Probing the physiological role of the plastid outer-envelope membrane using the oemiR plasmid collection.

Plastids are the site of complex biochemical pathways, most prominently photosynthesis. The organelle evolved through endosymbiosis with a cyanobacterium, which is exemplified by the outer envelope membrane that harbors more than 40 proteins in Arabidopsis. Their evolutionary conservation indicates high significance for plant cell function. While a few proteins are well-studied as part of the protein translocon complex the majority of outer envelope protein functions is unclear. Gaining a deeper functional understanding has been complicated by the lack of observable loss-of-function mutant phenotypes, which is often rooted in functional genetic redundancy. Therefore, we designed outer envelope-specific artificial micro RNAs (oemiRs) capable of downregulating transcripts from several loci simultaneously. We successfully tested oemiR function by performing a proof-of-concept screen for pale and cold-sensitive mutants. An in-depth analysis of pale mutant alleles deficient in the translocon component TOC75 using proteomics provided new insights into putative compensatory import pathways. The cold stress screen not only recapitulated 3 previously known phenotypes of cold-sensitive mutants but also identified 4 mutants of additional oemiR outer envelope loci. Altogether our study revealed a role of the outer envelope to tolerate cold conditions and showcasts the power of the oemiR collection to research the significance of outer envelope proteins.

The ABC transporter family efflux pump PvdRT-OpmQ of Pseudomonas putida KT2440: purification and initial characterization.

Tripartite efflux systems of the ABC-type family transport a variety of substrates and contribute to the antimicrobial resistance of Gram-negative bacteria. PvdRT-OpmQ, a member of this family, is thought to be involved in the secretion of the newly synthesized and recycled siderophore pyoverdine in Pseudomonas species. Here, we purified and characterized the inner membrane component PvdT and the periplasmic adapter protein PvdR of the plant growth-promoting soil bacterium Pseudomonas putida KT2440. We show that PvdT possesses an ATPase activity that is stimulated by the addition of PvdR. In addition, we provide the first biochemical evidence for direct interactions between pyoverdine and PvdRT.

Characterization of the preferred cation cofactors of chloroplast protein kinases in Arabidopsis thaliana.

Chloroplasts sense a variety of stimuli triggering several acclimation responses. One prominent response is the mechanism of state transitions, which enables rapid adaption to changes in illumination. Here, we investigated the link between divalent cations (calcium, magnesium, and manganese) and protein kinase activity in Arabidopsis chloroplasts. Our results show that manganese ions are the strongest activator of kinase activity in chloroplasts followed by magnesium ions, whereas calcium ions are not able to induce kinase activity. Additionally, the phosphorylation of specific protein bands is strongly reduced in chloroplasts of a cmt1 mutant, which is impaired in manganese import into chloroplasts, as compared to the wild-type. These findings provide insights for the future characterization of chloroplast protein kinase activity and potential target proteins.

Chloroplast Envelope Membrane Subfractionation from Arabidopsis and Pea.

Due to their endosymbiotic origin, chloroplasts harbor several subcompartments and membrane systems. Each of these has a different protein and lipid composition that dynamically changes either naturally during plant development or induced by environmental stimuli. Here, we describe a protocol for chloroplast envelope membrane subfractionation via discontinuous sucrose gradients, which offers the possibility to separate the different plastid subcompartments for several downstream applications. It is a strong tool for protein sublocalization studies as well as for tracking dynamic movement patterns. Furthermore, it can be combined with in vitro import studies of radioactively labeled proteins, which allows sublocalization of putative envelope proteins independent of the availability of specific antisera.

Chloroplasts are key players to cope with light and temperature stress.

Under natural environmental conditions, changes in light intensity and temperature are closely interwoven, and of all organelles, only chloroplasts react strongly upon alterations of these two parameters. We review increasing evidence indicating that changes in chloroplast metabolism are critical for the comprehensive cellular answer in a challenging environment. This cellular answer starts with rapid modifications of thylakoid-located processes, followed by modifications in the stroma and transport activities across the chloroplast envelope. We propose that the 'modulators' involved contribute to plant stress tolerance and that deciphering of their characteristics is essential to understand 'acclimation'. Especially in times of climatic changes, we must gain knowledge on physiological reactions that might become instrumental for directed breeding strategies aiming to develop stress-tolerant crop plants.

The TPR- and J-domain-containing proteins DJC31 and DJC62 are involved in abiotic stress responses in Arabidopsis thaliana.

