Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www. grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions. Molecular & Cellular Proteomics 9:1063-1084, 2010.Plastids are semiautonomous organelles that are ubiquitously found in plant cells. They are derived from an endosymbiotic event and are thought to have evolved from an ancient photosynthetic prokaryote related to present-day cyanobacteria. Following endosymbiosis, the plastid genome has been reduced to ϳ100 genes, mainly coding for housekeeping functions (translation and transcription of the plastid genome), proteins required for primary photosynthetic reactions, and a few, yet poorly characterized, gene products (1). The most conspicuous plastid type is the chloroplast, found in leaves and carrying out photosynthesis as its main function. Photosynthesis is an integrated biological process involving the coordinated functioning of ...

Plants respond to changes in light quality by regulating the absorption capacity of their photosystems. These short-term adaptations use redox-controlled, reversible phosphorylation of the lightharvesting complexes (LHCIIs) to regulate the relative absorption cross-section of the two photosystems (PSs), commonly referred to as state transitions. It is acknowledged that state transitions induce substantial reorganizations of the PSs. However, their consequences on the chloroplast structure are more controversial. Here, we investigate how state transitions affect the chloroplast structure and function using complementary approaches for the living cells of Chlamydomonas reinhardtii. Using small-angle neutron scattering, we found a strong periodicity of the thylakoids in state 1, with characteristic repeat distances of ∼200 Å, which was almost completely lost in state 2. As revealed by circular dichroism, changes in the thylakoid periodicity were paralleled by modifications in the long-range order arrangement of the photosynthetic complexes, which was reduced by ∼20% in state 2 compared with state 1, but was not abolished. Furthermore, absorption spectroscopy reveals that the enhancement of PSI antenna size during state 1 to state 2 transition (∼20%) is not commensurate to the decrease in PSII antenna size (∼70%), leading to the possibility that a large part of the phosphorylated LHCIIs do not bind to PSI, but instead form energetically quenched complexes, which were shown to be either associated with PSII supercomplexes or in a free form. Altogether these noninvasive in vivo approaches allow us to present a more likely scenario for state transitions that explains their molecular mechanism and physiological consequences.green algae | photosynthesis | thylakoid membrane T he efficient operation of the photosynthetic machinery of oxygenic photosynthetic organisms requires a balanced energy supply to the two photosystems (PSs) under changing environments. To avoid unbalanced excitations of the two PSs, a rapid acclimation mechanism, state transitions (STs), takes place, which allows redistributing the excitation energy between the two PSs. ST is seen in green algae and vascular plants and is modulated by the redox-controlled, reversible phosphorylation of LHCII, the light-harvesting chlorophyll a/b antenna complex. A serine-threonine protein kinase, STN7 in vascular plants (1) and Stt7 in green algae (2), is responsible for this phosphorylation. Stt7/STN7 activity depends on the redox state of the plastoquinone (PQ) pool, which is sensed by the cytochrome b 6 f complex (3).According to the current model of STs, preferential PSII excitation leads to PQ reduction, and thus to LHCII phosphorylation by the kinase. The PQ pool can also be reduced in dark anaerobic conditions in algal suspensions. In this state, called state 2 (S2), phosphorylated LHCII dissociates from PSII, thereby reducing its absorption cross-section, and associates to PSI, acting as an additional antenna for this complex. In the reverse process, prefere...

