Functional differentiation of bundle sheath and mesophyll maize chloroplasts determined by comparative proteomics

Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway.     

C4GEM, a genome-scale metabolic model to study C4 plant metabolism

Leaves of C(4) grasses (such as maize [Zea mays], sugarcane [Saccharum officinarum], and sorghum [Sorghum bicolor]) form a classical Kranz leaf anatomy. Unlike C(3) plants, where photosynthetic CO(2) fixation proceeds in the mesophyll (M), the fixation process in C(4) plants is distributed between two cell types, the M cell and the bundle sheath (BS) cell. Here, we develop a C(4) genome-scale model (C4GEM) for the investigation of flux distribution in M and BS cells during C(4) photosynthesis. C4GEM, to our knowledge, is the first large-scale metabolic model that encapsulates metabolic interactions between two different cell types. C4GEM is based on the Arabidopsis (Arabidopsis thaliana) model (AraGEM) but has been extended by adding reactions and transporters responsible to represent three different C(4) subtypes (NADP-ME [for malic enzyme], NAD-ME, and phosphoenolpyruvate carboxykinase). C4GEM has been validated for its ability to synthesize 47 biomass components and consists of 1,588 unique reactions, 1,755 metabolites, 83 interorganelle transporters, and 29 external transporters (including transport through plasmodesmata). Reactions in the common C(4) model have been associated with well-annotated C(4) species (NADP-ME subtypes): 3,557 genes in sorghum, 11,623 genes in maize, and 3,881 genes in sugarcane. The number of essential reactions not assigned to genes is 131, 135, and 156 in sorghum, maize, and sugarcane, respectively. Flux balance analysis was used to assess the metabolic activity in M and BS cells during C(4) photosynthesis. Our simulations were consistent with chloroplast proteomic studies, and C4GEM predicted the classical C(4) photosynthesis pathway and its major effect in organelle function in M and BS. The model also highlights differences in metabolic activities around photosystem I and photosystem II for three different C(4) subtypes. Effects of CO(2) leakage were also explored. C4GEM is a viable framework for in silico analysis of cell cooperation between M and BS cells during photosynthesis and can be used to explore C(4) plant metabolism.     

Comparative proteomics of chloroplast envelopes from C3 and C4 plants reveals specific adaptations of the plastid envelope to C4 photosynthesis and candidate proteins required for maintaining C4 metabolite fluxes

C(4) plants have up to 10-fold higher apparent CO(2) assimilation rates than the most productive C(3) plants. This requires higher fluxes of metabolic intermediates across the chloroplast envelope membranes of C(4) plants in comparison with those of C(3) plants. In particular, the fluxes of metabolites involved in the biochemical inorganic carbon pump of C(4) plants, such as malate, pyruvate, oxaloacetate, and phosphoenolpyruvate, must be considerably higher in C(4) plants because they exceed the apparent rate of photosynthetic CO(2) assimilation, whereas they represent relatively minor fluxes in C(3) plants. While the enzymatic steps involved in the C(4) biochemical inorganic carbon pump have been studied in much detail, little is known about the metabolite transporters in the envelope membranes of C(4) chloroplasts. In this study, we used comparative proteomics of chloroplast envelope membranes from the C(3) plant pea (Pisum sativum) and mesophyll cell chloroplast envelopes from the C(4) plant maize (Zea mays) to analyze the adaptation of the mesophyll cell chloroplast envelope proteome to the requirements of C(4) photosynthesis. We show that C(3)- and C(4)-type chloroplasts have qualitatively similar but quantitatively very different chloroplast envelope membrane proteomes. In particular, translocators involved in the transport of triosephosphate and phosphoenolpyruvate as well as two outer envelope porins are much more abundant in C(4) plants. Several putative transport proteins have been identified that are highly abundant in C(4) plants but relatively minor in C(3) envelopes. These represent prime candidates for the transport of C(4) photosynthetic intermediates, such as pyruvate, oxaloacetate, and malate.     

