In addition, we observed a reduction in light-harvesting antenna size during HL treatment that ceased after HL treatment in WT, but not in npq4-1 mutants. When plants were adapted to HL, more reactive oxygen species (ROS) were accumulated in npq4-1 mutants compared to WT. Gene expression studies indicated that npq4-1 mutants failed to express genes involved in plastoquinone biosynthesis. These results suggest that the PsbS protein regulates recovery processes such as electron transport and qL during long-term HL acclimation by maintaining plastoquinone biosynthetic gene expression and enhancing ROS homeostasis.

Role of Thylakoid Protein Phosphorylation in Energy-Dependent Quenching of Chlorophyll Fluorescence in Rice Plants.

Under natural environments, light quality and quantity are extremely varied. To respond and acclimate to such changes, plants have developed a multiplicity of molecular regulatory mechanisms. Non-photochemical quenching of chlorophyll fluorescence (NPQ) and thylakoid protein phosphorylation are two mechanisms that protect vascular plants. To clarify the role of thylakoid protein phosphorylation in energy-dependent quenching of chlorophyll fluorescence (qE) in rice plants, we used a direct Western blot assay after BN-PAGE to detect all phosphoproteins by P-Thr antibody as well as by P-Lhcb1 and P-Lhcb2 antibodies. Isolated thylakoids in either the dark- or the light-adapted state from wild type (WT) and PsbS-KO rice plants were used for this approach to detect light-dependent interactions between PsbS, PSII, and LHCII proteins. We observed that the bands corresponding to the phosphorylated Lhcb1 and Lhcb2 as well as the other phosphorylated proteins were enhanced in the PsbS-KO mutant after illumination. The qE relaxation became slower in WT plants after 10 min HL treatment, which correlated with Lhcb1 and Lhcb2 protein phosphorylation in the LHCII trimers under the same experimental conditions. Thus, we concluded that light-induced phosphorylation of PSII core and Lhcb1/Lhcb2 proteins is enhanced in rice PsbS-KO plants which might be due to more reactive-oxygen-species production in this mutant.

Dracocephalum palmatum Stephan extract induces caspase­ and mitochondria­dependent apoptosis via Myc inhibition in diffuse large B cell lymphoma.

Dracocephalum palmatum Stephan (DPS), a medicinal plant used by Russian nomads, has been known to exhibit antioxidant properties. However, to the best of our knowledge, its anticancer effect has not been elucidated. The present study aimed to evaluate the tumor­suppressive effect of DPS extract (DPSE) in diffuse large B cell lymphoma (DLBCL) and the underlying mechanism. MTS assays and Annexin V staining were performed to assess the anti­proliferative and apoptotic effects of DPSE, respectively. To reveal the underlying mechanisms, the levels of pro­ and anti­apoptotic Bcl­2 members were analyzed by western blotting. Rescue experiments were performed to investigate the potential involvement of Myc in DPSE­induced tumor­inhibitory effects. Additionally, high­performance liquid chromatography analysis was performed to analyze the components with anticancer effects. Exposure of multiple DLBCL cell lines to DPSE significantly decreased cell viability and increased apoptosis, whereas it had no effect on the survival of normal cells in vitro and in vivo. This indicates that its cytotoxic effect may be specific to cancer cells. Mechanistically, cell death induced by DPSE was dependent on the activation of caspase­3/7 and the disruption of mitochondrial membrane potential. Treatment with the extract ameliorated the expression of anti­apoptotic Bcl­2 members Bcl­xL and Mcl­1, and upregulated that of pro­apoptotic Bcl­2 members Bax and Bak. These modulations led to the disruption of mitochondrial membrane potential, which culminated in the activation of executioner caspases­3 and ­7. Notably, overexpression of Myc inhibited DPSE­induced cell killing, indicating the involvement of Myc in this process. Given that dysregulation of Myc is strongly associated with the pathobiology of DLBCL, the present study highlights the potential therapeutic efficacy of DPSE in patients with DLBCL with aberrant Myc expression. Furthermore, fractionation of DPSE by thin layer chromatography and liquid chromatography/mass spectrometry­based investigation of the fraction with bioactive compounds demonstrated that flavonoids may be responsible for most, if not all, of the anti­lymphoma effect. Efforts to identify the bioactive flavonoids is currently underway.

