Characteristics of photosynthesis and functions of the water-water cycle in rice (Oryza sativa) leaves in response to potassium deficiency

The mechanisms of photoprotection of photosynthesis and dissipation of excitation energy in rice leaves in response to potassium (K) deficiency were investigated. Net photosynthetic rate and the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase decreased under K deficiency. Compared with the control, non-photochemical quenching of Chl fluorescence increased in K-deficient plant, whereas the efficiency of excitation transfer (F'(v)/F'(m)) and the photochemical quenching coefficient (q(P)) decreased. Thus, thermal dissipation of excitation energy increased as more excess electrons were accumulated in the photosynthetic chain. The electron transport rate through PSII (J(f)) was more sensitive to O2 concentration, and the fraction of electron transport rate required to sustain CO2 assimilation and photorespiration (J(g)/J(f)) was significantly decreased under K deficiency compared with the control. Furthermore, the alternative electron transport (J(a)/J(f)) was increased, indicating that a considerable amount of electrons had been transported to O2 during the water-water cycle in the K-deficient leaves. Although the fraction of electron transport to photorespiration (J(o)/J(f)) was also increased in the K-deficient leaves, it was less sensitive than that of the water-water cycle. With the generation of reactive oxygen species level, the activities of superoxide dismutase and ascorbate peroxidase, two of the key enzymes involved in scavenging of active oxygen species in the water-water cycle, also increased in K-deficient rice. Therefore, it is likely that a series of photoprotective mechanisms were initiated in rice plants in response to K deficiency and the water-water cycle might be critical for protecting photosynthetic apparatus under K deficiency in rice.     

The biogenesis and physiological function of chloroplast superoxide dismutases

Iron-superoxide dismutase (FeSOD) and copper/zinc-superoxide dismutase (Cu/ZnSOD) are evolutionarily conserved proteins in higher plant chloroplasts. These enzymes are responsible for the efficient removal of the superoxide formed during photosynthetic electron transport and function in reactive oxygen species metabolism. The availability of copper is a major determinant of Cu/ZnSOD and FeSOD expression. Analysis of the phenotypes of plants that over-express superoxide dismutases in chloroplasts has given support for the proposed roles of these enzymes in reactive oxygen species scavenging. However, over-production of chloroplast superoxide dismutase gives only limited protection to environmental stress and does not result in greatly improved whole plant performance. Surprisingly, plant lines that lack the most abundant Cu/ZnSOD or FeSOD activities perform as well as the wild-type under most conditions tested, indicating that these superoxide dismutases are not limiting to photoprotection or the prevention of oxidative damage. In contrast, a strong defect in chloroplast gene expression and development was seen in plants that lack the two minor FeSOD isoforms, which are expressed predominantly in seedlings and that associate closely with the chloroplast genome. These findings implicate reactive oxygen species metabolism in signaling and emphasize the critical role of sub-cellular superoxide dismutase location. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

The water-water cycle as alternative photon and electron sinks

The water-water cycle in chloroplasts is the photoreduction of dioxygen to water in photosystem I (PS I) by the electrons generated in photosystem II (PS II) from water. In the water-water cycle, the rate of photoreduction of dioxygen in PS I is several orders of magnitude lower than those of the disproportionation of superoxide catalysed by superoxide dismutase, the reduction of hydrogen peroxide to water catalysed by ascorbate peroxidase, and the reduction of the resulting oxidized forms of ascorbate by reduced ferredoxin or catalysed by either dehydroascorbate reductase or monodehydroascorbate reductase. The water-water cycle therefore effectively shortens the lifetimes of photoproduced superoxide and hydrogen peroxide to suppress the production of hydroxyl radicals, their interactions with the target molecules in chloroplasts, and resulting photoinhibition. When leaves are exposed to photon intensities of sunlight in excess of that required to support the fixation of CO2, the intersystem electron carriers are over-reduced, resulting in photoinhibition. Under such conditions, the water-water cycle not only scavenges active oxygens, but also safely dissipates excess photon energy and electrons, in addition to downregulation of PS II and photorespiration. The dual functions of the water-water cycle for protection from photoinhibition under photon excess stress are discussed, along with its functional evolution.     