Molecular chaperones play an important role during the response to different stresses. Since plants are sessile organisms, they need to be able to adapt quickly to different conditions. To do so, plants possess a complex chaperone machinery, composed of HSP70, HSP90, J proteins and other factors. In this study we characterized DJC31 (also known as TPR16) and DJC62 (also known as TPR15) of Arabidopsis thaliana, two J proteins that additionally carry clamp-type tetratricopeptide repeat domains. Using cell fractionation and split GFP, we could show that both proteins are attached to the cytosolic side of the endoplasmic reticulum membrane. Moreover, an interaction with cytosolic HSP70.1 and HSP90.2 could be shown using bimolecular fluorescence complementation. Knockout of both DJC31 and DJC62 caused severe defects in growth and development, which affected almost all organs. Furthermore, it could be shown that the double mutant is more sensitive to osmotic stress and treatment with abscisic acid, but surprisingly exhibited enhanced tolerance to drought. Taken together, these findings indicate that DJC31 and DJC62 might act as important regulators of chaperone-dependent signaling pathways involved in plant development and stress responses.

Loss of inner-envelope K+/H+ exchangers impairs plastid rRNA maturation and gene expression.

The inner-envelope K+ EFFLUX ANTIPORTERS (KEA) 1 and 2 are critical for chloroplast development, ion homeostasis, and photosynthesis. However, the mechanisms by which changes in ion flux across the envelope affect organelle biogenesis remained elusive. Chloroplast development requires intricate coordination between the nuclear genome and the plastome. Many mutants compromised in plastid gene expression (PGE) display a virescent phenotype, that is delayed greening. The phenotypic appearance of Arabidopsis thaliana kea1 kea2 double mutants fulfills this criterion, yet a link to PGE has not been explored. Here, we show that a simultaneous loss of KEA1 and KEA2 results in maturation defects of the plastid ribosomal RNAs. This may be caused by secondary structure changes of rRNA transcripts and concomitant reduced binding of RNA-processing proteins, which we documented in the presence of skewed ion homeostasis in kea1 kea2. Consequently, protein synthesis and steady-state levels of plastome-encoded proteins remain low in mutants. Disturbance in PGE and other signs of plastid malfunction activate GENOMES UNCOUPLED 1-dependent retrograde signaling in kea1 kea2, resulting in a dramatic downregulation of GOLDEN2-LIKE transcription factors to halt expression of photosynthesis-associated nuclear-encoded genes (PhANGs). PhANG suppression delays the development of fully photosynthesizing kea1 kea2 chloroplasts, probably to avoid progressing photo-oxidative damage. Overall, our results reveal that KEA1/KEA2 function impacts plastid development via effects on RNA-metabolism and PGE.

The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs Involved in Chloroplast Development.

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7, or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

Molecular changes of Arabidopsis thaliana plastoglobules facilitate thylakoid membrane remodeling under high light stress.

Plants require rapid responses to adapt to environmental stresses. This includes dramatic changes in the size and number of plastoglobule lipid droplets within chloroplasts. Although the morphological changes of plastoglobules are well documented, little is known about the corresponding molecular changes. To address this gap, we have compared the quantitative proteome, oligomeric state, prenyl-lipid content and kinase activities of Arabidopsis thaliana plastoglobules under unstressed and 5-day light-stressed conditions. Our results show a specific recruitment of proteins related to leaf senescence and jasmonic acid biosynthesis under light stress, and identify nearly half of the plastoglobule proteins in high native molecular weight masses. Additionally, a specific increase in plastoglobule carotenoid abundance under the light stress was consistent with enhanced thylakoid disassembly and leaf senescence, supporting a specific role for plastoglobules in senescence and thylakoid remodeling as an intermediate storage site for photosynthetic pigments. In vitro kinase assays of isolated plastoglobules demonstrated kinase activity towards multiple target proteins, which was more pronounced in the plastoglobules of unstressed than light-stressed leaf tissue, and which was diminished in plastoglobules of the abc1k1/abc1k3 double-mutant. These results strongly suggest that plastoglobule-localized ABC1 kinases hold endogenous kinase activity, as these were the only known or putative kinases identified in the isolated plastoglobules by deep bottom-up proteomics. Collectively, our study reveals targeted changes to the protein and prenyl-lipid composition of plastoglobules under light stress that present strategies by which plastoglobules appear to facilitate stress adaptation within 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.

The ER luminal C-terminus of AtSec62 is critical for male fertility and plant growth in Arabidopsis thaliana.