Cytosolic events involved in chloroplast protein targeting

Chloroplasts are unique organelles that are responsible for photosynthesis. Although chloroplasts contain their own genome, the majority of chloroplast proteins are encoded by the nuclear genome. These proteins are transported to the chloroplasts after translation in the cytosol. Chloroplasts contain three membrane systems (outer/inner envelope and thylakoid membranes) that subdivide the interior into three soluble compartments known as the intermembrane space, stroma, and thylakoid lumen. Several targeting mechanisms are required to deliver proteins to the correct chloroplast membrane or soluble compartment. These mechanisms have been extensively studied using purified chloroplasts in vitro. Prior to targeting these proteins to the various compartments of the chloroplast, they must be correctly sorted in the cytosol. To date, it is not clear how these proteins are sorted in the cytosol and then targeted to the chloroplasts. Recently, the cytosolic carrier protein AKR2 and its associated cofactor Hsp17.8 for outer envelope membrane proteins of chloroplasts were identified. Additionally, a mechanism for controlling unimported plastid precursors in the cytosol has been discovered. This review will mainly focus on recent findings concerning the possible cytosolic events that occur prior to protein targeting to the chloroplasts. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.     

Integrated proteome and metabolite analysis of the de-etiolation process in plastids from rice (Oryza sativa L.)

We have analyzed the dynamics of the rice etioplast membrane proteome during the early phase of de-etiolation using iTRAQ-based relative protein quantification. Several hundred plastid proteins were identified from enriched membranes, including 36 putative transporters. Hierarchical clustering revealed the coordinated light induction of thylakoid membrane proteins with proteins involved in translation and fatty acid metabolism. No other functional category of identified proteins showed a similarly consistent light induction, and no consistent changes were observed for the identified transporters. This suggests that the etioplast metabolism is already primed to accommodate the metabolic changes that occur during the onset of photosynthesis. This hypothesis was further tested in metabolite profiling experiments. Here, the changes upon illumination are mostly restricted to a decrease in the concentration of some amino acids and an increase in the concentrations of aspartic acid, malic acid, fumaric acid, and succinic acid. These changes are consistent with a rapid activation of photosynthesis and subsequent rapid production of storage carbohydrates and proteins. The information at the proteome level and the parallel measurements of metabolite accumulation both support the view that only minor metabolic network reconstruction and modification of enzyme levels occurs during the first 4?h of etioplast to chloroplast differentiation.     

A role for lipid trafficking in chloroplast biogenesis

Chloroplasts are the defining plant organelle carrying out photosynthesis. Photosynthetic complexes are embedded into the thylakoid membrane which forms an intricate system of membrane lamellae and cisternae. The chloroplast boundary consists of two envelope membranes controlling the exchange of metabolites between the plastid and the extraplastidic compartments of the cell. The plastid internal matrix (stroma) is the primary location for fatty acid biosynthesis in plants. Fatty acids can be assembled into glycerolipids at the envelope membranes of plastids or they can be exported and assembled into lipids at the endoplasmic reticulum (ER) to provide building blocks for extraplastidic membranes. Some of these glycerolipids, assembled at the ER, return to the plastid where they are remodeled into the plastid typical glycerolipids. As a result of this cooperation of different subcellular membrane systems, a rich complement of lipid trafficking phenomena contributes to the biogenesis of chloroplasts. Considerable progress has been made in recent years towards a better mechanistic understanding of lipid transport across plastid envelopes. Lipid transporters of bacteria and plants have been discovered and their study begins to provide detailed mechanistic insights into lipid trafficking phenomena relevant to chloroplast biogenesis.     

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.     

Without a little help from 'my' friends: direct insertion of proteins into chloroplast membranes?

