Photosynthesis: Genetic Strategies Adopted to Gain Higher Efficiency.

The global challenge of feeding an ever-increasing population to maintain food security requires novel approaches to increase crop yields. Photosynthesis, the fundamental energy and material basis for plant life on Earth, is highly responsive to environmental conditions. Evaluating the operational status of the photosynthetic mechanism provides insights into plants' capacity to adapt to their surroundings. Despite immense effort, photosynthesis still falls short of its theoretical maximum efficiency, indicating significant potential for improvement. In this review, we provide background information on the various genetic aspects of photosynthesis, explain its complexity, and survey relevant genetic engineering approaches employed to improve the efficiency of photosynthesis. We discuss the latest success stories of gene-editing tools like CRISPR-Cas9 and synthetic biology in achieving precise refinements in targeted photosynthesis pathways, such as the Calvin-Benson cycle, electron transport chain, and photorespiration. We also discuss the genetic markers crucial for mitigating the impact of rapidly changing environmental conditions, such as extreme temperatures or drought, on photosynthesis and growth. This review aims to pinpoint optimization opportunities for photosynthesis, discuss recent advancements, and address the challenges in improving this critical process, fostering a globally food-secure future through sustainable food crop production.

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.

Formation of light-harvesting complex II aggregates from LHCII-PSI-LHCI complexes in rice plants under high light.

During low light- (LL) induced state transitions in dark-adapted rice (Oryza sativa) leaves, light-harvesting complex (LHC) II become phosphorylated and associate with PSI complexes to form LHCII-PSI-LHCI supercomplexes. When the leaves are subsequently transferred to high light (HL) conditions, phosphorylated LHCII complexes are no longer phosphorylated. Under the HL-induced transition in LHC phosphorylation status, we observed a new green band in the stacking gel of native green-PAGE, which was determined to be LHCII aggregates by immunoblotting and 77K chlorophyll fluorescence analysis. Knockout mutants of protein phosphatase 1 (PPH1) which dephosphorylates LHCII failed to form these LHCII aggregates. In addition, the ability to develop non-photochemical quenching in the PPH1 mutant under HL was less than for wild-type plants. As determined by immunoblotting analysis, LHCII proteins present in LHCII-PSI-LHCI supercomplexes included the Lhcb1 and Lhcb2 proteins. In this study, we provide evidence suggesting that LHCII in the LHCII-PSI-LHCI supercomplexes are dephosphorylated and subsequently form aggregates to dissipate excess light energy under HL conditions. We propose that this LHCII aggregation, involving LHCII L-trimers, is a newly observed photoprotective light-quenching process operating in the early stage of acclimation to HL in rice plants.

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.

The STN8 kinase-PBCP phosphatase system is responsible for high-light-induced reversible phosphorylation of the PSII inner antenna subunit CP29 in rice.

Reversible phosphorylation of thylakoid light-harvesting proteins is a mechanism to compensate for unbalanced excitation of photosystem I (PSI) versus photosystem II (PSII) under limiting light. In monocots, an additional phosphorylation event on the PSII antenna CP29 occurs upon exposure to excess light, enhancing resistance to light stress. Different from the case of the major LHCII antenna complex, the STN7 kinase and its related PPH1 phosphatase were proven not to be involved in CP29 phosphorylation, indicating that a different set of enzymes act in the high-light (HL) response. Here, we analyze a rice stn8 mutant in which both PSII core proteins and CP29 phosphorylation are suppressed in HL, implying that STN8 is the kinase catalyzing this reaction. In order to identify the phosphatase involved, we produced a recombinant enzyme encoded by the rice ortholog of AtPBCP, antagonist of AtSTN8, which catalyzes the dephosphorylation of PSII core proteins. The recombinant protein was active in dephosphorylating P-CP29. Based on these data, we propose that the activities of the OsSTN8 kinase and the antagonistic OsPBCP phosphatase, in addition to being involved in the repair of photo-damaged PSII, are also responsible for the HL-dependent reversible phosphorylation of the inner antenna CP29.

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.

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.

Functional implication of ß-carotene hydroxylases in soybean nodulation.

Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative ß-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.

Age-dependent changes in the functions and compositions of photosynthetic complexes in the thylakoid membranes of Arabidopsis thaliana.

