The function of the chloroplast 2-cysteine peroxiredoxin in peroxide detoxification and its regulation

The Arabidopsis genome contains nine open reading frames with homology to members of the peroxiredoxin (prx) family: one 1-Cys-prx, two 2-Cys-prx, five type II-prx, and one peroxiredoxin Q. The function of the peroxiredoxins in plant metabolism is only slowly emerging. They are assumed to reduce toxic peroxides to their corresponding alcohols with a rather broad substrate specificity. The 2-Cys peroxiredoxins (2-CP) were recently identified as members of the antioxidant defence system of chloroplasts. Knock-out mutants of Synechocystis and antisense mutants of Arabidopsis have provided insight into the function of 2-CPs in the photosynthetic antioxidant network. This review summarizes present knowledge on the enzymatic mechanism, the physiological context and the genetic regulation of the 2-CPs in plants and cyanobacteria. In addition, an extrapolation on the metabolic role of the chloroplast 2-CP is attempted based on the molecular features of 2-CPs from other organisms.     

Overexpression of 2-cysteine peroxiredoxin enhances tolerance to methyl viologen-mediated oxidative stress and high temperature in potato plants

Oxidative stress is one of the major causative factors for injury to plants exposed to environmental stresses. Plants have developed diverse defense mechanisms for scavenging oxidative stress-inducing molecules. The antioxidative enzyme 2-cysteine peroxiredoxin (2-Cys Prx) removes peroxides and protects the photosynthetic membrane from oxidative damage. In this study, transgenic potato (Solanum tuberosum L. cv. Atlantic) expressing At2-Cys Prx under control of the oxidative stress-inducible SWPA2 promoter or enhanced CaMV 35S promoter (referred to as SP and EP plants, respectively) was generated using Agrobacterium-mediated transformation. The transgenic plants were tested for tolerance to stress. Following treatment with 3 μM methyl viologen (MV), leaf discs from SP and EP plants showed approximately 33 and 15% less damage than non-transformed (NT) plants. When 300 μM MV was sprayed onto whole plants, the photosynthetic activity of SP plants decreased by 25%, whereas that of NT plants decreased by 60%. In addition, SP plants showed enhanced tolerance to high temperature at 42 °C. After treatment at high temperature, the photosynthetic activity of SP plants decreased by about 7% compared to plants grown at 25 °C, whereas it declined by 31% in NT plants. These results indicate that transgenic potato can efficiently regulate oxidative stress from various environmental stresses via overexpression of At2-Cys Prx under control of the stress-inducible SWPA2 promoter.     

Poplar peroxiredoxin Q. A thioredoxin-linked chloroplast antioxidant functional in pathogen defense

Peroxiredoxins are ubiquitous thioredoxin- or glutaredoxin-dependent peroxidases, the function of which is to destroy peroxides. Peroxiredoxin Q, one of the four plant subtypes, is a homolog of the bacterial bacterioferritin comigratory proteins. We show here that the poplar (Populus tremula x Populus tremuloides) protein acts as a monomer with an intramolecular disulfide bridge between two conserved cysteines. A wide range of electron donors and substrates was tested. Unlike type II peroxiredoxin, peroxiredoxin Q cannot use the glutaredoxin or cyclophilin isoforms tested, but various cytosolic, chloroplastic, and mitochondrial thioredoxins are efficient electron donors with no marked specificities. The redox midpoint potential of the peroxiredoxin Q catalytic disulfide is -325 mV at pH 7.0, explaining why the wild-type protein is reduced by thioredoxin but not by glutaredoxin. Additional evidence that thioredoxin serves as a donor comes from the formation of heterodimers between peroxiredoxin Q and monocysteinic mutants of spinach (Spinacia oleracea) thioredoxin m. Peroxiredoxin Q can reduce various alkyl hydroperoxides, but with a better efficiency for cumene hydroperoxide than hydrogen peroxide and tertiary butyl hydroperoxide. The use of immunolocalization and of a green fluorescence protein fusion construct indicates that the transit sequence efficiently targets peroxiredoxin Q to the chloroplasts and especially to those of the guard cells. The expression of this protein and of type II peroxiredoxin is modified in response to an infection by two races of Melampsora larici-populina, the causative agent of the poplar rust. In the case of an hypersensitive response, the peroxiredoxin expression increased, whereas it decreased during a compatible interaction.     

