PAA1, a P-type ATPase of Arabidopsis,functions in copper transport in chloroplasts

Copper (Cu) is an essential trace element with important roles as a cofactor in many plant functions, including photosynthesis. However, free Cu ions can cause toxicity, necessitating precise Cu delivery systems. Relatively little is known about Cu transport in plant cells, and no components of the Cu transport machinery in chloroplasts have been identified previously. Cu transport into chloroplasts provides the cofactor for the stromal enzyme copper/zinc superoxide dismutase (Cu/ZnSOD) and for the thylakoid lumen protein plastocyanin, which functions in photosynthetic electron transport from the cytochrome b(6)f complex to photosystem I. Here, we characterized six Arabidopsis mutants that are defective in the PAA1 gene, which encodes a member of the metal-transporting P-type ATPase family with a functional N-terminal chloroplast transit peptide. paa1 mutants exhibited a high-chlorophyll-fluorescence phenotype as a result of an impairment of photosynthetic electron transport that could be ascribed to decreased levels of holoplastocyanin. The paa1-1 mutant had a lower chloroplast Cu content, despite having wild-type levels in leaves. The electron transport defect of paa1 mutants was evident on medium containing <1 micro M Cu, but it was suppressed by the addition of 10 micro M Cu. Chloroplastic Cu/ZnSOD activity also was reduced in paa1 mutants, suggesting that PAA1 mediates Cu transfer across the plastid envelope. Thus, PAA1 is a critical component of a Cu transport system in chloroplasts responsible for cofactor delivery to plastocyanin and Cu/ZnSOD.     

HMA1, a new Cu-ATPase of the chloroplast envelope, is essential for growth under adverse light conditions

Although ions play important roles in the cell and chloroplast metabolism, little is known about ion transport across the chloroplast envelope. Using a proteomic approach specifically targeted to the Arabidopsis chloroplast envelope, we have identified HMA1, which belongs to the metal-transporting P1B-type ATPases family. HMA1 is mainly expressed in green tissues, and we validated its chloroplast envelope localization. Yeast expression experiments demonstrated that HMA1 is involved in copper homeostasis and that deletion of its N-terminal His-domain partially affects the metal transport. Characterization of hma1 Arabidopsis mutants revealed a lower chloroplast copper content and a diminution of the total chloroplast superoxide dismutase activity. No effect was observed on the plastocyanin content in these lines. The hma1 insertional mutants grew like WT plants in standard condition but presented a photosensitivity phenotype under high light. Finally, direct biochemical ATPase assays performed on purified chloroplast envelope membranes showed that the ATPase activity of HMA1 is specifically stimulated by copper. Our results demonstrate that HMA1 offers an additional way to the previously characterized chloroplast envelope Cu-ATPase PAA1 to import copper in the chloroplast.     

Protein Import into and Sorting inside the Chloroplast Are Independent Processes

Plastocyanin is a nuclear-encoded chloroplast thylakoid lumen protein that is synthesized in the cytoplasm with a large N-terminal extension (66 amino acids). Transport of plastocyanin involves two steps: import across the chloroplast envelope into the stroma, followed by transfer across the thylakoid membrane into the lumen. During transport the N-terminal extension is removed in two parts by two different processing proteases. In this study we examined the functions of the two cleaved parts, C1 and C2, in the transport pathway of plastocyanin. The results show that C1 mediates import into the chloroplast. C1 is sufficient to direct chloroplast import of mutant proteins that lack C2. It is also sufficient to direct import of a nonplastid protein and can be replaced functionally by the transit peptide of an imported stromal protein. C2 is a prerequisite for intraorganellar routing but is not required for chloroplast import. Deletions in C2 result in accumulation of intermediates in the stroma or on the outside of the thylakoids. The fact that C1 is functionally equivalent to a stromal-targeting transit peptide shows that plastocyanin is imported into the chloroplast by way of the same mechanism as stromal proteins, and that import into and routing inside the chloroplasts are independent processes.     

Biochemical characterization of AtHMA6/PAA1, a chloroplast envelope Cu(I)-ATPase

Copper is an essential plant micronutrient playing key roles in cellular processes, among them photosynthesis. In Arabidopsis thaliana, copper delivery to chloroplasts, mainly studied by genetic approaches, is thought to involve two P(IB)-type ATPases: AtHMA1 and AtHMA6/PAA1. The lack of biochemical characterization of AtHMA1 and PAA1, and more generally of plant P(IB)-type ATPases, is due to the difficulty of getting high amounts of these membrane proteins in an active form, either from their native environment or after expression in heterologous systems. In this study, we report the first biochemical characterization of PAA1, a plant copper-transporting ATPase. PAA1 produced in Lactococcus lactis is active, forming an aspartyl phosphate intermediate in the presence of ATP and the adequate metal ion. PAA1 can also be phosphorylated using inorganic phosphate in the absence of transition metal. Both phosphorylation types allowed us to demonstrate that PAA1 is activated by monovalent copper ions (and to a lower extent by silver ions) with an apparent affinity in the micromolar range. In agreement with these biochemical data, we also demonstrate that when expressed in yeast, PAA1 induces increased sensitivities to copper and silver. These data provide the first enzymatic characterization of a P(IB-1)-type plant ATPase and clearly identify PAA1 as a high affinity Cu(I) transporter of the chloroplast envelope.     