Protein translocation into the endoplasmic reticulum (ER) occurs either co- or post-translationally through the Sec translocation system. The Arabidopsis Sec post-translocon is composed of the protein-conducting Sec61 complex, the chaperone-docking protein AtTPR7, the J-domain-containing proteins AtERdj2A/B and the yet uncharacterized AtSec62. Yeast Sec62p is suggested to mainly function in post-translational translocation, whereas mammalian Sec62 also interacts with ribosomes. In Arabidopsis, loss of AtSec62 leads to impaired growth and drastically reduced male fertility indicating the importance of AtSec62 in protein translocation and subsequent secretion in male gametophyte development. Moreover, AtSec62 seems to be divergent in function as compared with yeast Sec62p, since we were not able to complement the thermosensitive yeast mutant sec62-ts. Interestingly, AtSec62 has an additional third transmembrane domain in contrast to its yeast and mammalian counterparts resulting in an altered topology with the C-terminus facing the ER lumen instead of the cytosol. In addition, the AtSec62 C-terminus has proven to be indispensable for AtSec62 function, since a construct lacking the C-terminal region was not able to rescue the mutant phenotype in Arabidopsis. We thus propose that Sec62 acquired a unique topology and function in protein translocation into the ER in plants.

The topology of plastid inner envelope potassium cation efflux antiporter KEA1 provides new insights into its regulatory features.

The plastid potassium cation efflux antiporters (KEAs) are important for chloroplast function, development, and photosynthesis. To understand their regulation at the protein level is therefore of fundamental importance. Prior studies have focused on the regulatory K+ transport and NAD-binding (KTN) domain in the C-terminus of the thylakoid carrier KEA3 but the localization of this domain remains unclear. While all three plastid KEA members are highly conserved in their transmembrane region and the C-terminal KTN domain, only the inner envelope KEA family members KEA1 and KEA2 carry a long soluble N-terminus. Interestingly, this region is acetylated at lysine 168 by the stromal acetyltransferase enzyme NSI. If an odd number of transmembrane domains existed for inner envelope KEAs, as it was suggested for all three plastid KEA carriers, regulatory domains and consequently protein regulation would occur on opposing sides of the inner envelope. In this study we therefore set out to investigate the topology of inner envelope KEA proteins. Using a newly designed antibody specific to the envelope KEA1 N-terminus and transgenic Arabidopsis plants expressing a C-terminal KEA1-YFP fusion protein, we show that both, the N-terminal and C-terminal, regulatory domains of KEA1 reside in the chloroplast stroma and not in the intermembrane space. Considering the high homology between KEA1 and KEA2, we therefore reason that envelope KEAs must consist of an even number of transmembrane domains.

High Light Acclimation Induces Chloroplast Precursor Phosphorylation and Reduces Import Efficiency.

Acclimation is an essential process in plants on many levels, but especially in chloroplasts under changing light conditions. It is partially known how the photosynthetic machinery reacts upon exposure to high light intensities, including rearrangement of numerous protein complexes. Since the majority of proteins residing within chloroplasts needs to be posttranslationally imported into the organelles, we endeavored to study how this important process is regulated upon subjecting plants from pea and Arabidopsis to high light. Our results reveal that acclimation takes place on the one hand in the cytosol by differential phosphorylation of preproteins and resulting from the altered expression of the responsible kinases, and on the other hand at the level of the translocation machineries in the outer (TOC) and inner (TIC) envelope membranes. Intriguingly, while phosphorylation is more pronounced under high light, import itself shows a lower efficiency, along with a reduced accumulation of the Toc receptor proteins Toc34 and Toc159.

The ACT domain in chloroplast precursor-phosphorylating STY kinases binds metabolites and allosterically regulates kinase activity.

Protein import of nucleus-encoded proteins into plant chloroplasts is a highly regulated process, requiring fine-tuning mechanisms especially during chloroplast differentiation. One way of altering import efficiency is phosphorylation of chloroplast transit peptides in the cytosol. We recently investigated the role of three serine/threonine/tyrosine (STY) kinases, STY8, STY17, and STY46, in precursor phosphorylation. These three kinases have a high degree of similarity and harbor a conserved aspartate kinase-chorismate mutase-tyrA (prephenate dehydrogenase) (ACT) domain upstream of the kinase domain. The ACT domain is a widely distributed structural motif known to be important for allosteric regulation of many enzymes. In this work, using biochemical and biophysical techniques in vitro and in planta, including kinase assays, microscale thermophoresis, size exclusion chromatography, as well as site-directed mutagenesis approaches, we show that the ACT domain regulates autophosphorylation and substrate phosphorylation of the STY kinases. We found that isoleucine and S-adenosylmethionine bind to the ACT domain, negatively influencing its autophosphorylation ability. Moreover, we investigated the role of the ACT domain in planta and confirmed its involvement in chloroplast differentiation in vivo Our results provide detailed insights into the regulation of enzyme activity by ACT domains and establish that it has a role in binding amino acid ligands during chloroplast biogenesis.