Diurnal changes of the ascorbate-glutathione cycle components in wheat genotypes exposed to drought.

The ascorbate-glutathione (AsA-GSH) cycle is a major pathway of H2O2 scavenging in plants. The effect of diurnal variations in hydrogen peroxide (H2O2) content, the intensity of lipid peroxidation (malondialdehyde, MDA), photosynthesis, antioxidants and antioxidative enzyme activities involved in AsA-GSH metabolism has been studied comparatively in leaves of durum (Triticum durum Desf.) and bread (Triticum aestivum L.) wheat genotypes exposed to soil drought. Drought stress caused an increase in the content of H2O2, MDA, alterations in the activities of AsA-GSH cycle enzymes and quantitative changes in AsA and GSH content during the day. PSII efficiency was significantly lower in the control and drought exposed leaves at the highest temperature in the afternoon. The ascorbate peroxidase activity was found to increase and ascorbic acid amount decreased with increasing temperature during the day. Further, the glutathione amount and glutathione reductase activity increased at the expense of the regeneration of the oxidised form of glutathione. Our results revealed that wheat can tolerate drought stress by enhancing the antioxidant enzyme activities and alteration of the concentration of ascorbate and glutathione.

Effect of oxygen on the non-photochemical quenching of vascular plants and potential oxygen deficiency in the stroma of PsbS-knock-out rice.

The excessive and harmful light energy absorbed by the photosystem (PS) II of higher plants is dissipated as heat through a protective mechanism termed non-photochemical quenching (NPQ) of chlorophyll fluorescence. PsbS-knock-out (KO) mutants lack the trans-thylakoid proton gradient (ΔpH)-dependent part of NPQ. To elucidate the molecular mechanism of NPQ, we investigated its dependency on oxygen. The development of NPQ in wild-type (WT) rice under low-oxygen (LO) conditions was reduced to more than 50% of its original value. However, under high-oxygen (HO) conditions, the NPQ of both WT and PsbS-KO mutants recovered. Moreover, WT and PsbS-KO mutant leaves infiltrated with the ΔpH dissipating uncoupler nigericin showed increased NPQ values under HO conditions. The experiments using intact chloroplasts and protoplasts of Arabidopsis thaliana supported that the LO effects observed in rice leaves were not due to carbon dioxide deficiency. There was a noticeable 90% reduction in the half-time of P700 oxidation rate in LO-treated leaves compared with that of WT control leaves, but the HO treatment did not significantly change the half-time of P700 oxidation rate. Overall, the results obtained here indicate that the stroma of the PsbS-KO plants could be potentially under O2 deficiency. Because the functions of PsbS in rice leaves are likely to be similar to those in other higher plants, our findings offer novel insights into the role of oxygen in the development of NPQ.

Angelica gigas Nakai and Decursin Downregulate Myc Expression to Promote Cell Death in B-cell Lymphoma.

Angelica gigas Nakai (AGN) is an oriental traditional medicine to treat anemia, dysmenorrhea, and migraine. However, its anti-lymphoma effect is yet to be tested. Here, we demonstrated that AGN and its major component decursin target Myc to suppress lymphomagenesis in vitro and in vivo. AGN inhibited cell viability in multiple B lymphoma cells, while sparing normal splenocytes and bone marrow cells. Increased cleaved PARP level and caspase 3/7 activity and the repression of survival-promoting AKT/mTOR and MAPK pathways downstream of BCR, were responsible for the pro-apoptotic effects of AGN. We found that Myc, a prominent downstream target of these signaling pathways, contributes to AGN-induced cell death. Moreover, co-treatment with AGN and a Myc inhibitor, JQ1 or 10058-F4 yielded synergistic cytotoxic activities against cancer cells with markedly reduced Myc expression. AGN downregulated Myc expression and suppressed tumorigenesis in Eµ-myc transgenic mice. The proapoptotic activities of AGN were recapitulated by decursin, indicating that the anti-tumor effect of AGN was mainly caused by decursin. These findings suggest that AGN and decursin possess potent anti-lymphoma activity, and combination therapies with AGN/decursin and a Myc inhibitor to target Myc more efficiently could be a valuable avenue to explore in the treatment of B-cell lymphoma.