The chloroplast membranes are highly regulated and biological active regions of the living plant cell, which carry numerous essential proteinaceous components. For example, in the thylakoid membrane the photosynthesis apparatus, one of the most life-relevant biological machineries, is located. How these membrane proteins are targeted to and inserted into their target membranes was one of the questions we aimed to understand in the last few years. Fifteen years ago little to nothing was known about the targeting and translocation of outer envelope proteins (G.W. Schmidt and L.M. Mishkind, Annu. Rev. Biochem. 55 (1986)). Although several protein assisted pathways for translocation of proteins across the membranes have been characterised, only recent results gave insight into how membrane proteins are inserted into the chloroplast membranes. Here we will focus on the mode of insertion of a class of proteins into the outer envelope and the thylakoid membranes, which share a unique feature: they insert apparently directly into the lipid bilayer, i.e. without the help of a proteinaceous translocation pore.     

Recognition and envelope translocation of chloroplast preproteins

Plastids are a diverse group of plant organelles that perform essential functions including important steps in many biosynthetic pathways. Chloroplasts are the best characterized type of plastid, and constitute the site of oxygenic photosynthesis in plants, a process essential to all higher life forms. It is well established that the majority (>90%) of chloroplast proteins are nucleus-encoded and must be post-translationally imported into these envelope-bound compartments. Most nucleus-encoded chloroplast proteins are translated in precursor form on cytosolic ribosomes, targeted to the chloroplast surface, and then imported across the double-membrane envelope by translocons in the outer and inner envelope membranes of the chloroplast, termed TOC and TIC, respectively. Recently, significant progress has been made in our understanding of how proteins are targeted to the chloroplast surface and translocated across the chloroplast envelope into the stroma. Evidence suggesting the existence of multiple import pathways at the outer envelope membrane for different classes of precursor proteins has been presented. These pathways appear to utilize similar TOC complexes equipped with different combinations of homologous GTPase receptors, providing preprotein recognition specificity.     

Plastid protein import and sorting: different paths to the same compartments

Chloroplasts contain several thousand different proteins, of which more than 95% are encoded on nuclear genes, synthesized in the cytosol as precursor proteins, and imported into the organelle. The major pathways for import and routing have been described; a general import apparatus in the chloroplast envelope and several ancestral translocases in the thylakoid membranes. In this update we focus on some interesting and emerging areas: the Tat translocase, which operates in parallel with the Sec system but transports folded proteins; different routes to the envelope membranes, which promises an understanding of the ways the Tic apparatus sorts transmembrane domains (TMDs) and may also uncover developmental relationships between envelope and thylakoids; and novel routes for proteins into chloroplasts including delivery from the secretory system.     

The role of the transmembrane domain in determining the targeting of membrane proteins to either the inner envelope or thylakoid membrane

Chloroplastic membrane proteins can be targeted to any of three distinct membrane systems, i.e., the outer envelope membrane (OEM), inner envelope membrane (IEM), and thylakoid membrane. This complex structure of chloroplasts adds significantly to the challenge of studying protein targeting to various membrane sub-compartments within a chloroplast. In this investigation, we examined the role played by the transmembrane domain (TMD) in directing membrane proteins to either the IEM or thylakoid membrane. Using the IEM protein, Arc6 (Accumulation and Replication of Chloroplasts 6), we exchanged the stop-transfer TMD of Arc6 with various TMDs derived from different IEM and thylakoid membrane proteins and monitored the subcellular localization of these Arc6-hybrid proteins. We showed that when the Arc6 TMD was replaced with a TMD derived from various thylakoid membrane proteins, these Arc6(thylTMD) hybrid proteins could be directed to the thylakoid membrane rather than to the IEM. Conversely, when the TMD of the thylakoid membrane proteins, STN8 (State Transition protein kinase 8) or Plsp1 (Plastidic type I signal peptidase 1), was replaced with the stop-transfer TMD of Arc6, STN8 and Plsp1 were halted at the IEM. From our investigation, we conclude that the TMD plays a critical role in targeting integral membrane proteins to either the IEM or thylakoid membrane.     

