The phosphorylation state of chloroplast transit peptides regulates preprotein import

Import of nuclear encoded proteins into chloroplast is an essential and well-regulated mechanism. The cytosolic kinases STY8, STY17 and STY46 have been shown to phosphorylate chloroplast preprotein transit peptides advantaging the binding of a 14-3-3 dimer. Analyses of sty8 sty17 sty46 mutant plants revealed a role for the kinases in chloroplast differentiation, possibly due to lack of transit peptide phosphorylation. Moreover we could show that not only phosphorylation but also transit peptide dephosphorylation appears to be required for the fine regulation of the back-transport of nuclear encoded proteins to the chloroplast.     

Effect of salt stress on genes encoding translation-associated proteins in Arabidopsis thaliana

Salinity negatively affects plant growth and disturbs chloroplast integrity. Here, we aimed at identifying salt-responsive translation-related genes in Arabidopsis thaliana with an emphasis on those encoding plastid-located proteins. We used quantitative real-time PCR to test the expression of 170 genes after short-term salt stress (up to 24 h) and identified several genes affected by the stress including: PRPL11, encoding plastid ribosomal protein L11, ATAB2, encoding a chloroplast-located RNA-binding protein presumably functioning as an activator of translation, and PDF1B, encoding a peptide deformylase involved in N-formyl group removal from nascent proteins synthesized in chloroplasts. These genes were previously shown to have important functions in chloroplast biology and may therefore represent new targets for biotechnological optimization of salinity tolerance.     

Tandem duplications of a degenerated GTP-binding domain at the origin of GTPase receptors Toc159 and thylakoidal SRP

The evolutionary origin of some nuclear encoded proteins that translocate proteins across the chloroplast envelope remains unknown. Therefore, sequences of GTPase proteins constituting the Arabidopsis thaliana translocon at the outer membrane of chloroplast (atToc) complexes were analyzed by means of HCA. In particular, atToc159 and related proteins (atToc132, atToc120, and atToc90) do not have proven homologues of prokaryotic or eukaryotic ancestry. We established that the three domains commonly referred to as A, G, and M originate from the GTPase G domain, tandemly repeated, and probably evolving toward an unstructured conformation in the case of the A domain. It resulted from this study a putative common ancestor for these proteins and a new domain definition, in particular the splitting of A into three domains (A1, A2, and A3), has been proposed. The family of Toc159, previously containing A. thaliana and Pisum sativum, has been extended to Medicago truncatula and Populus trichocarpa and it has been revised for Oryza sativa. They have also been compared to GTPase subunits involved in the cpSRP system. A distant homology has been revealed among Toc and cpSRP GTP-hydrolyzing proteins of A. thaliana, and repetitions of a GTPase domain were also found in cpSRP protein receptors, by means of HCA analysis.     

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.     

Light-induced displacement of PLASTID MOVEMENT IMPAIRED1 precedes light-dependent chloroplast movements

Light-dependent chloroplast movements are an actin-dependent cellular response to changes in the light environment that help plants maximize photosynthetic potential and reduce photodamage. Over a dozen proteins are known to be required for normal chloroplast movements, but the molecular mechanisms regulating the transformation of light perception into chloroplast motility are not fully understood. Here, we show that in Arabidopsis (Arabidopsis thaliana) the actin-bundling plasma membrane-associated proteins THRUMIN1, PLASTID MOVEMENT IMPAIRED1 (PMI1), and KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT1 (KAC1) interact through the 14-3-3 proteins KAPPA and OMEGA. We also show that the interaction of PMI1 with 14-3-3 KAPPA and OMEGA is regulated by blue light activation of the Phototropin2 photoreceptor. Live-cell confocal microscopy revealed light-induced dynamic changes in the cellular localizations of PMI1 and KAC1. In particular, PMI1 was relocated away from irradiated areas of the plasma membrane in less than a minute after blue light exposure, consistent with PMI1 playing a critical role in initiating light-dependent chloroplast movements. We present a modified conceptual model for high light-dependent chloroplast movements in which PMI1 acts as the mobile signal that initiates a coordinated sequence of changes in protein–protein and protein–plasma membrane interactions that initiate the chloroplast movement response and determine where in the cell chloroplasts are able to anchor to the plasma membrane.