Identification and subcellular localization of the soybean copper P1B-ATPase GmHMA8 transporter

We have identified a copper P(1B)-ATPase transporter in soybean (Glycine max), named as GmHMA8, homologue to cyanobacterial PacS and Arabidopsis thaliana AtHMA8 (PAA2) transporters. A novel specific polyclonal anti-GmHMA8 antibody raised against a synthetic peptide reacted with a protein of an apparent mass of around 180-200 kDa in chloroplast and thylakoid membrane preparations isolated from soybean cell suspensions. Immunoblot analysis with this antibody also showed a band with similar apparent molecular mass in chloroplasts from Lotus corniculatus. Immunofluorescence labelling with the anti-GmHMA8 antibody and double immunofluorescence labelling with anti-GmHMA8 and anti-RuBisCo antibodies revealed the localization of the GmHMA8 transporter within the chloroplast organelle. Furthermore, the precise ultrastructural distribution of GmHMA8 within the chloroplast subcompartments was demonstrated by using electron microscopy immunogold labelling. The GmHMA8 copper transporter from soybean was localized in the thylakoid membranes showing a heterogeneous distribution in small clusters.     

Structural Insights into the Nucleotide-Binding Domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana

Copper is a crucial ion in cells, but needs to be closely controlled due to its toxic potential and ability to catalyse the formation of radicals. In chloroplasts, an important step for the proper functioning of the photosynthetic electron transfer chain is the delivery of copper to plastocyanin in the thylakoid lumen. The main route for copper transport to the thylakoid lumen is driven by two P_IB-type ATPases, Heavy Metal ATPase 6 (HMA6) and HMA8, located in the inner membrane of the chloroplast envelope and in the thylakoid membrane, respectively. Here, the crystal structures of the nucleotide binding domain of HMA6 and HMA8 from Arabidopsis thaliana are reported at 1.5Å and 1.75Å resolution, respectively, providing the first structural information on plants Cu⁺-ATPases. The structures reveal a compact domain, with two short helices on both sides of a twisted beta-sheet. A double mutant, aiding in the crystallization, provides a new crystal contact, but also avoids an internal clash highlighting the benefits of construct modifications. Finally, the histidine in the HP motif of the isolated domains, unable to bind ATP, shows a side chain conformation distinct from nucleotide bound structures.     

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.     

AtCCS is a functional homolog of the yeast copper chaperone Ccs1/Lys7

In plant chloroplasts two superoxide dismutase (SOD) activities occur, FeSOD and Cu/ZnSOD, with reciprocal regulation in response to copper availability. This system presents a unique model to study the regulation of metal-cofactor delivery to an organelle. The Arabidopsis thaliana gene AtCCS encodes a functional homolog to yeast Ccs1p/Lys7p, a copper chaperone for SOD. The AtCCS protein was localized to chloroplasts where it may supply copper to the stromal Cu/ZnSOD. AtCCS mRNA expression levels are upregulated in response to Cu-feeding and senescence. We propose that AtCCS expression is regulated to allow the most optimal use of Cu for photosynthesis.     

Full-length plastocyanin precursor is translocated across isolated thylakoid membranes

In higher plants, the chloroplastic protein plastocyanin is synthesized as a transit peptide-containing precursor by cytosolic ribosomes and posttranslationally transported to the thylakoid lumen. En route to the lumen, a plastocyanin precursor is first imported into chloroplasts and then further directed across the thylakoid membrane by a second distinct transport event. A partially processed form of plastocyanin is observed in the stroma during import experiments using intact chloroplasts and has been proposed to be the translocation substrate for the second step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). To further characterize this second step, we have reconstituted thylakoid transport in a system containing in vitro-synthesized precursor proteins and isolated thylakoid membranes. This system was specific for lumenal proteins since stromal proteins lacking the appropriate targeting information did not accumulate in the thylakoid lumen. Plastocyanin precursor was taken up by isolated thylakoids, proteolytically processed to mature size, and converted to holo form. Translocation was temperature-dependent and was stimulated by millimolar levels of ATP but did not strictly require the addition of stromal factors. We have examined the substrate requirements of thylakoid translocation by testing the ability of different processed forms of plastocyanin to transport in the in vitro system. Interestingly, only the full-length plastocyanin precursor, not the partially processed intermediate form, was competent for transport in this in vitro system.     