Photosynthetic apparatus of chilling-sensitive plants. XI. Reversibility by light of cold- and dark-induced inactivation of cyanide-sensitive superoxide dismutase activity in tomato leaf chloroplasts

(1) The inactivation of cyanide-sensitive, copper- and zinc-containing superoxide dismutase activity in chloroplasts following cold and dark storage of both detached leaves and growing tomato plants is accompanied by a decrease in copper and zinc content in both chloroplast preparations and butanol extracts of the enzyme. In contrast, this treatment of chloroplast preparations affects neither superoxide dismutase activity nor copper and zinc content. (2) Copper- and zinc-containing superoxide dismutase is not reactivated following the 2–3 h illumination of cold- and dark-stored detached leaves. However, prolonged illumination of growing seedlings results in the restoration of both the enzyme activity and copper and zinc content in chloroplasts. (3) The data suggest that the dissociation of copper, and probably of zinc, from the enzyme during cold and dark treatment of either detached leaves or growing plants and reincorporation of the metals following the illumination of intact plants are responsible for the reversible inactivation of chloroplast cyanide-sensitive superoxide dismutase of chilling-sensitive plants.     

Cellular response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice exposed to hyperoxia

Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.     

Impairment of glutamate transport and increased vulnerability to oxidative stress in neuroblastoma SH-SY5Y cells expressing a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis

Human neuroblastoma SH-SY5Y cells transfected with either familial amyotrophic lateral sclerosis-typical G93A mutant or wild-type copper/zinc superoxide dismutase were compared to untransfected cells in term of glutamate transport. Vmax of glutamate uptake was reduced in mutant cells, with no change in Km. No difference in EAAT1, EAAT2 and EAAT3 glutamate transporter mRNAs and immunoreactive proteins was found, suggesting that one or more transporters are functionally inactivated, possibly due to increased oxidative stress induced by the G93A mutation. Mutant cells showed a marked sensitivity to oxidants, resulting in a more pronounced reduction of glutamate uptake. Short-term antioxidant treatment did not reverse the impairment of glutamate uptake in G93A cells. Interestlingly, N-acetylcysteine was partially effective in preventing glutamate uptake reduction due to exogenous oxidative insults. Since the inhibition of the EAAT2 transporter subtype had no effect on glutamate re-uptake in this model, our study suggests an impaired function of the EAAT1/3 transporter subtypes, possibly due to oxidative inactivation, in the presence of mutant copper/zinc superoxide dismutase. Therefore, this model might prove to be a valuable tool to study the effects of mutant copper/zinc superoxide dismutase associated with amyotrophic lateral sclerosis on glutamate transport in neuronal cells, without the specific contribution of glial cells. These findings might lead to the identification of new therapeutic strategies aimed at preventing the damage associated with ALS.     

Water-water cycle involved in dissipation of excess photon energy in phosphorus deficient rice leaves

The water-water cycle which may be helpful for dissipating the excitation pressure over electron transport chain and minimizing the risk of photoinhibition and photodamage was investigated in rice after 10-d P-deficient treatment. Net photosynthetic rate decreased under P-deficiency, thus the absorption of photon energy exceeded the energy required for CO₂ assimilation. A more sensitive response of effective quantum yield of photosystem 2 (Φ_PS2) to O₂ concentration was observed in plants that suffered P starvation, indicating that more electrons were transported to O₂ in the P-deficient leaves. The electron transport rate through photosystem 2 (PS 2) (J_f) was stable, and the fraction of electron transport rate required to sustain CO₂ assimilation and photorespiration (J_g/J_f) was significantly decreased accompanied by an increase in the alternative electron transport (J_a/J_f), indicating that a considerable electron amount had been transported to O₂ during the water-water cycle in the P-deficient leaves. However, the fraction of electron transport to photorespiration (J_o/J_f) was also increased in the P-deficient leaves and it was less sensitive than that of water-water cycle. Therefore, water-water cycle could serve as an efficient electron sink. The higher non-photochemical fluorescence quenching (q_N) in the P-deficient leaves depended on O₂ concentration, suggesting that the water-water cycle might also contribute to non-radiative energy dissipation. Hence, the enhanced activity of the water-water cycle is important for protecting photosynthetic apparatus under P-deficiency in rice.     

Two P-type ATPases are required for copper delivery in Arabidopsis thaliana chloroplasts