Reconstruction of Metabolic Pathways,Protein Expression,and Homeostasis Machineries across Maize Bundle Sheath and Mesophyll Chloroplasts: Large-Scale Quantitative Proteomics Using the First Maize Genome Assembly

Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C₄ photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization. The experimental analysis was based on high-accuracy mass spectrometry, protein quantification by spectral counting, and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families, and gene duplications related to the polyploidy of maize; this avoided overidentification of proteins and resulted in more accurate protein quantification. A total of 1,105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Nearly complete coverage of primary carbon, starch, and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen, and amino acid metabolism, was obtained. This showed, for example, quantitative and qualitative cell type-specific specialization in starch biosynthesis, arginine synthesis, nitrogen assimilation, and initial steps in sulfur assimilation. An extensive overview of BS and M chloroplast protein expression and homeostasis machineries (more than 200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundance, and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database.Plants can be classified as C₃ or C₄ species based on the primary product of carbon fixation in photosynthesis. The primary product of carbon fixation is a four-carbon compound (oxaloacetate [OAA]) in C₄ plants but a three-carbon compound (3-phosphoglycerate [3PGA]) in C₃ plants. In leaves of C₄ grasses such as maize (Zea mays), photosynthetic activities are partitioned between two anatomically and biochemically distinct bundle sheath (BS) and mesophyll (M) cells. A single ring of BS cells surrounds the vascular bundle, followed by a concentric ring of specialized M cells, creating the classical Kranz anatomy. Active carbon transport (in the form of C₄ organic acids) from M cell to BS cells and specific expression of Rubisco in the BS cells allows Rubisco, the carboxylating enzyme in the Calvin cycle, to operate in a high CO₂ concentration. The high CO₂ concentration suppresses the oxygenation reaction by Rubisco (and the subsequent energy-wasteful photorespiratory pathway), resulting in increased photosynthetic yield and more efficient use of water and nitrogen. The history of C₄ research has been described (Nelson and Langdale, 1992; Sage and Monson, 1999; Edwards et al., 2001). At present, there is renewed interest in C₄ photosynthesis, stimulated in part by the potential use of C₄ plants as a source of biofuels (Carpita and McCann, 2008) and the genetic engineering of C₄ rice (Oryza sativa; Sheehy et al., 2007; Hibberd et al., 2008; Taniguchi et al., 2008). The use of new genomics and/or proteomics tools has resulted in new insights into cellular differentiation in C₄ plants (Majeran and Van Wijk, 2009).Proteins are responsible for most cellular functions, and knowing their abundance, cell-type specific expression patterns, and subcellular localization is essential to understand C₄ differentiation. Previously, we published a quantitative analysis of purified M and BS chloroplast (soluble) stromal proteomes in which BS-M protein accumulation ratios for 125 accessions were determined; this covered a limited range of plastid functions, although it enabled the integration of information from previous studies (Majeran et al., 2005). A subsequent complementary quantitative proteomics study, using nano-liquid chromatography (LC)-LTQ-Orbitrap mass spectrometry (MS) and label-free spectral counting complemented with other techniques, identified proteins in BS and M thylakoid and envelope membranes of maize chloroplasts and determined cell type-specific differences in (1) the protein assembly state and composition of the four photosynthetic complexes and of a new type of NADPH dehydrogenase (NDH) complex; (2) the auxiliary functions of the thylakoid proteome; and (3) protein and metabolite transport functions of M and BS chloroplast envelopes (Majeran et al., 2008). Comparative MS analysis of chloroplast envelope membranes from leaves of pea (Pisum sativum), a C₃ species, and from M chloroplasts of maize showed an enrichment of several known and putative translocators in the maize M envelopes (Brautigam et al., 2008). The conclusions of these proteome analyses are summarized by Majeran and van Wijk, 2009.Whereas