JASSY, a chloroplast outer membrane protein required for jasmonate biosynthesis.

Jasmonates are vital plant hormones that not only act in the stress response to biotic and abiotic influences, such as wounding, pathogen attack, and cold acclimation, but also drive developmental processes in cooperation with other plant hormones. The biogenesis of jasmonates starts in the chloroplast, where several enzymatic steps produce the jasmonate precursor 12-oxophytodienoic acid (OPDA) from α-linolenic acid. OPDA in turn is exported into the cytosol for further conversion into active jasmonates, which subsequently induces the expression of multiple genes in the nucleus. Despite its obvious importance, the export of OPDA across the chloroplast membranes has remained elusive. In this study, we characterized a protein residing in the chloroplast outer membrane, JASSY, which has proven indispensable for the export of OPDA from the chloroplast. We provide evidence that JASSY has channel-like properties and propose that it thereby facilitates OPDA transport. Consequently, a lack of JASSY in Arabidopsis leads to a deficiency in accumulation of jasmonic acids, which results in impaired expression of jasmonate target genes on exposure to various stresses. This results in plants that are more susceptible to pathogen attack and also exhibit defects in cold acclimation.

Plastoglobular protein 18 is involved in chloroplast function and thylakoid formation.

Plastoglobules are lipoprotein particles that are found in different types of plastids. They contain a very specific and specialized set of lipids and proteins. Plastoglobules are highly dynamic in size and shape, and are therefore thought to participate in adaptation processes during either abiotic or biotic stresses or transitions between developmental stages. They are suggested to function in thylakoid biogenesis, isoprenoid metabolism, and chlorophyll degradation. While several plastoglobular proteins contain identifiable domains, others provide no structural clues to their function. In this study, we investigate the role of plastoglobular protein 18 (PG18), which is conserved from cyanobacteria to higher plants. Analysis of a PG18 loss-of-function mutant in Arabidopsis thaliana demonstrated that PG18 plays an important role in thylakoid formation; the loss of PG18 results in impaired accumulation, assembly, and function of thylakoid membrane complexes. Interestingly, the mutant accumulated less chlorophyll and carotenoids, whereas xanthophyll cycle pigments were increased. Accumulation of photosynthetic complexes is similarly affected in both a Synechocystis and an Arabidopsis PG18 mutant. However, the ultrastructure of cyanobacterial thylakoids is not compromised by the lack of PG18, probably due to its less complex architecture.

A brief history of thylakoid biogenesis.

The thylakoid membrane network inside chloroplasts harbours the protein complexes that are necessary for the light-dependent reactions of photosynthesis. Cellular processes for building and altering this membrane network are therefore essential for life on Earth. Nevertheless, detailed molecular processes concerning the origin and synthesis of the thylakoids remain elusive. Thylakoid biogenesis is strongly coupled to the processes of chloroplast differentiation. Chloroplasts develop from special progenitors called proplastids. As many of the needed building blocks such as lipids and pigments derive from the inner envelope, the question arises how these components are recruited to their target membrane. This review travels back in time to the beginnings of thylakoid membrane research to summarize findings, facts and fictions on thylakoid biogenesis and structure up to the present state, including new insights and future developments in this field.

Phosphorylation of the outer membrane mitochondrial protein OM64 influences protein import into mitochondria.

Mitochondrial localized proteins are mostly synthesized in the cytosol and translocated across the outer mitochondrial membrane via the translocase of the outer membrane (TOM) complex. Although the channel protein is conserved among eukaryotes, the receptor proteins are more divergent and show features specific to the plant lineage. OM64, which is a paralogue of the chloroplast docking protein Toc64, is unique to plants. However, due to the presence of a cytosolic exposed TPR domain it might functionally replace yeast/mammalian Tom70, which is not found in plant mitochondria, by interacting with the C-terminal (M)EEVD motif of the heat shock proteins Hsp90 and Hsp70. In this study, we show that OM64 is phosphorylated within its TPR domain. Using isothermal titration calorimetry it could be demonstrated that phosphorylation reduces the binding affinity of OM64 to Hsp90. Moreover, in vivo expression of genes encoding different OM64 variants in planta revealed that phosphorylation of OM64 impairs the import efficiency of the mitochondrial preprotein pFAD, a subunits of the mitochondrial ATP synthase. In summary, our data provide significant insight into the fine-tuning mechanisms of mitochondrial protein import mediated by phosphorylation of the cytosolic exposed receptor protein OM64.