Copper delivery to the thylakoid lumen protein plastocyanin and the stromal enzyme Cu/Zn superoxide dismutase in chloroplasts is required for photosynthesis and oxidative stress protection. The copper delivery system in chloroplasts was characterized by analyzing the function of copper transporter genes in Arabidopsis thaliana. Two mutant alleles were identified of a previously uncharacterized gene, PAA2 (for P-type ATPase of Arabidopsis), which is required for efficient photosynthetic electron transport. PAA2 encodes a copper-transporting P-type ATPase with sequence similarity to PAA1, which functions in copper transport in chloroplasts. Both proteins localized to the chloroplast, as indicated by fusions to green fluorescent protein. The PAA1 fusions were found in the chloroplast periphery, whereas PAA2 fusions were localized in thylakoid membranes. The phenotypes of paa1 and paa2 mutants indicated that the two transporters have distinct functions: whereas both transporters are required for copper delivery to plastocyanin, copper delivery to the stroma is inhibited only in paa1 but not in paa2. The effects of paa1 and paa2 on superoxide dismutase isoform expression levels suggest that stromal copper levels regulate expression of the nuclear genes IRON SUPEROXIDE DISMUTASE1 and COPPER/ZINC SUPEROXIDE DISMUTASE2. A paa1 paa2 double mutant was seedling-lethal, underscoring the importance of copper to photosynthesis. We propose that PAA1 and PAA2 function sequentially in copper transport over the envelope and thylakoid membrane, respectively.     

Characterization of photosystem II in transgenic tobacco plants with decreased iron superoxide dismutase

Iron superoxide dismutases (FeSODs) play an important role in preventing the oxidative damage associated with photosynthesis. To investigate the mechanisms of FeSOD in protection against photooxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with severely decreased FeSOD by using a gene encoding tobacco chloroplastic FeSOD for the RNAi construct. Transgenic plants were highly sensitive to photooxidative stress and accumulated increased levels of O??? under normal light conditions. Spectroscopic analysis and electron transport measurements showed that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed that there was a slow electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses showed that PSII protein accumulation was also decreased in transgenic plants. PSII photodamage and D1 protein degradation under high light treatment was increased in transgenic plants, whereas the PSII repair was not affected, indicating that the stability of the PSII complex was decreased in transgenic plants. The results in this study suggest that FeSOD plays an important role in maintaining PSII function by stabilizing PSII complexes in tobacco plants.     

Regulation of superoxide dismutase expression by copper availability

The most abundant copper proteins in green tissues are plastocyanin (PC) in thylakoids and copper/zinc superoxide dismutase (Cu/ZnSOD) of which the major isoforms are found in the cytosol and in the chloroplast stroma. An iron superoxide dismutase (FeSOD) can also be found in the stroma. The expression of superoxide dismutases (SODs) has been studied mainly in the context of abiotic stress. However, the availability of metal cofactors may also determine SOD expression patterns. Indeed, in Arabidopsis thaliana , Cu/ZnSOD enzymes were only expressed when copper was sufficient. This observation was made for plants grown on sucrose-containing tissue culture media and regulation of SOD expression by copper has not been tested for other species. To investigate the effect of copper on SOD expression, we used a hydroponic set-up in which plants grew without any evident stress symptoms. We observed that A. thaliana , Brassica juncea , Lycopersicum lycopersicum , Zea mays and Oryza sativa , downregulated Cu/ZnSOD in response to copper limitation. Under this condition, FeSOD expression was upregulated to replace Cu/ZnSOD in the stroma in all plants except Z. mays , in which FeSOD was not detectable. Copper limitation did not affect PC accumulation in any of the plants except Z. mays . Comparisons of leaf copper contents and SOD expression suggest that Cu/ZnSOD and FeSOD expression levels are good indicators of impending copper deficiency. Plants that downregulate Cu/ZnSOD and upregulate FeSOD under copper limitation can maintain superoxide scavenging and save copper for use in PC, which is essential for photosynthesis.     

Copper deprivation potentiates oxidative stress in HL-60 cell mitochondria.

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.     

The copper transport protein Atox1 promotes neuronal survival

Atox1, a copper transport protein, was recently identified as a copper-dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.     

Enhanced tolerance to methyl viologen‐induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD,APX and NDPK2 genes

Oxidative stress is a major threat for plants exposed to various environmental stresses. Previous studies found that transgenic potato plants expressing both copper zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) (referred to as SSA plants), or nucleoside diphosphate kinase 2 (NDPK2) (SN plants), showed enhanced tolerance to methyl viologen (MV)‐induced oxidative stress and high temperature. This study aimed to develop transgenic plants that were more tolerant of oxidative stress by introducing the NDPK2 gene into SSA potato plants under the control of an oxidative stress‐inducible peroxidase (SWPA2) promoter to create SSAN plants. SSAN leaf discs and whole plants showed enhanced tolerance to MV, as compared to SSA, SN or non‐transgenic (NT) plants. SSAN plants sprayed with 400 µM MV exhibited about 53 and 83% less visible damage than did SSA and SN plants, respectively. The expression levels of the CuZnSOD, APX and NDPK2 genes in SSAN plants following MV treatment correlated well with MV tolerance. SOD, APX, NDPK and catalase antioxidant enzyme activities were also increased in MV‐treated SSAN plants. In addition, SSAN plants were more tolerant to high temperature stress at 42°C, exhibiting a 6.2% reduction in photosynthetic activity as compared to plants grown at 25°C. In contrast, the photosynthetic activities of SN and SSA plants decreased by 50 and 18%, respectively. These results indicate that the simultaneous overexpression of CuZnSOD, APX and NDPK2 is more effective than single or double transgene expression for developing plants with enhanced tolerance to various environmental stresses.     