these proteomics studies provide significant progress in understanding the organization of C₄ metabolism in maize, three aspects have not been adequately addressed: (1) the stromal proteomes of BS and M chloroplasts likely each contain more than 1,500 proteins, but the BS-M ratios for only approximately 125 proteins were quantified, resulting in very limited coverage of several important secondary metabolic pathways such as sulfur, fatty acid, amino acid, and nucleotide metabolism; (2) information about relative concentrations of stromal proteins in BS and M chloroplasts is lacking but is needed as a basis for quantitative modeling and metabolic engineering of C₄ photosynthesis and other metabolic pathways; the growing “toolbox” of proteomics and MS now allows for such quantitative analyses (Bantscheff et al., 2007; Kumar and Mann, 2009); (3) the soluble (Majeran et al., 2005) and membrane (Majeran et al., 2008) proteome data sets were analyzed by different techniques and mass spectrometers, mostly due to the improvement of commercial mass spectrometers in that time frame. Therefore, it is difficult to understand the quantitative relationships between these data sets. This study addresses these three aspects.So far, maize proteome analyses used essentially ZmGI maize assemblies (for the Z. mays Gene Index) based on ESTs, combined with a limited amount of additional DNA sequence information. The ZmGI was originally generated by The Institute for Genome Research and subsequently supported by the computational Biology and Functional Genomics Laboratory (http://compbio.dfci.harvard.edu/index.html). This ZmGI database did not have annotated gene models (for proteome analysis, the DNA sequences were searched in all six reading frames), and low expressed genes were likely underrepresented. In our most recent BS-M chloroplast analyses (Majeran et al., 2008) as well as a maize envelope analysis (Brautigam et al., 2008), the MS data were searched against ZmGI version 16.0 or 17.0. Since that time, the maize genome has been sequenced (using a bacterial artificial chromosome approach), a physical map was created (the maize accessioned golden path AGP version 1), and its first assembly with gene coordinates and predicted proteins was very recently released (June 2009; http://ftp.maizesequence.org/release-4a.53/sequences/) and published (Schnable et al., 2009). This release contains 32,540 genes with 53,764 gene models; most of the gene models are evidence based. The new maize genome assembly is expected to improve maize proteome analysis with more accurate protein identification and quantitative assessment of protein expression patterns. This also allows for the determination of N-terminal localization signals, which was rarely possible from EST assemblies, as N termini were often lacking.This study presents a quantitative protein expression atlas of differentiated maize leaf M and BS chloroplasts using high-resolution and mass-accuracy MS (using a LTQ-Orbitrap) and the new maize genome assembly. Three biological replicates of stromal proteomes of isolated BS and M chloroplasts were analyzed. Quantification was carried out based on the “spectral counting” method (Zybailov et al., 2005, 2008; Bantscheff et al., 2007; Choi et al., 2008) using a sophisticated bioinformatics “workflow” in particular to deal with gene duplications and extended gene families observed in polyploids such as maize. These new stromal data sets were combined with a reanalysis of our recent BS and M membrane proteome data sets (Majeran et al., 2008) against genome 4a53. Compared with previous maize leaf proteome analyses, this study provides an integrated overview of both primary and especially secondary metabolism, as well as chloroplast gene expression and protein biogenesis, in far greater depth. The reconstructed pathways are presented as figures that include quantitative protein information; pathways include primary carbon metabolism, starch metabolism, nucleotide metabolism, fatty acid and lipid biosynthesis, chlorophyll, heme, and carotenoid synthesis, and nitrogen assimilation. We briefly comment on the use of the new maize genome assembly for proteome analysis. All matched peptides are projected on the predicted protein models via the Plant Proteomics Database (PPDB; http://ppdb.tc.cornell.edu/). Interactive functional annotation, chloroplast localization assignments, as well as details of protein identification are also available via PPDB.     