Photosynthesis has shaped atmospheric and ocean chemistries and probably changed the climate as well, as oxygen is released from water as part of the photosynthetic process. In photosynthetic eukaryotes, this process occurs in the chloroplast, an organelle containing the most abundant biological membrane, the thylakoids. The thylakoids of plants and some green algae are structurally inhomogeneous, consisting of two main domains: the grana, which are piles of membranes gathered by stacking forces, and the stroma-lamellae, which are unstacked thylakoids connecting the grana. The major photosynthetic complexes are unevenly distributed within these compartments because of steric and electrostatic constraints. Although proteomic analysis of thylakoids has been instrumental to define its protein components, no extensive proteomic study of subthylakoid localization of proteins in the BBY (grana) and the stroma-lamellae fractions has been achieved so far. To fill this gap, we performed a complete survey of the protein composition of these thylakoid subcompartments using thylakoid membrane fractionations. We employed semiquantitative proteomics coupled with a data analysis pipeline and manual annotation to differentiate genuine BBY and stroma-lamellae proteins from possible contaminants. About 300 thylakoid (or potentially thylakoid) proteins were shown to be enriched in either the BBY or the stroma-lamellae fractions. Overall, present findings corroborate previous observations obtained for photosynthetic proteins that used nonproteomic approaches. The originality of the present proteomic relies in the identification of photosynthetic proteins whose differential distribution in the thylakoid subcompartments might explain already observed phenomenon such as LHCII docking. Besides, from the present localization results we can suggest new molecular actors for photosynthesis-linked activities. For instance, most PsbP-like subunits being differently localized in stroma-lamellae, these proteins could be linked to the PSI-NDH complex in the context of cyclic electron flow around PSI. In addition, we could identify about a hundred new likely minor thylakoid (or chloroplast) proteins, some of them being potential regulators of the chloroplast physiology. Molecular & Cellular Proteomics 13: 10.1074/mcp.M114.040923, 2147-2167, 2014.As primary producers, plants are at the basis of the food chain for most ecosystems and control the planet atmosphere via photosynthesis. In eukaryotes, this process, responsible for carbon fixation and for the release of gaseous oxygen into the atmosphere, takes place in a specialized compartment, the chloroplast. This semiautonomous organelle plays a number of essential functions, including photosynthesis, nitrogen assimilation, sulfur reduction and assimilation, synthesis of amino acids, fatty acids, and many secondary metabolites (1-6). The chloroplast is not the only type of plastids. Plastids are indeed found in every plant tissue (with a very few exceptions such as angiosperm pollen grains) and in...

BackgroundDespite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.Methodology/Principal FindingsThe aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.Conclusions/SignificanceAltogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.

By quantitatively comparing the proteomes of total leaf (crude cell extract) from Arabidopsis and purified chloroplast envelope fractions, this study makes available a novel parameter (calculated Enrichment Factor) for each putative envelope protein. This parameter provides important information to enable the more confident identification of genuine envelope components, distinguishing them from contaminants from other cellular/chloroplast compartments.

The physiological role of the plastid terminal oxidase (PTOX) involved in plastoquinol oxidation in chloroplasts has been investigated in vivo in tomato leaves. Enzyme activity was assessed by non-invasive methods based on the analysis of the kinetics of chlorophyll fluorescence changes. In the dark, the maximum PTOX rate was smaller than 1 electron per second per PSII. This value was further decreased upon light acclimation, and became almost negligible upon inhibition of the photosynthetic performances by reducing the CO(2) availability. In contrast, prolonged exposure to high light resulted in an increase of the overall PTOX activity, which was paralleled by an increased protein accumulation. Under all the conditions tested the enzyme activity always remained about two orders of magnitude lower than that of electron flux through the linear photosynthetic electron pathway. Therefore, PTOX cannot have a role of a safety valve for photogenerated electrons, while it could be involved in acclimation to high light. Moreover, by playing a major role in the control of the stromal redox poise, PTOX is also capable of modulating the balance between linear and cyclic electron flow around PSI during the deactivation phase of carbon assimilation that follows a light to dark transition.

The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1β in tested α- and β-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the β-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.

Global warming and drought stress are expected to have a negative impact on agricultural productivity. Desiccation-tolerant species, which are able to tolerate the almost complete desiccation of their vegetative tissues, are appropriate models to study extreme drought tolerance and identify novel approaches to improve the resistance of crops to drought stress. In the present study, to better understand what makes resurrection plants extremely tolerant to drought, we performed transmission electron microscopy and integrative large-scale proteomics, including organellar and phosphorylation proteomics, and combined these investigations with previously published transcriptomic and metabolomics data from the resurrection plant Haberlea rhodopensis. The results revealed new evidence about organelle and cell preservation, posttranscriptional and posttranslational regulation, photosynthesis, primary metabolism, autophagy, and cell death in response to desiccation in H. rhodopensis. Different protective intrinsically disordered proteins, such as late embryogenesis abundant (LEA) proteins, thaumatin-like proteins (TLPs), and heat shock proteins (HSPs), were detected. We also found a constitutively abundant dehydrin in H. rhodopensis whose phosphorylation levels increased under stress in the chloroplast fraction. This integrative multi-omics analysis revealed a systemic response to desiccation in H. rhodopensis and certain targets for further genomic and evolutionary studies on DT mechanisms and genetic engineering towards the improvement of drought tolerance in crops.