Rice mutants deficient in ω-3 fatty acid desaturase (FAD8) fail to acclimate to cold temperatures.

To investigate the role of ω-3 fatty acid (FA) desaturase (FAD8) during cold acclimation in higher plants, we characterized three independent T-DNA insertional knock-out mutants of OsFAD8 from rice (Oryza sativa L.). At room temperature (28 °C), osfad8 plants exhibited significant alterations in fatty acid (FA) unsaturation for all four investigated plastidic lipid classes. During a 5-d acclimation period at 4 °C, further changes in FA unsaturation in both wild-type (WT) and mutant plants varied according to the type of lipid. We also monitored the fluidity of the thylakoid membrane using a threshold temperature to represent the change in fluorescence. The values were altered significantly by both FAD8 mutation and cold acclimation, suggesting that factors other than FAD8 are involved in C18 FA unsaturation and fluctuations in membrane fluidity. Similarly, significant changes were noted for both the mutant and WT samples in terms of their FA compositions as well as activities related to photosystem (PS) I, PSII, and photoprotection. This included the development of non-photochemical quenching and increased zeaxanthin accumulation. Despite the relatively small changes in FA composition during cold acclimation, cold-inducible FAD8 knock-out mutants displayed strong differences in photoprotective activities and a further drop in membrane fluidity. The mutants were more sensitive than WT to short-term low-temperature stress that resulted in increased production of reactive oxygen species after 5 d of chilling. Taken together, our findings suggest that FA unsaturation by OsFAD8 is crucial for the acclimation of higher plants to low-temperature stress.

Production of superoxide from photosystem II-light harvesting complex II supercomplex in STN8 kinase knock-out rice mutants under photoinhibitory illumination.

When phosphorylation of Photosystem (PS) II core proteins is blocked in STN8 knock-out mutants of rice (Oryza sativa) under photoinhibitory illumination, the mobilization of PSII supercomplex is prevented. We have previously proposed that more superoxide (O2(-)) is produced from PSII in the mutant (Nath et al., 2013, Plant J. 76, 675-686). Here, we clarify the type and site for the generation of reactive oxygen species (ROS). Using both histochemical and fluorescence probes, we observed that, compared with wild-type (WT) leaves, levels of ROS, including O2(-) and hydrogen peroxide (H2O2), were increased when leaves from mutant plants were illuminated with excess light. However, singlet oxygen production was not enhanced under such conditions. When superoxide dismutase was inhibited, O2(-) production was increased, indicating that it is the initial event prior to H2O2 production. In thylakoids isolated from WT leaves, kinase was active in the presence of ATP, and spectrophotometric analysis of nitrobluetetrazolium absorbance for O2(-) confirmed that PSII-driven superoxide production was greater in the mutant thylakoids than in the WT. This contrast in levels of PSII-driven superoxide production between the mutants and the WT plants was confirmed by conducting protein oxidation assays of PSII particles from osstn8 leaves under strong illumination. Those assays also demonstrated that PSII-LHCII supercomplex proteins were oxidized more in the mutant, thereby implying that PSII particles incur greater damage even though D1 degradation during PSII-supercomplex mobilization is partially blocked in the mutant. These results suggest that O2(-) is the major form of ROS produced in the mutant, and that the damaged PSII in the supercomplex is the primary source of O2(-).

Production of superoxide from Photosystem II in a rice (Oryza sativa L.) mutant lacking PsbS.

BACKGROUND: PsbS is a 22-kDa Photosystem (PS) II protein involved in non-photochemical quenching (NPQ) of chlorophyll fluorescence. Rice (Oryza sativa L.) has two PsbS genes, PsbS1 and PsbS2. However, only inactivation of PsbS1, through a knockout (PsbS1-KO) or in RNAi transgenic plants, results in plants deficient in qE, the energy-dependent component of NPQ. RESULTS: In studies presented here, under fluctuating high light, growth of young seedlings lacking PsbS is retarded, and PSII in detached leaves of the mutants is more sensitive to photoinhibitory illumination compared with the wild type. Using both histochemical and fluorescent probes, we determined the levels of reactive oxygen species, including singlet oxygen, superoxide, and hydrogen peroxide, in leaves and thylakoids. The PsbS-deficient plants generated more superoxide and hydrogen peroxide in their chloroplasts. PSII complexes isolated from them produced more superoxide compared with the wild type, and PSII-driven superoxide production was higher in the mutants. However, we could not observe such differences either in isolated PSI complexes or through PSI-driven electron transport. Time-course experiments using isolated thylakoids showed that superoxide production was the initial event, and that production of hydrogen peroxide proceeded from that. CONCLUSION: These results indicate that at least some of the photoprotection provided by PsbS and qE is mediated by preventing production of superoxide released from PSII under conditions of excess excitation energy.