Multiple pathways for protein transport into or across the thylakoid membrane.

Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over-expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen-resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein-specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor-specific thylakoid transport pathways.     

A balanced PGR5 level is required for chloroplast development and optimum operation of cyclic electron transport around photosystem I

PSI cyclic electron transport contributes markedly to photosynthesis and photoprotection in flowering plants. Although the thylakoid protein PGR5 (Proton Gradient Regulation 5) has been shown to be essential for the main route of PSI cyclic electron transport, its exact function remains unclear. In transgenic Arabidopsis plants overaccumulating PGR5 in the thylakoid membrane, chloroplast development was delayed, especially in the cotyledons. Although photosynthetic electron transport was not affected during steady-state photosynthesis, a high level of non-photochemical quenching (NPQ) was transiently induced after a shift of light conditions. This phenotype was explained by elevated activity of PSI cyclic electron transport, which was monitored in an in vitro system using ruptured chloroplasts, and also in leaves. The effect of overaccumulation of PGR5 was specific to the antimycin A-sensitive pathway of PSI cyclic electron transport but not to the NAD(P)H dehydrogenase (NDH) pathway. We propose that a balanced PGR5 level is required for efficient regulation of the rate of antimycin A-sensitive PSI cyclic electron transport, although the rate of PSI cyclic electron transport is probably also regulated by other factors during steady-state photosynthesis.     

A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis

There are three iron superoxide dismutases in Arabidopsis thaliana: FE SUPEROXIDE DISMUTASE1 (FSD1), FSD2, and FSD3. Their biological roles in chloroplast development are unknown. Here, we show that FSD2 and FSD3 play essential roles in early chloroplast development, whereas FSD1, which is found in the cytoplasm, does not. An fsd2-1 fsd3-1 double mutant had a severe albino phenotype on agar plates, whereas fsd2 and fsd3 single knockout mutants had pale green phenotypes. Chloroplast development was arrested in young seedlings of the double mutant. The mutant plants were highly sensitive to oxidative stress and developed increased levels of reactive oxygen species (ROS) during extended darkness. The FSD2 and FSD3 proteins formed a heteromeric protein complex in the chloroplast nucleoids. Furthermore, transgenic Arabidopsis plants overexpressing both the FSD2 and FSD3 genes showed greater tolerance to oxidative stress induced by methyl viologen than did the wild type or single FSD2- or FSD3-overexpressing lines. We propose that heteromeric FSD2 and FSD3 act as ROS scavengers in the maintenance of early chloroplast development by protecting the chloroplast nucleoids from ROS.     

The involvement of stromal ATP in maintaining the pH gradient across the chloroplast envelope in the light

The possible involvement of ATP in maintaining the pH gradient across the chloroplast envelope membrane was investigated by simultaneously measuring the stromal ATP concentration and the pH of the stroma and intrathylakoid spaces in intact isolated chloroplasts. Addition of exogenous ATP in the dark increased stromal pH by 0.3–0.4 pH units and increased the pH gradient across the thylakoid membrane by a similar amount. In the dark, dihydroxyacetone phosphate plus oxaloacetate increased stromal ATP to levels equal to those obtained in illuminated chloroplasts, but stromal pH was only increased by 0.1–0.3 pH units compared to an increase of 0.8–1.0 units in the light. The energy-transfer inhibitor, phlorizin, decreased stromal ATP in illuminated chloroplasts almost to dark levels, but did not decrease stromal pH. Inorganic pyrophosphate and an analog of ATP were used to exchange endogenous adenine nucleotides out of chloroplasts, and this also decreased the stromal ATP to dark levels without decreasing stromal pH in the light. Addition of 15–20 μM 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) reduced both the stromal pH and ATP content of illuminated chloroplasts to dark levels but lower concentrations of DCMU preferentially decreased stromal pH. It is concluded that the pH gradient across the chloroplast envelope is unlikely to be maintained by an electrogenic proton pump driven by ATP hydrolysis. Photosynthetic electron transport is required to maintain the pH gradients across both the chloroplast thylakoid and chloroplast envelope membranes.     