Photosynthetic complexes in the thylakoid membrane of plant leaves primarily function as energy-harvesting machinery during the growth period. However, leaves undergo developmental and functional transitions along aging and, at the senescence stage, these complexes become major sources for nutrients to be remobilized to other organs such as developing seeds. Here, we investigated age-dependent changes in the functions and compositions of photosynthetic complexes during natural leaf senescence in Arabidopsis thaliana. We found that Chl a/b ratios decreased during the natural leaf senescence along with decrease of the total chlorophyll content. The photosynthetic parameters measured by the chlorophyll fluorescence, photochemical efficiency (F v/F m) of photosystem II, non-photochemical quenching, and the electron transfer rate, showed a differential decline in the senescing part of the leaves. The CO2 assimilation rate and the activity of PSI activity measured from whole senescing leaves remained relatively intact until 28 days of leaf age but declined sharply thereafter. Examination of the behaviors of the individual components in the photosynthetic complex showed that the components on the whole are decreased, but again showed differential decline during leaf senescence. Notably, D1, a PSII reaction center protein, was almost not present but PsaA/B, a PSI reaction center protein is still remained at the senescence stage. Taken together, our results indicate that the compositions and structures of the photosynthetic complexes are differentially utilized at different stages of leaf, but the most dramatic change was observed at the senescence stage, possibly to comply with the physiological states of the senescence process.

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.

Biological water-oxidizing complex: a nano-sized manganese-calcium oxide in a protein environment.

The resolution of Photosystem II (PS II) crystals has been improved using isolated PS II from the thermophilic cyanobacterium Thermosynechococcus vulcanus. The new 1.9 Å resolution data have provided detailed information on the structure of the water-oxidizing complex (Umena et al. Nature 473: 55-61, 2011). The atomic level structure of the manganese-calcium cluster is important for understanding the mechanism of water oxidation and to design an efficient catalyst for water oxidation in artificial photosynthetic systems. Here, we have briefly reviewed our knowledge of the structure and function of the cluster.

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.

Over-Expression of Dehydroascorbate Reductase Improves Salt Tolerance, Environmental Adaptability and Productivity in Oryza sativa.

Abiotic stress induces reactive oxygen species (ROS) generation in plants, and high ROS levels can cause partial or severe oxidative damage to cellular components that regulate the redox status. Here, we developed salt-tolerant transgenic rice plants that overexpressed the dehydroascorbate reductase gene (OsDHAR1) under the control of a stress-inducible sweet potato promoter (SWPA2). OsDHAR1-expressing transgenic plants exhibited improved environmental adaptability compared to wild-type plants, owing to enhanced ascorbate levels, redox homeostasis, photosynthetic ability, and membrane stability through cross-activation of ascorbate-glutathione cycle enzymes under paddy-field conditions, which enhanced various agronomic traits, including root development, panicle number, spikelet number per panicle, and total grain yield. dhar2-knockdown plants were susceptible to salt stress, and owing to poor seed maturation, exhibited reduced biomass (root growth) and grain yield under paddy field conditions. Microarray revealed that transgenic plants highly expressed genes associated with cell growth, plant growth, leaf senescence, root development, ROS and heavy metal detoxification systems, lipid metabolism, isoflavone and ascorbate recycling, and photosynthesis. We identified the genetic source of functional genomics-based molecular breeding in crop plants and provided new insights into the physiological processes underlying environmental adaptability, which will enable improvement of stress tolerance and crop species productivity in response to climate change.

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.

AtERF71/HRE2 transcription factor mediates osmotic stress response as well as hypoxia response in Arabidopsis.

Various transcription factors are involved in the response to environmental stresses in plants. In this study, we characterized AtERF71/HRE2, a member of the Arabidopsis AP2/ERF family, as an important regulator of the osmotic and hypoxic stress responses in plants. Transcript level of AtERF71/HRE2 was highly increased by anoxia, NaCl, mannitol, ABA, and MV treatments. aterf71/hre2 loss-of-function mutants displayed higher sensitivity to osmotic stress such as high salt and mannitol, accumulating higher levels of ROS under high salt treatment. In contrast, AtERF71/HRE2-overexpressing transgenic plants showed tolerance to salt and mannitol as well as flooding and MV stresses, exhibiting lower levels of ROS under high salt treatment. AtERF71/HRE2 protein was localized in the nucleus, and the C-terminal region of AtERF71/HRE2 was required for transcription activation activity. Taken together, our results suggest that AtERF71/HRE2 might function as a transcription factor involved in the response to osmotic stress as well as hypoxia.