Acceleration of cyclic electron flow in rice plants (Oryza sativa L.) deficient in the PsbS protein of Photosystem II.

When compared with Photosystem I (PSI) in wild-type (WT) rice plants, PSI in PsbS-knockout (KO) plants that lack the energy-dependent component of nonphotochemical quenching (NPQ) was less sensitive to photoinhibition. Therefore, we investigated the relationship between NPQ and cyclic electron flow (CEF) around PSI as a photoprotective mechanism. Activities of two CEF routes (PGR5-dependent or NDH-dependent) were compared between those genotypes by using both dark-adapted plants and pre-illuminated plants, i.e., those in which the Calvin-Benson cycle is de-activated and activated, respectively. In dark-adapted leaves activity of the PGR5-dependent route was determined as the rate of P700 photooxidation. Activity was higher in the mutants than in the WT. However, no difference was noted when plants of either genotype were pre-illuminated. When the electron transport pathway was switched to the cyclic mode by infiltrating leaf segments with 150 mM sorbitol, 40 µM DCMU, and 2 mM hydroxylamine, the rate of P700 oxidation was faster in the mutant. That difference disappeared when leaves were infiltrated with antimycin A to inhibit the PGR5-dependent route. Chlorophyll fluorescence (Fo) was also evaluated. To achieve an Fo level comparable to that of the WT, activation of the NDH-dependent route in the mutant required pre-illumination at a certain dose. Therefore, we propose that, as an alternate pathway for the photoprotection of photosystems in the absence of energy-dependent quenching, this PGR5-dependent route is more highly activated in the PsbS-KO mutants than in the WT. Moreover, that stronger activity is probably responsible for slower activation of the NDH-dependent route in the mutant.

Loss-of-function of OsSTN8 suppresses the photosystem II core protein phosphorylation and interferes with the photosystem II repair mechanism in rice (Oryza sativa).

STN8 kinase is involved in photosystem II (PSII) core protein phosphorylation (PCPP). To examine the role of PCPP in PSII repair during high light (HL) illumination, we characterized a T-DNA insertional knockout mutant of the rice (Oryza sativa) STN8 gene. In this osstn8 mutant, PCPP was significantly suppressed, and the grana were thin and elongated. Upon HL illumination, PSII was strongly inactivated in the mutants, but the D1 protein was degraded more slowly than in wild-type, and mobilization of the PSII supercomplexes from the grana to the stromal lamellae for repair was also suppressed. In addition, higher accumulation of reactive oxygen species and preferential oxidation of PSII reaction center core proteins in thylakoid membranes were observed in the mutants during HL illumination. Taken together, our current data show that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants and to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8-mediated PCPP in PSII repair.

Correlations between the temperature dependence of chlorophyll fluorescence and the fluidity of thylakoid membranes.

To monitor changes in membrane fluidity in Arabidopsis leaves and thylakoid membranes, we investigated the temperature dependence of a chlorophyll fluorescence parameter, minimum fluorescence (Fo), and calculated the threshold temperature [T(Fo)] at which the rise of the fluorescence level Fo was considered to be started. For the modification of membrane fluidity we took three different approaches: (1) an examination of wild-type leaves initially cultured at room temperature (22°C), then exposed to either a lower (4°C) or higher (35°C) temperature for 5 days; (2) measurements of the shift in T(Fo) by two mutants deficient in fatty acid desaturase genes - fad7 and fad7fad8 and (3) an evaluation of the performance of wild-type plants when leaves were infiltrated with chemicals that modify fluidity. When wild-type plants were grown at 22°C, the T(Fo) was 48.3 ± 0.3°C. Plants that were then transferred to a chamber set at 4 or 35°C showed a shift in their T(Fo) to 42.7 ± 0.9°C or 48.9 ± 0.1°C, respectively. Under low-temperature acclimation, the decline in this putative transition temperature was significantly less in fad7 and fad7fad8 mutants compared with the wild-type. In both leaf and thylakoid samples, values for T(Fo) were reduced in samples treated with benzyl alcohol, a membrane fluidizer, whereas T(Fo) rose in samples treated with dimethylsulfoxide, a membrane rigidifier. These results indicate that the heat-induced rise of chlorophyll fluorescence is strongly correlated with the fluidity of thylakoid membranes.