Differential accumulation of antioxidant mRNAs in Arabidopsis thaliana exposed to ozone.

Antioxidant isoenzymes function to eliminate free radicals and are localized to several different subcellular compartments within the plant cell. In Arabidopsis thaliana exposed to ozone (O3), we have monitored the accumulation of mRNAs encoding both cytosolic and chloroplastic antioxidant isoenzymes. Two different O3 exposure protocols yielded similar results. Upon O3 exposure, the steady-state levels of three mRNAs encoding cytosolic antioxidant isoenzymes (ascorbate peroxidase, copper/zinc superoxide dismutase, and glutathione S-transferase) increase. The glutathione S-transferase mRNA responds very quickly to the oxidative stress (2-fold increase in 30 min) and is elevated to very high levels, especially in plants grown with a 16-h photoperiod. In contrast, O3 exposure causes a decline in the levels of two chloroplastic antioxidant mRNAs (iron superoxide dismutase and glutathione reductase) and two photosynthetic protein mRNAs (chlorophyll a/b-binding protein and ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit). We show that this decline does not include all mRNAs encoding chloroplast-targeted proteins, since O3 causes an elevation of mRNA encoding the chloroplast-localized tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase. Two alternative hypotheses that could explain this differential mRNA accumulation in response to O3 are discussed.     

The amino-acid sequence of copper/zinc superoxide dismutase from swordfish liver. Comparison of copper/zinc superoxide dismutase sequences

The amino acid sequence of copper/zinc superoxide dismutase from swordfish (Xiphias gladius) liver has been determined by alignment of the tryptic peptides according to the known sequence of bovine erythrocyte copper/zinc superoxide dismutase. This alignment has resulted in the ligands to the copper (His-47, 49, 76 and 94) and the zinc (His-76, 85, 134 and Asp-97) being conserved in all the copper/zinc superoxide dismutases sequenced so far. Also conserved in the sequences are the cysteines forming the intrachain disulphide bridge (Cys-58 and 160) and the essential arginine (Arg-157). Comparison of the amino acid sequence of swordfish liver copper/zinc superoxide dismutase with the bovine, human, horse, yeast and Photobacterium leiognathi indicates that the swordfish enzyme has a high homology with the other eukaryotic enzymes. Low homology is, however, observed with the P. leiognathi enzyme.     

Antioxidants and antioxidative enzymes in wild-type and transgenic Lycopersicon genotypes of different chilling tolerance

The Mehler–Ascorbate–Peroxidase cycle is a protection system against reactive oxygen species (ROS) occurring during over-excitation of the photosynthetic apparatus. In the cultivated tomato, Lycopersicon esculentum, long-term chilling under moderate light leads to oxidation of the Calvin cycle key enzyme, ribulose-1,5-bisphosphate carboxylase (rubisco), presumably by generation of ROS. In contrast, high-altitude lines of the wild tomato species L. peruvianum were tolerant against the same chilling stress. In the present study, we analysed leaf contents of antioxidants (ascorbate, glutathione) and activities of enzymes of the Mehler–Ascorbate–Peroxidase cycle in the two Lycopersicon species. While antioxidant levels and activities of chloroplast superoxide dismutase (SOD) and ascorbate peroxidase (APX), both inducible by chilling stress, were similar in chilling-tolerant and chilling-sensitive genotypes, chilled L. esculentum showed lower glutathione reductase (GR) activities than high-altitude L. peruvianum. We constructed transgenic plants overexpressing an Escherichia coli GR in the chloroplast (approximately 60-fold of the wild-type (WT) activity). However, these plants resembled identical chilling sensitivity of the photosynthetic apparatus as WT plants as measured after a photoinhibition treatment and by the effect of long-term chilling on rubisco activity. We conclude that the Mehler–Ascorbate–Peroxidase cycle is not the limiting factor for the sensitivity of the photosynthetic apparatus of L. esculentum towards long-term chilling under moderate light. We suggest that a possible cause for the higher chilling tolerance of L. peruvianum is prevention of ROS formation by better conversion of light energy to photochemistry at suboptimal temperatures.