High light intensity-induced chloroplast movements in Arabidopsis thaliana mesophyll cells involves protein interaction changes and requires movement of the protein PMI1 from the plasma membrane.     

Chloroplast protein PLGG1 is involved in abscisic acid-regulated lateral root development and stomatal movement in Arabidopsis

The plant hormone abscisic acid (ABA) plays a crucial role in root architecture; however, the molecular mechanism of ABA-regulated lateral root (LR) growth is not well known. We screened an Arabidopsis thaliana mutant with LR growth that was sensitive to ABA from a T-DNA insertion mutant library, which was an allelic mutant of plgg1-1, termed plgg1-2. PLGG1 encodes a chloroplast protein that transports plastidic glycolate and glycerate. The length and number of LRs at the root-hypocotyl junction of plgg1-1 and plgg1-2 were significantly impaired under exogenous ABA treatment, and the transgenic plant complementary lines of plgg1-2 restored LR growth in response to ABA. In addition, we found that PLGG1 is involved in other major ABA responses, including ABA-inhibited seed germination, ABA-mediated stomatal movement, and drought tolerance. These findings open new perspectives on elucidating the mechanism of ABA response, and provide clues for analysing the functions of chloroplast proteins in regulating root growth.     

Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.     

Regulation of protein phosphorylation by nitric oxide in cell culture of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Effect of nitric oxide (NO) on phosphorylation of soluble proteins in the cell culture of wild-type Arabidopsis thaliana (L.) Heynh. (ecoptype Columbia, Col-0) was studied. Among the identified proteins whose phosphorylation was affected by the NO donor treatment, the enzymes of primary metabolism (glyceraldehyde-3-phosphate dehydrogenase, enolase) and regulatory proteins (14-3-3-like protein GF14ω, protein-disulfide isomerase-like protein, chaperonin-60α) were detected. The results clarify possible mechanisms of NO action on primary metabolism, cell cycle, and stress-induced responses of cultured plant cells.     

Exploring ligand recognition,selectivity and dynamics of TPR domains of chloroplast Toc64 and mitochondria Om64 from Arabidopsis thaliana

The study aims to gain insight into the mode of ligand recognition by tetratricopeptide repeat (TPR) domains of chloroplast translocon at the outer envelope of chloroplast (Toc64) and mitochondrial Om64, two paralogous proteins that mediate import of proteins into chloroplast and mitochondria, respectively. Chaperone proteins associate with precursor proteins in the cytosol to maintain them in a translocation competent conformation and are recognized by Toc64 and Om64 that are located on the outer membrane of the target organelle. Heat shock proteins (Hsp70) and Hsp90 are two chaperones, which are known to play import roles in protein import. The C‐termini of these chaperones are known to interact with the TPR domain of chloroplast Toc64 and mitochondrial Om64 in Arabidopsis thaliana (At). Using a molecular dynamics approach and binding energy calculations, we identify important residues involved in the interactions. Our findings suggest that the TPR domain from AtToc64 has higher affinity towards C‐terminal residues of Hsp70. The interaction occurs as the terminal helices move towards each other enclosing the cradle on interaction of AtHsp70 with the TPR domain. In contrast, the TPR domain from AtOm64 does not discriminate between the C‐termini of Hsp70 and Hsp90. These binding affinities are discussed with respect to our knowledge of protein targeting and specificity of protein import into endosymbiotic organelles in plant cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Enrichment and Analysis of Intact Phosphoproteins in Arabidopsis Seedlings