PsbS-specific zeaxanthin-independent changes in fluorescence emission spectrum as a signature of energy-dependent non-photochemical quenching in higher plants.

The PsbS protein of photosystem II is necessary for the development of energy-dependent quenching of chlorophyll (Chl) fluorescence (qE), and PsbS-deficient Arabidopsis plant leaves failed to show qE-specific changes in the steady-state 77 K fluorescence emission spectra observed in wild-type leaves. The difference spectrum between the quenched and un-quenched states showed a negative peak at 682 nm. Although the level of qE development in the zeaxanthin-less npq1-2 mutant plants, which lacked violaxanthin de-epoxidase enzyme, was only half that of wild type, there were no noticeable changes in this qE-dependent difference spectrum. This zeaxanthin-independent DeltaF682 signal was not dependent on state transition, and the signal was not due to photobleaching of pigments either. These results suggest that DeltaF682 signal is formed due to PsbS-specific conformational changes in the quenching site of qE and is a new signature of qE generation in higher plants.

ZEBRA-NECROSIS, a thylakoid-bound protein, is critical for the photoprotection of developing chloroplasts during early leaf development.

The zebra-necrosis (zn) mutant of rice (Oryza sativa) produces transversely green/yellow-striped leaves. The mutant phenotype is formed by unequal impairment of chloroplast biogenesis before emergence from the leaf sheath under alternate light/dark or high/low temperatures (restrictive), but not under constant light and temperature (permissive) conditions. Map-based cloning revealed that ZN encodes a thylakoid-bound protein of unknown function. Virus-induced gene silencing of a ZN homolog in Nicotiana benthamiana causes leaf variegation with sporadic green/yellow sectors, indicating that ZN is essential for chloroplast biogenesis during early leaf development. Necrotic lesions often occur in the yellow sectors as a result of an excessive accumulation of reactive oxygen species (ROS). The phenotypic severity (leaf variegation and necrosis) and ROS levels are positively correlated with an increase in light intensity under restrictive conditions. In the mutant leaves, chlorophyll (Chl) metabolism, ROS scavenging activities, maximum quantum yield of photosystem II (PSII), and structures and functions of the photosynthetic complexes are normal in the Chl-containing cells, suggesting that ROS are mainly generated from the defective plastids of the Chl-free cells. The PSII activity of normal chloroplasts is hypersensitive to photoinhibition because the recovery rates of PSII are much slower. In the PSII repair, the degradation of damaged D1 is not impaired, suggesting a reduced activity of new D1 synthesis, possibly because of higher levels of ROS generated from the Chl-free cells by excess light. Together, we propose that ZN is required for protecting developing chloroplasts, especially during the assembly of thylakoid protein complexes, from incidental light after darkness.

Loss of peripheral polypeptides in the stromal side of photosystem I by light-chilling in cucumber leaves.

Photosystem I (PSI) is severely damaged by chilling at 4 degrees C in low light, especially in the chilling sensitive plant cucumber. To investigate the early events in PSI photoinhibition, we examined structural changes in the level of pigment-protein complexes in cucumber leaves in comparison with pea leaves. The complexes were separated on a native green gel and an increase in the intensity of a band was observed only in light-chilled cucumber leaves. The 77 K fluorescence emission spectrum of this green band indicated that the band was mainly composed of PSI with light-harvesting complex I. Each lane was cut from the green gel and separated on a fully denaturing SDS-PAGE in the second dimension. The new green gel band observed after light-chilling in cucumber leaves lacked 19, 18, and 16.5 kDa polypeptides. These results suggest that light-chilling facilitates the release of three peripheral polypeptides as an early event of chilling stress in vivo, which results in the inactivation of PSI in intact cucumber leaves.

Phytochromes promote seedling light responses by inhibiting four negatively-acting phytochrome-interacting factors.

