Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge.

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens. While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation. Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5.

Structural features of chloroplast trigger factor determined at 2.6†Šresolution.

The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl cis-trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their bacterial orthologs and their molecular mechanism in plant organelles has been little investigated to date. Here, the crystal structure of the plastidic trigger factor from the green alga Chlamydomonas reinhardtii is presented at 2.6 Šresolution. Due to the high intramolecular flexibility of the protein, diffraction to this resolution was only achieved using a protein that lacked the N-terminal ribosome-binding domain. The eukaryotic trigger factor from C. reinhardtii exhibits a comparable dragon-shaped conformation to its bacterial counterpart. However, the C-terminal chaperone domain displays distinct charge distributions, with altered positioning of the helical arms and a specifically altered charge distribution along the surface responsible for substrate binding. While the PPIase domain shows a highly conserved structure compared with other PPIases, its rather weak activity and an unusual orientation towards the C-terminal domain points to specific adaptations of eukaryotic trigger factor for function in chloroplasts.

Acclimation in plants - the Green Hub consortium.

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.

Equilibrative nucleoside transporter 1 (ENT1) is critical for pollen germination and vegetative growth in Arabidopsis.

ENT1 of Arabidopsis thaliana was the first member of the equilibrative nucleoside transporter (ENT) family to be identified in plants and characterized as a cellular, high-affinity nucleoside importer. Evidence is presented here for a tonoplast localization of ENT1 based on proteome data and Western blot analyses. Increased export of adenosine from reconstituted tonoplast preparations from 35S:ENT1 mutants compared with those from the wild type and ENT1-RNAi mutants support this view. Furthermore, increased vacuolar adenosine and vacuolar 2'3'-cAMP (an intermediate of RNA catabolism) contents in ENT1-RNAi mutants, but decreased contents of these metabolites in 35S:ENT1 over-expresser mutants, were observed. An up-regulation of the salvage pathway was detected in the latter mutants, leading to the conclusion that draining the vacuolar adenosine storage by ENT1 over-expression interferes with cellular nucleotide metabolism. As a consequence of the observed metabolic alterations 35S:ENT1 over-expresser mutants exhibited a smaller phenotypic appearance compared with wild-type plants. In addition, ENT1:RNAi mutants exhibited significantly lower in vitro germination of pollen and contained reduced internal and external ATP levels. This indicates that ENT1-mediated nucleosides, especially adenosine transport, is important for nucleotide metabolism, thus influencing growth and pollen germination.

Nonmitochondrial ATP/ADP transporters accept phosphate as third substrate.

Chlamydiales and Rickettsiales as metabolically impaired, intracellular pathogenic bacteria essentially rely on "energy parasitism" by the help of nucleotide transporters (NTTs). Also in plant plastids NTT-type carriers catalyze ATP/ADP exchange to fuel metabolic processes. The uptake of ATP4-, followed by energy consumption and the release of ADP3-, would lead to a metabolically disadvantageous accumulation of negative charges in form of inorganic phosphate (Pi) in the bacterium or organelle if no interacting Pi export system exists. We identified that Pi is a third substrate of several NTT-type ATP/ADP transporters. During adenine nucleotide hetero-exchange, Pi is cotransported with ADP in a one-to-one stoichiometry. Additionally, Pi can be transported in exchange with solely Pi. This Pi homo-exchange depends on the presence of ADP and provides a first indication for only one binding center involved in import and export. Furthermore, analyses of mutant proteins revealed that Pi interacts with the same amino acid residue as the gamma-phosphate of ATP. Import of ATP in exchange with ADP plus Pi is obviously an efficient way to couple energy provision with the export of the two metabolic products (ADP plus Pi) and to maintain cellular phosphate homeostasis in intracellular living "energy parasites" and plant plastids. The additional Pi transport capacity of NTT-type ATP/ADP transporters makes the existence of an interacting Pi exporter dispensable and might explain why a corresponding protein so far has not been identified.

Enlightening energy parasitism by analysis of an ATP/ADP transporter from chlamydiae.

Energy parasitism by ATP/ADP transport proteins is an essential, common feature of intracellular bacteria such as chlamydiae and rickettsiae, which are major pathogens of humans. Although several ATP/ADP transport proteins have so far been characterized, some fundamental questions regarding their function remained unaddressed. In this study, we focused on the detailed biochemical analysis of a representative ATP/ADP transporter (PamNTT1), from the amoeba symbiont Protochlamydia amoebophila (UWE25) to further clarify the principle of energy exploitation. We succeeded in the purification of the first bacterial nucleotide transporter (NTT) and its functional reconstitution into artificial lipid vesicles. Reconstituted PamNTT1 revealed high import velocities for ATP and an unexpected and previously unobserved stimulating effect of the luminal ADP on nucleotide import affinities. Latter preference of the nucleotide hetero-exchange is independent of the membrane potential, and therefore, PamNTT1 not only structurally but also functionally differs from the well-characterized mitochondrial ADP/ATP carriers. Reconstituted PamNTT1 exhibits a bidirectional orientation in lipid vesicles, but interestingly, only carriers inserted with the N-terminus directed to the proteoliposomal interior are functional. The data presented here comprehensively explain the functional basis of how the intracellular P. amoebophila manages to exploit the energy pool of its host cell effectively by using the nucleotide transporter PamNTT1. This membrane protein mediates a preferred import of ATP, which is additionally stimulated by a high internal (bacterial) ADP/ATP ratio, and the orientation-dependent functionality of the transporter ensures that it is not working in a mode that is detrimental to P. amoebophila. Heterologous expression and purification of high amounts of PamNTT1 provides the basis for its crystallization and detailed structure/function analyses. Furthermore, functional reconstitution of this essential chlamydial protein paves the way for high-throughput uptake studies in order to screen for specific inhibitors potentially suitable as anti-chlamydial drugs.