Role of Arabidopsis CHL27 protein for photosynthesis, chloroplast development and gene expression profiling

In Chl biosynthesis, aerobic Mg-protoporphyrin IX monomethyl ester (MPE) cyclase is a key enzyme involved in the synthesis of protochlorophyllide a, and its membrane-bound component is known to be encoded by homologs of CHL27 in photosynthetic bacteria, green algae and plants. Here, we report that the Arabidopsis chl27-t knock-down mutant exhibits retarded growth and chloroplast developmental defects that are caused by damage to PSII reaction centers. The mutant contains a T-DNA insertion within the CHL27 promoter that dramatically reduces the CHL27 mRNA level. chl27-t mutant plants grew slowly with a pale green appearance, suggesting that they are defective in Chl biosynthesis. Chl fluorescence analysis showed significantly low photosynthetic activity in chl27-t mutants, indicating damage in their PSII reaction centers. The chl27-t mutation also conferred severe defects in chloroplast development, including the unstacking of thylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis, including those encoding components of light-harvesting complex I (LHCI) and LHCII, and PSI and PSII, which accounts for the defects in photosynthetic activity and chloroplast development. In addition, the microarray data also revealed the significant repression of genes such as PORA and AtFRO6 for Chl biosynthesis and iron acquisition, respectively, and, furthermore, implied that there is cross-talk in the Chl biosynthetic pathway among the PORA, AtFRO6 and CHL27 proteins.     

Anionic lipids are required for chloroplast structure and function in Arabidopsis

Photosynthetic membranes of plants primarily contain non-phosphorous glycolipids. The exception is phosphatidylglycerol (PG), which is an acidic/anionic phospholipid. A second major anionic lipid in chloroplasts is the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). It is hypothesized that under severe phosphate limitation, SQDG substitutes for PG, ensuring a constant proportion of anionic lipids even under adverse conditions. A newly constructed SQDG and PG-deficient double mutant supports this hypothesis. This mutant, sqd2 pgp1-1, carries a T-DNA insertion in the structural gene for SQDG synthase (SQD2) and a point mutation in the structural gene for phosphatidylglycerolphosphate synthase (PGP1). In the sqd2 pgp1-1 double mutant, the fraction of total anionic lipids is reduced by approximately one-third, resulting in pale yellow cotyledons and leaves with reduced chlorophyll content. Photoautotrophic growth of the double mutant is severely compromised, and its photosynthetic capacity is impaired. In particular, photosynthetic electron transfer at the level of photosystem II (PSII) is affected. Besides these physiological changes, the mutant shows altered leaf structure, a reduced number of mesophyll cells, and ultrastructural changes of the chloroplasts. All observations on the sqd2 pgp1-1 mutant lead to the conclusion that the total content of anionic thylakoid lipids is limiting for chloroplast structure and function, and is critical for overall photoautotrophic growth and plant development.     

Chloroplast targeting of chloroplast division FtsZ2 proteins in Arabidopsis

Plant nuclear genomes encode chloroplast division proteins homologous to the eubacterial cell division protein FtsZ. In higher plants, FtsZ genes constitute a small gene family that consists of two subgroups, FtsZ1 and FtsZ2. It was previously hypothesized that members of one family (FtsZ1) targeted chloroplasts, while members of the other family (FtsZ2) localized in the cytoplasm. We determined the full-length cDNA sequences of two FtsZ2 genes from Arabidopsis thaliana (AtFtsZ2-1 and AtFtsZ2-2) and found that the genes encode polypeptides of 478 and 473 amino acids, respectively, and both contain N-terminal extensions beyond what have previously been predicted. The N-terminal regions of both AtFtsZ2-1 and AtFtsZ2-2 were expressed as green fluorescent protein (GFP) fusions under the cauliflower mosaic virus 35S promoter in bombarded tobacco cells. Confocal laser scanning microscopy revealed both fusions exclusively localized to chloroplasts, demonstrating that the N-terminal regions function as chloroplast-targeting signals in vivo. Thus, FtsZ2 proteins function within chloroplasts.     