Protein phosphorylation regulates diverse cellular functions and plays a key role in the early development of plants. To complement and expand upon previous investigations of protein phosphorylation in Arabidopsis seedlings we used an alternative approach that combines protein extraction under non-denaturing conditions with immobilized metal-ion affinity chromatography (IMAC) enrichment of intact phosphoproteins in Rubisco-depleted extracts, followed by identification using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-gel trypsin digestion and analysis of selected gel spots identified 144 phosphorylated peptides and residues, of which only18 phosphopeptides and 8 phosphosites were found in the PhosPhAt 4.0 and P³DB Arabidopsis thaliana phosphorylation site databases. More than half of the 82 identified phosphoproteins were involved in carbohydrate metabolism, photosynthesis/respiration or oxidative stress response mechanisms. Enrichment of intact phosphoproteins prior to 2-DE and LC-MS/MS appears to enhance detection of phosphorylated threonine and tyrosine residues compared with methods that utilize peptide-level enrichment, suggesting that the two approaches are somewhat complementary in terms of phosphorylation site coverage. Comparing results for young seedlings with those obtained previously for mature Arabidopsis leaves identified five proteins that are differentially phosphorylated in these tissues, demonstrating the potential of this technique for investigating the dynamics of protein phosphorylation during plant development.     

Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis

Plants have varying abilities to tolerate chilling (low but not freezing temperatures), and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance.     

Identification and characterization of SlVKOR,a disulfide bond formation protein from Solanum lycopersicum,and bioinformatic analysis of plant VKORs

Homologs of vitamin K epoxide reductase (VKOR) exist widely in plants. However, only VKOR of Arabidopsis thaliana has been the subject of many studies to date. In the present study, the coding region of a VKOR from Solanum lyco-persicum (JF951971 in GenBank) was cloned; it contained a membrane domain (VKOR domain) and an additional soluble thioredoxin-like (Trx-like) domain. Bioinformatic analysis showed that the first 47 amino acids in the N-terminus should act as a transit peptide targeting the protein to the chloroplast. Western blot demonstrated that the protein is localized in thylakoid membrane with the Trx-like domain facing the lumen. Modeling of three-dimensional structure showed that SlVKOR has a similar conformation with Arabidopsis and cyanobacterial VKORs, with five transmembrane segments in the VKOR domain and a typical Trx-like domain in the lumen. Functional assay showed that the full-length of SlVKOR with Trx-like domain without the transit peptide could catalyze the formation of disulfide bonds. Similar transit peptides at the N-terminus commonly exist in plant VKORs, most of them targeting to chloroplast according to prediction. Comparison of sequences and structures from different plants indicated that all plant VKORs possess two domains, a transmembrane VKOR domain and a soluble Trx-like domain, each having four conservative cysteines. The cysteines were predicted to be related to the function of catalyzing the formation of disulfide bonds.     

Photosynthetic Control of Arabidopsis Leaf Cytoplasmic Translation Initiation by Protein Phosphorylation

Photosynthetic CO₂ assimilation is the carbon source for plant anabolism, including amino acid production and protein synthesis. The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to Arabidopsis thaliana rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO₂-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating (e.g. RPS6, eIF4B phosphorylation) and inhibiting (e.g. eIF4G phosphorylation) molecular events.     

Functional Proteomics: A Promising Approach to Find Novel Components of the Circadian System

In the postgenome era, the analysis of entire subproteomes in correlation with their function has emerged due to high throughput technologies. Early approaches have been initiated to identify novel components of the circadian system. For example, in the marine dinoflagellate Lingulodinium polyedra, a chronobiological proteome assay was performed, which resulted in the identification of already known circadian expressed proteins as well as novel temporal controlled proteins involved in metabolic pathways. In the green alga Chlamydomonas reinhardtii, two circadian expressed proteins (a protein disulfide isomerase and a tetratricopeptide repeat protein) were identified by functional proteomics. Also, the first hints of temporal control within chloroplast proteins of Arabidopsis thaliana were identified by proteome analysis.     