Effect of Doubled-CO2 Concentration on the Ultrastructure of Chloroplasts from Medicago sativa and Setaria italica

It has been reported in quite a number of literatures that doubled CO2 concentration increased the photosynthetic rate and dry matter production of C3 plants, but substantially affected C4 plants little. However, why may CO2 enrichment promote growth and either no change or decrease reproductive allocation of the C3 species, but havinag no effects on growth characteristics of the C4 plants? So far, there has been no satisfactory explanation on that mentioned above, except the differences in their CO2 compensatory points. In the past, although some studies on ultrastructure of the chloroplasts under doubled CO2 concentration were limitedly conducted. Almost all the relevant experimental materials were only from C3 plants not from C4 plants, and even though the results were of inconsistancy. Thereby, it needs to verify whether the differences in photosynthesis of C3 and C4 plants at doubled CO2 level is caused by the difference in their chloroplast deterioration. Experiments to this subject were conducted at the Botanical Garden of Institute of Botany, Academia Sinica in 1993 and 1994. Both experimental materials from C3 plant alfalfa (Medicago sativa) and C4 plant foxtail millet (Setaria italica) were cultivated in the cylindrical open-top chambers (2.2 m in diameter × 2.4 m in height) with aluminum frames covered by polyethylene film. Natural air or air with 350× 10-6 CO2 were blown from the bottom of the chamber space with constant temperature between inside and outside of the chamber 〈0.2℃〉. Electron microscopic observation revealed that the ultrastructure of the chloroplasts from C3 plant Medicago sativa and C4 plant Seteria italica growing under the same doubled CO2 concentration were quite different from each other. The differential characteristics in ultrastructure of chloro plasts displayed mainly in the configuration of thylakoid membrances and the accumulation of starch grains. They were as follows: 1. The most striking feature was the building up of starch grains in the chloroplasts of the bundle sheath cells (BSCs) and the mesophyll cells (MCs) at doubled CO2 concentra tion. The starch grains appeared centrifugally first in the BSCs and then in the chloroplast of the other MCs. It was worthy to note that the starch grains in the chloroplasts of C4 plant Setaria ira/ica were much more than those of the C3 plant Medicago sativa . The decline of photosynthesis in the doubled CO2-grown C4 plants might be caused by an over accumulation of starch grains, that deformed the chloroplast even demaged the stroma thylakoids and grana. There might exsist a correlation between the comformation of thylakoid system and starch grain accumulation, namely conversion and transfer of starch need energy from ATP, and coupling factor (CF) for ATP formation distributed mainly on protoplastic surface (PSu) of stroma thylakoid membranes, as well as end and margin membranes of grana thylakoids. Thereby, these results could provide a conclusive evidence for the reason of non effectiveness on growth characteristics of C4 plant. 2. Under normal condition , the mature chlolroplats of higher plants usually develop complete and regularly arranged photosynthetic membrane systems . Chloroplasts from the C4 plant Setaria italica, however, exerted significant changes on stacking degree, grana width and stroma thylakoid length under doubled CO2 concentration; In these changes, the grana stacks were smaller and more numerous, and the number of thylakoids per granum was greatly increased, and the stroma thylakoid was greatly lengthened as compared to those of the control chloroplasts. But the grana were mutually intertwined by stroma thylakoid. The integrity of some of the grana were damaged due to the augmentation of the intrathylakoid space . Similarly, the stroma thylakoids were also expanded. In case. the plant was seriously effected by doubled CO2 concentration as observed in C4 plant Setaria italica , its chloroplasts contained merely the stroma (matrix) with abundant starch grains, while grana and stroma thylakoid membranes were unrecognizable, or occasionally a few residuous pieces of thylakoid membranes could be visualized, leaving a situation which appeared likely to be chloroplast deterioration. However, under the same condition the C3 plant Medicago sativa possessed normally developed chloroplasts, with intact grana and stroma thylakoid membranes. Its chloroplasts contained grana intertwined with stroma thylakoid membranes, and increased in stacking degree and granum width, in spite of more accumulated starch grains within the chloroplasts. These configuration changes of the thylakoid system were in consistant with the results of the authors another study on chloroplast function, viz. the increased capacity of chloroplasts for light absorption and efficiency of PSⅡ.     