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C_16:1 and C_18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.     

ATP and Mg2+ promote the reversible oligomerization and aggregation of chloroplast 2-Cys peroxiredoxin

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous peroxidases with important roles in cellular antioxidant defense and hydrogen peroxide-mediated signaling. Post-translational modifications of conserved cysteines cause the transition from low to high molecular weight oligomers, triggering the functional change from peroxidase to molecular chaperone. However, it remains unclear how non-covalent interactions of 2-Cys Prx with metabolites modulate the quaternary structure. Here, we disclose that ATP and Mg(2+) (ATP/Mg) promote the self-polymerization of chloroplast 2-Cys Prx (polypeptide 23.5 kDa) into soluble higher order assemblies (>2 MDa) that proceed to insoluble aggregates beyond 5 mM ATP. Remarkably, the withdrawal of ATP or Mg(2+) brings soluble oligomers and insoluble aggregates back to the native conformation without compromising the associated functions. As confirmed by transmission electron microscopy, ATP/Mg drive the toroid-like decamers (diameter 13 nm) to the formation of large sphere-like particles (diameter ～30 nm). Circular dichroism studies on ATP-labeled 2-Cys Prx reveal that ATP/Mg enhance the proportion of β-sheets with the concurrent decrease in the content of α-helices. In line with this observation, the formation of insoluble aggregates is strongly prevented by 2,2,2-trifluoroethanol, a cosolvent employed to induce α-helical conformations. We further find that the response of self-polymerization to ATP/Mg departs abruptly from that of the associated peroxidase and chaperone activities when two highly conserved residues, Arg(129) and Arg(152), are mutated. Collectively, our data uncover that non-covalent interactions of ATP/Mg with 2-Cys Prx modulate dynamically the quaternary structure, thereby coupling the non-redox chemistry of cell energy with redox transformations at cysteine residues.     

The C-terminal extension of chloroplast 2-Cys peroxiredoxin is critical for interaction with ATP

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous enzymes that have been implicated in peroxide-mediated signaling of markedly different processes, such as cancer and photosynthesis. A highly conserved C-terminal extension of eukaryotic homologues modulates both the overoxidation of cysteines and the formation of oligomers. Here, we reveal that the plant counterpart regulates the self-polymerization of 2-Cys Prx triggered by ATP and Mg(2+). This feature is of particular importance under oxidative stress because the interaction of ATP with 2-Cys Prx rapidly integrates nonredox chemistry of signaling pathways into a network hub governed by multiple redox transformations at cysteine residues.     

The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis

A mutant of Arabidopsis thaliana with reduced content of C_18:3 and C_16:3 fatty acids in membrane lipids exhibited a 45% reduction in the cross-sectional area of chloroplasts and had a decrease of similar magnitude in the amount of chloroplast lamellar membranes. The reduction in chloroplast size was partially compensated by a 45% increase in the number of chloroplasts per cell in the mutant. When expressed on a chlorophyll basis the rates of CO₂-fixation and photosynthetic electron transport were not affected by these changes. Fluorescence polarization measurements indicated that the fluidity of the thylakoid membranes was not significantly altered by the mutation. Similarly, on the basis of temperature-induced fluorescence yield enhancement measurements, there was no significant effect on the thermal stability of chlorophyll-protein complexes in the mutant. These observations suggest that the high content of trienoic fatty acids in chloroplast lipids may be an important factor regulating organelle biogenesis but is not required to support normal levels of the photosynthetic activities associated with the thylakoid membranes.     

Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana

The development of chloroplasts and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting envelope membranes. To provide the most exhaustive view of the protein repertoire of chloroplast envelope membranes, we analyzed this membrane system using proteomics. To this purpose, we first developed a procedure to prepare highly purified envelope membranes from Arabidopsis chloroplasts. We then extracted envelope proteins using different methods, i.e. chloroform/methanol extraction and alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to least hydrophobic ones. Liquid chromatography tandem mass spectrometry analyses were then performed on each envelope membrane subfraction, leading to the identification of more than 100 proteins. About 80% of the identified proteins are known to be, or are very likely, located in the chloroplast envelope. The validation of localization in the envelope of two phosphate transporters exemplifies the need for a combination of strategies to perform the most exhaustive identification of genuine chloroplast envelope proteins. Interestingly, some of the identified proteins are found to be Nalpha-acetylated, which indicates the accurate location of the N terminus of the corresponding mature protein. With regard to function, more than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are a) involved in ion and metabolite transport, b) components of the protein import machinery, and c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism, or proteins involved in responses to oxidative stress, were associated with envelope membranes. Almost one-third of the proteins we identified have no known function. The present work helps understanding chloroplast envelope metabolism at the molecular level and provides a new overview of the biochemical machinery of the chloroplast envelope membranes.     

ABA-responsive RNA-binding proteins are involved in chloroplast and stromule function in Arabidopsis seedlings

The phytohormone abscisic acid (ABA) regulates essential growth and developmental processes in plants. Recently, RNA-binding proteins have been described as components of ABA signaling during germination. We have identified ten ABA-regulated RNA-binding proteins in Arabidopsis seedlings. Among those genes, AtCSP41B and cpRNP29 are highly expressed in seedlings. Using promoter:reporter gene analyses, we showed that both AtCSP41B and cpRNP29 were in particular expressed in photosynthetically active organs like green cotyledons, leaves, and petioles. The analysis of CFP-fusion proteins demonstrates that cpRNP29 localized to chloroplasts and AtCSP41B to chloroplasts and stromules. Whereas RNA-binding of cpRNP29 has previously been shown, we demonstrated through in vitro RNA-binding assays that recombinant AtCSP41B binds to RNA, and that chloroplast petD RNA can serve as a target of AtCSP41B. Developmental or environmental stimuli affected the expression of AtCSP41B and cpRNP29 in seedlings. Both genes were repressed during senescence, but only AtCSP41B was significantly repressed upon water stress. In addition, AtCSP41B and cpRNP29 exhibited low expression in etiolated seedlings compared to green seedlings, and cpRNP29 was regulated during the day photoperiod. Homozygous T-DNA insertion lines were isolated, characterized on the molecular level, and monitored for phenotypic changes. Taken together, the data show that both proteins are regulated during processes that are known to involve ABA signaling. Their localization in chloroplasts and RNA-binding activity suggest a role in chloroplast RNA metabolism in Arabidopsis seedlings.     

The plastidic 2-cysteine peroxiredoxin is a target for a thioredoxin involved in the protection of the photosynthetic apparatus against oxidative damage

The chloroplastic drought-induced stress protein of 32 kD (CDSP32) is composed of two thioredoxin modules and is induced by environmental and oxidative stress conditions. We investigated whether the plastidic protein BAS1, which is related to eubacterial 2-Cys peroxiredoxin, is a target for CDSP32. Using a CDSP32 active-site mutant, we showed that the BAS1 and CDSP32 proteins form a mixed disulfide complex in vitro. Moreover, affinity chromatography indicated that BAS1 is a major target for CDSP32 in chloroplasts. CDSP32 was able to reduce BAS1 in vitro, and BAS1 displayed CDSP32-dependent peroxidase activity. The function of CDSP32 was investigated in transgenic potato lines without detectable levels of the protein as a result of cosuppression. Under conditions of photooxidative stress induced by incubation with either methyl viologen or t-butyl hydroperoxide or by exposure to low temperature under high light, plants lacking CDSP32 exhibited decreased maximal photosystem II photochemical efficiencies compared with the wild type and transgenic controls. In addition, plants without CDSP32 retained much less chlorophyll than controls under stress, indicating increased damage to photosynthetic membranes. We conclude that CDSP32 is a thioredoxin with a critical role in plastid defense against oxidative damage and that this role is related to its function as a physiological electron donor to the BAS1 peroxiredoxin.     