PIF3 is a phytochrome-interacting basic helix-loop-helix transcription factor that negatively regulates light responses, including hypocotyl elongation, cotyledon opening, and hypocotyl negative gravitropism. However, the role of PIF3 in chlorophyll biosynthesis has not been clearly defined. Here, we show that PIF3 also negatively regulates chlorophyll biosynthesis by repressing biosynthetic genes in the dark. Consistent with the gene expression patterns, the etiolated pif3 mutant accumulated a higher amount of protochlorophyllide and was bleached severely when transferred into light. The photobleaching phenotype of pif3 could be suppressed by the gun5 mutation and mimicked by overexpression of GUN5. When 4 negative phytochrome-interacting protein genes (PIF1, PIF3, PIF4, and PIF5) were mutated, the resulting quadruple mutant seedlings displayed constitutive photomorphogenic phenotypes, including short hypocotyls, open cotyledons, and disrupted hypocotyl gravitropism in the dark. Microarray analysis further confirmed that the dark-grown quadruple mutant has a gene expression pattern similar to that of red light-grown WT. Together, our data indicate that 4 phytochrome-interacting proteins are required for skotomorphogenesis and phytochromes activate photomorphogenesis by inhibiting these factors.

Abnormal chloroplast development and growth inhibition in rice thioredoxin m knock-down plants.

Plant cells contain several thioredoxin isoforms that are characterized by subcellular localization and substrate specificity. Here, we describe the functional characterization of a rice (Oryza sativa) thioredoxin m isoform (Ostrxm) using a reverse genetics technique. Ostrxm showed green tissue-specific and light-responsive mRNA expression. Ostrxm was localized in chloroplasts of rice mesophyll cells, and the recombinant protein showed dithiothreitol-dependent insulin beta-chain reduction activity in vitro. RNA interference (RNAi) of Ostrxm resulted in rice plants with developmental defects, including semidwarfism, pale-green leaves, abnormal chloroplast structure, and reduced carotenoid and chlorophyll content. Ostrxm RNAi plants showed remarkably decreased F(v)/F(m) values under high irradiance conditions (1,000 micromol m(-2) s(-1)) with delayed recovery. Two-dimensional electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight analysis showed that the levels of several chloroplast proteins critical for photosynthesis and biogenesis were significantly decreased in Ostrxm RNAi plants. Furthermore, 2-Cys peroxiredoxin, a known target of thioredoxin, was present in oxidized forms, and hydrogen peroxide levels were increased in Ostrxm RNAi plants. The pleiotropic effects of Ostrxm RNAi suggest that Ostrxm plays an important role in the redox regulation of chloroplast target proteins involved in diverse physiological functions.

Dependence of reaction center-type energy-dependent quenching on photosystem II antenna size.

The effects of photosystem II antenna size on reaction center-type energy-dependent quenching (qE) were examined in rice plants grown under two different light intensities using both wild type and qE-less (OsPsbS knockout) mutant plants. Reaction center-type qE was detected by measuring non-photochemical quenching at 50 micromol photons m(-2) s(-1) white light intensity. We observed that in low light-grown rice plants, reaction center-type qE was higher than in high light-grown plants, and the amount of reaction center-type qE did not depend on zeaxanthin accumulation. This was confirmed in Arabidopsis npq1-2 mutant plants that lack zeaxanthin due to a mutation in the violaxanthin de-epoxidase enzyme. Although the electron transport rate measured at a light intensity of 50 micromol photons m(-2) s(-1) was the same in high light- and low light-grown wild type and mutant plants lacking PsbS protein, the generation of energy-dependent quenching was completely impaired only in mutant plants. Analyses of the pigment content, Lhcb proteins and D1 protein of PSII showed that the antenna size was larger in low light-grown plants, and this correlated with the amount of reaction center-type qE. Our results mark the first time that the reaction center-type qE has been shown to depend on photosystem II antenna size and, although it depends on the existence of PsbS protein, the extent of reaction center-type qE does not correlate with the transcript levels of PsbS protein. The presence of reaction center-type energy-dependent quenching, in addition to antenna-type quenching, in higher plants for dissipation of excess light energy demonstrates the complexity and flexibility of the photosynthetic apparatus of higher plants to respond to different environmental conditions.