Evidence for two-step processing of nuclear-encoded chloroplast proteins during membrane assembly

A plastome (chloroplast genome) mutant of tobacco, lutescens-1, displays abnormal degradation of the chloroplast-encoded polypeptides which form the core complex of photosystem II (PSII). Two nuclear-encoded proteins (present in polymorphic forms), which normally function in the water oxidation process of PSII, accumulate as larger size-class polypeptides in mutant thylakoid membranes. These accumulated proteins are intermediate in size between the full-length primary protein synthesized in the cytoplasm and the proteolytically processed mature polypeptides. Trypsin treatment of unstacked mutant thylakoids and of inside-out vesicle (PSII-enriched) preparations indicated that the intermediate size forms were correctly localized on the inner surface of the thylakoid membrane, but not surface-exposed in the same way as the mature proteins. Only one of the intermediate size-class proteins could be extracted by salt washes. We interpret these data to be consistent with the idea that the two imported proteins that function in the water oxidation step of photosynthesis and are localized in the loculus (the space within the thylakoid vesicles) undergo two-step processing. The second step in proteolytic processing may be related to transport through a second membrane (the first transport step through the chloroplast envelope having been completed); this step may be arrested in the mutant due to the absence of the PSII core complex.     

高等植物铁氧还蛋白-NADP＋氧化还原酶研究进展

高等植物叶绿体定位的铁氧还蛋白-NADP＋氧化还原酶（LFNR）负责催化光合线性电子传递的最后一步反应,催化电子由还原态的铁氧还蛋白（Fd）传递给NADP＋。LFNR分布在叶绿体的3个不同的组分中,即叶绿体基质中、类囊体膜上和叶绿体内膜上。最近的研究表明,大多数膜定位的LFNR并非光合作用所必需的,叶绿体基质中的LFNR足以维持光合作用的正常进行。叶绿体中的两个蛋白——Tic62和TROL作为LFNR的锚定蛋白,可以与LFNR在类囊体膜上形成高分子量的蛋白复合体。Tic62-LFNR复合体主要负责在夜间保护LFNR的活性,但它不直接在光合作用中起作用。然而,TROL-LFNR复合体对植物的光合作用有一定的影响。本文将概述植物LFNR的最新研究进展。     

Determining the limitations and regulation of photosynthetic energy transduction in leaves

The light-dependent production of ATP and reductants by the photosynthetic apparatus in vivo involves a series of electron and proton transfers. Consideration is given as to how electron fluxes through photosystem I (PSI), using absorption spectroscopy, and through photosystem II (PSII), using chlorophyll fluorescence analyses, can be estimated in vivo. Measurements of light-induced electrochromic shifts using absorption spectroscopy provide a means of analyzing the proton fluxes across the thylakoid membranes in vivo. Regulation of these electron and proton fluxes is required for the thylakoids to meet the fluctuating metabolic demands of the cell. Chloroplasts exhibit a wide and flexible range of mechanisms to regulate electron and proton fluxes that enable chloroplasts to match light use for ATP and reductant production with the prevailing metabolic requirements. Non-invasive probing of electron fluxes through PSI and PSII, and proton fluxes across the thylakoid membranes can provide insights into the operation of such regulatory processes in vivo.     

Chloroplast-targeted ferredoxin-NADP(+) oxidoreductase (FNR): structure, function and location

Ferredoxin-NADP(+) oxidoreductase (FNR) is a ubiquitous flavin adenine dinucleotide (FAD)-binding enzyme encoded by a small nuclear gene family in higher plants. The chloroplast targeted FNR isoforms are known to be responsible for the final step of linear electron flow transferring electrons from ferredoxin to NADP(+), while the putative role of FNR in cyclic electron transfer has been under discussion for decades. FNR has been found from three distinct chloroplast compartments (i) at the thylakoid membrane, (ii) in the soluble stroma, and (iii) at chloroplast inner envelope. Recent in vivo studies have indicated that besides the membrane-bound FNR, also the soluble FNR is photosynthetically active. Two chloroplast proteins, Tic62 and TROL, were recently identified and shown to form high molecular weight protein complexes with FNR at the thylakoid membrane, and thus seem to act as the long-sought molecular anchors of FNR to the thylakoid membrane. Tic62-FNR complexes are not directly involved in photosynthetic reactions, but Tic62 protects FNR from inactivation during the dark periods. TROL-FNR complexes, however, have an impact on the photosynthetic performance of the plants. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.