Roles of the nuclear-encoded chloroplast SMR domain-containing PPR protein SVR7 in photosynthesis and oxidative stress tolerance in Arabidopsis

The mutations of the plastid SMR domaincontaining PPR protein SVR7 were previously reported to cause a specific reduction in the chloroplast ATP synthase levels. Here, we isolated a new mutant allele of SVR7, named svr7-4, in which T-DNA is inserted into the initiation codon of SVR7. The rosette leaves of svr7-4, especially in the juvenile stage, showed a pale green phenotype as a result of a reduction in the chlorophyll levels. The values of P700 and Fv/Fm indicated that the photosynthetic capacities of both PSI and PSII were damaged in svr7-4. Furthermore, we found that the svr7-4 accumulated more reactive oxygen species (ROS) and showed lower photo-oxidative stress tolerance by histochemical staining and hydrogen peroxide bleaching experiments, respectively. The leaves of svr7-4 also had increased anthocyanins accumulation compared to that of wild-type (WT) when floated on water under light. Finally, we found that the expression levels of four abiotic stress-responsive genes including ZAT10, AtAPX1, CAT1 and AtGPX2 were up-regulated in svr7-4. SVR7 was expressed ubiquitously during plant development. These results indicate that SVR7 is important for normal photosynthesis and photo-oxidative stress responses in chloroplasts.     

Response of soybean photosynthesis and chloroplast membrane function to canopy development and mutual shading

The effect of natural shading on photosynthetic capacity and chloroplast thylakoid membrane function was examined in soybean (Glycine max. cv Young) under field conditions using a randomized complete block design. Seedlings were thinned to 15 plants per square meter at 20 days after planting. Leaves destined to function in the shaded regions of the canopy were tagged during early expansion at 40 days after planting. To investigate the response of shaded leaves to an increase in available light, plants were removed from certain plots at 29 or 37 days after tagging to reduce the population from 15 to three plants per square meter and alter the irradiance and spectral quality of light. During the transition from a sun to a shade environment, maximum photosynthesis and chloroplast electron transport of control leaves decreased by two- to threefold over a period of 40 days followed by rapid senescence and abscission. Senescence and abscission of tagged leaves were delayed by more than 4 weeks in plots where plant populations were reduced to three plants per square meter. Maximum photosynthesis and chloroplast electron transport activity were stabilized or elevated in response to increased light when plant populations were reduced from 15 to three plants per square meter. Several chloroplast thylakoid membrane components were affected by light environment. Cytochrome f and coupling factor protein decreased by 40% and 80%, respectively, as control leaves became shaded and then increased when shaded leaves acclimated to high light. The concentrations of photosystem I (PSI) and photosystem II (PSII) reaction centers were not affected by light environment or leaf age in field grown plants, resulting in a constant PSII/PSI ratio of 1.6 ± 0.3. Analysis of the chlorophyll-protein composition revealed a shift in chlorophyll from PSI to PSII as leaves became shaded and a reversal of this process when shaded leaves were provided with increased light. These results were in contrast to those of soybeans grown in a growth chamber where the PSII/PSI ratio as well as cytochrome f and coupling factor protein levels were dependent on growth irradiance. To summarize, light environment regulated both the photosynthetic characteristics and the timing of senescence in soybean leaves grown under field conditions.