The bZIP transcription factor OsABF1 is an ABA responsive element binding factor that enhances abiotic stress signaling in rice.

A number of basic leucine zipper (bZIP) transcription factors are known to function in stress signaling in plants but few have thus far been functionally characterized in rice. In our current study in rice, we have newly isolated and characterized the OsABF1 (Oryza sativa ABA responsive element binding factor 1) gene that encodes a bZIP transcription factor. Its expression in seedling shoots and roots was found to be induced by various abiotic stress treatments such as anoxia, salinity, drought, oxidative stress, cold and abscisic acid (ABA). Subcellular localization analysis in maize protoplasts using GFP fusion vectors indicated that OsABF1 is a nuclear protein. In a yeast experiment, OsABF1 was shown to bind to ABA responsive elements (ABREs) and its N-terminal region was necessary to transactivate the downstream reporter gene. The homozygous T-DNA insertional mutants Osabf1-1 and Osabf1-2 were more sensitive in response to drought and salinity treatments than wild type plants. Furthermore, the upregulated expression of some ABA/stress-regulated genes in response to ABA treatment was suppressed in these Osabf1 mutants. Our current results thus suggest that OsABF1 is involved in abiotic stress responses and ABA signaling in rice.

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.

Identification of the Optimal Light Harvesting Antenna Size for High-Light Stress Mitigation in Plants.

One of the major constraints limiting biomass production in autotrophs is the low thermodynamic efficiency of photosynthesis, ranging from 1 to 4%. Given the absorption spectrum of photosynthetic pigments and the spectral distribution of sunlight, photosynthetic efficiencies as high as 11% are possible. It is well-recognized that the greatest thermodynamic inefficiencies in photosynthesis are associated with light absorption and conversion of excited states into chemical energy. This is due to the fact that photosynthesis light saturates at one quarter full sunlight intensity in plants resulting in the dissipation of excess energy as heat, fluorescence and through the production of damaging reactive oxygen species. Recently, it has been demonstrated that it is possible to adjust the size of the light harvesting antenna over a broad range of optical cross sections through targeted reductions in chlorophyll b content, selectively resulting in reductions of the peripheral light harvesting antenna size, especially in the content of Lhcb3 and Lhcb6. We have examined the impact of alterations in light harvesting antenna size on the amplitude of photoprotective activity and the evolutionary fitness or seed production in Camelina grown at super-saturating and sub-saturating light intensities to gain an understanding of the driving forces that lead to the selection for light harvesting antenna sizes best fit for a range of light intensities. We demonstrate that plants having light harvesting antenna sizes engineered for the greatest photosynthetic efficiency also have the greatest capacity to mitigate high light stress through non-photochemical quenching and reduction of reactive oxygen associated damage. Under sub-saturating growth light intensities, we demonstrate that the optimal light harvesting antenna size for photosynthesis and seed production is larger than that for plants grown at super-saturating light intensities and is more similar to the antenna size of wild-type plants. These results suggest that the light harvesting antenna size of plants is designed to maximize fitness under low light conditions such as occurs in shaded environments and in light competition with other plants.

OsDEG10 encoding a small RNA-binding protein is involved in abiotic stress signaling.

Excessive light can be harmful to photosynthetic apparatus since it causes photoinhibition and photooxidation, and plants often encounter hypoxic or anoxic environments when they become submerged by heavy rain or an ensuing flood. In this study, Oryza sativa Differentially Expressed Genes (OsDEGs) from rice under photooxidation and anoxia conditions were isolated using DD-PCR. Among them, OsDEG10 is predicted to encode a small RNA-binding protein (RBP) and the transcript levels of OsDEG10 strongly increased under most of abiotic stress treatments such as high light, anoxia, NaCl, ABA, MV and cold. However, the transcript levels of two rice OsDEG10 homologs were not changed under those treatments. OsDEG10 RNAi transgenic plants were more sensitive to high light and cold stresses compared to wild-type plants. Our results suggest that OsDEG10 is a small RBP involved in the response to various abiotic stresses.

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.