PIC1, an ancient permease in Arabidopsis chloroplasts, mediates iron transport

In chloroplasts, the transition metals iron and copper play an essential role in photosynthetic electron transport and act as cofactors for superoxide dismutases. Iron is essential for chlorophyll biosynthesis, and ferritin clusters in plastids store iron during germination, development, and iron stress. Thus, plastidic homeostasis of transition metals, in particular of iron, is crucial for chloroplast as well as plant development. However, very little is known about iron uptake by chloroplasts. Arabidopsis thaliana PERMEASE IN CHLOROPLASTS1 (PIC1), identified in a screen for metal transporters in plastids, contains four predicted alpha-helices, is targeted to the inner envelope, and displays homology with cyanobacterial permease-like proteins. Knockout mutants of PIC1 grew only heterotrophically and were characterized by a chlorotic and dwarfish phenotype reminiscent of iron-deficient plants. Ultrastructural analysis of plastids revealed severely impaired chloroplast development and a striking increase in ferritin clusters. Besides upregulation of ferritin, pic1 mutants showed differential regulation of genes and proteins related to iron stress or transport, photosynthesis, and Fe-S cluster biogenesis. Furthermore, PIC1 and its cyanobacterial homolog mediated iron accumulation in an iron uptake-defective yeast mutant. These observations suggest that PIC1 functions in iron transport across the inner envelope of chloroplasts and hence in cellular metal homeostasis.     

Identification of a Ca2+/H+ antiport in the plant chloroplast thylakoid membrane

To assess the availability of Ca2+ in the lumen of the thylakoid membrane that is required to support the assembly of the oxygen-evolving complex of photosystem II, we have investigated the mechanism of 45Ca2+ transport into the lumen of pea (Pisum sativum) thylakoid membranes using silicone-oil centrifugation. Trans-thylakoid Ca2+ transport is dependent on light or, in the dark, on exogenously added ATP. Both light and ATP hydrolysis are coupled to Ca2+ transport through the formation of a transthylakoid pH gradient. The H+-transporting ionophores nigericin/K+ and carbonyl cyanide 3-chlorophenylhydrazone inhibit the transport of Ca2+. Thylakoid membranes are capable of accumulating up to 30 nmol Ca2+ mg-1 chlorophyll from external concentrations of 15 μM over the course of a 15-min reaction. These results are consistent with the presence of an active Ca2+/H+ antiport in the thylakoid membrane. Ca2+ transport across the thylakoid membrane has significant implications for chloroplast and plant Ca2+ homeostasis. We propose a model of chloroplast Ca2+ regulation whereby the activity of the Ca2+/H+ antiporter facilitates the light-dependent uptake of Ca2+ by chloroplasts and reduces stromal Ca2+ levels.     

定位于类囊体膜的PPF1在转基因拟南芥中的表达影响叶绿体的发育

PPF1是一个与植物营养生长相关的基因。它编码的产物可能是一个膜蛋白并与拟南芥叶绿体中的类囊体蛋白ALB3有很高的同源性。免疫电镜分析表明PPF1蛋白同样主要定位于类囊体膜 ,而且在短日照G2豌豆开花两周后仍发育良好的叶绿体中有很高的表达 ,在长日照豌豆同时期非正常叶绿体中丰度非常低。对转基因拟南芥和野生型植株的叶片衰老进程比较发现 ,PPF1在拟南芥中的过量表达可以延缓叶片的衰老 ,而用PPF1反义mRNA抑制拟南芥中的同源基因ALB3则明显加快叶片衰老速度。对转基因拟南芥的超微结构分析显示 ,PPF1在拟南芥中过量表达时 ,转基因植株的叶绿体比野生型植株的叶绿体大并含有更多的基粒和基质类囊体膜 ;相反 ,反义PPF1表达抑制其在拟南芥中的同源物时 ,转基因植株的叶绿体比野生型植株的叶绿体小并含有较少的基粒和发育较差的类囊体膜系统。这些数据表明叶绿体的发育状况与PPF1或拟南芥同源物ALB3的表达水平呈正相关。我们的结果提示PPF1基因可能通过控制叶绿体的发育状况来调节植物的发育。     

A systemic proteomic analysis of <Emphasis Type="Italic">Populus</Emphasis> chloroplast by using shotgun method

The chloroplast is one of the most important organelles in plants. Proteomic investigations of chloroplasts have been undertaken for many herb plant species, but to date no such investigation has been reported for woody plant chloroplasts. In the present study we initiated a systematic proteomic study of Populus chloroplasts using a shotgun proteomic method. After isolation of chloroplasts and tryptic digestion of the proteins, the protein fragments were separated via HPLC using an SCX column, and the peptides were analyzed by LC-MS/MS; 119 proteins were successfully identified. Based on annotation information in the UniProtKB/Swiss-Prot database, these proteins were identified as being localized in the chloroplast thylakoid membrane, chloroplast stroma, chloroplast thylakoid lumen, and plastoglobules. Over 50% of all identified proteins were confirmed as chloroplast thylakoid proteins, and 85 are encoded by the chloroplast genome with the remaining proteins encoded by the nuclear genome. Based on functional annotation, these proteins were classified into four functional categories, including photosynthesis, redox regulation and stress, primary and secondary metabolism, transport and signaling. These data provide a valuable basis for further studies on photosynthesis in poplar species.