Role of Plastid Protein Phosphatase TAP38 in LHCII Dephosphorylation and Thylakoid Electron Flow

Short-term changes in illumination elicit alterations in thylakoid protein phosphorylation and reorganization of the photosynthetic machinery. Phosphorylation of LHCII, the light-harvesting complex of photosystem II, facilitates its relocation to photosystem I and permits excitation energy redistribution between the photosystems (state transitions). The protein kinase STN7 is required for LHCII phosphorylation and state transitions in the flowering plant Arabidopsis thaliana. LHCII phosphorylation is reversible, but extensive efforts to identify the protein phosphatase(s) that dephosphorylate LHCII have been unsuccessful. Here, we show that the thylakoid-associated phosphatase TAP38 is required for LHCII dephosphorylation and for the transition from state 2 to state 1 in A. thaliana. In tap38 mutants, thylakoid electron flow is enhanced, resulting in more rapid growth under constant low-light regimes. TAP38 gene overexpression markedly decreases LHCII phosphorylation and inhibits state 1→2 transition, thus mimicking the stn7 phenotype. Furthermore, the recombinant TAP38 protein is able, in an in vitro assay, to directly dephosphorylate LHCII. The dependence of LHCII dephosphorylation upon TAP38 dosage, together with the in vitro TAP38-mediated dephosphorylation of LHCII, suggests that TAP38 directly acts on LHCII. Although reversible phosphorylation of LHCII and state transitions are crucial for plant fitness under natural light conditions, LHCII hyperphosphorylation associated with an arrest of photosynthesis in state 2 due to inactivation of TAP38 improves photosynthetic performance and plant growth under state 2-favoring light conditions.     

A homolog of GuidedEntry of Tail-anchored proteins3 functions in membrane-specific protein targeting in chloroplasts of Arabidopsis

The chloroplasts and mitochondria of photosynthetic eukaryotes contain proteins that are closely related to cytosolic Guided Entry of Tail-anchored proteins3 (Get3). Get3 is a targeting factor that efficiently escorts tail-anchored (TA) proteins to the ER. Because other components of the cytosolic-targeting pathway appear to be absent in organelles, previous investigators have asserted that organellar Get3 homologs are unlikely to act as targeting factors. However, we show here both that the Arabidopsis thaliana chloroplast homolog designated as GET3B is structurally similar to cytosolic Get3 proteins and that it selectively binds a thylakoid-localized TA protein. Based on genetic interactions between a get3b mutation and mutations affecting the chloroplast signal recognition particle-targeting pathway, as well as changes in the abundance of photosynthesis-related proteins in mutant plants, we propose that GET3B acts primarily to direct proteins to the thylakoids. Furthermore, through molecular complementation experiments, we show that function of GET3B depends on its ability to hydrolyze ATP, and this is consistent with action as a targeting factor. We propose that GET3B and related organellar Get3 homologs play a role that is analogous to that of cytosolic Get3 proteins, and that GET3B acts as a targeting factor in the chloroplast stroma to deliver TA proteins in a membrane-specific manner.

A chloroplast homolog of a cytosolic protein involved in posttranslational targeting of proteins to the ER is a targeting factor in chloroplasts that directs proteins to the thylakoids.     

<Emphasis Type="Italic">SulP</Emphasis>, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.     

A MORN-domain protein regulates growth and seed production and enhances freezing tolerance in Arabidopsis

AtRGP (AT4G17080, Arabidopsis thaliana reduction in growth and productivity) contains two N-terminal transmembrane helices and seven membrane occupation and recognition nexus motifs at its C-terminus, and associates with phosphatidylinositol phosphate kinase. To elucidate the function of AtRGP, we employed mutant plants to analyze gene expression, plant phenotypes, protein localization, structure and function of the chloroplast, and freezing tolerance. Overexpression of AtRGP increased growth rate, hypocotyl elongation, leaf size, seed production, photosynthetic rate, and freezing tolerance, and promoted chloroplast organization and stacking of grana. By contrast, Atrgp null mutants exhibited a smaller plant size, reduced seed production, photosynthetic rate, and freezing tolerance, and displayed abnormal chloroplast organization with insufficient stacking of grana. Considering these data, we postulate that AtRGP may bind transiently to the chloroplast envelope and interact with other proteins under certain conditions, thereby regulating cellular processes involved in growth and abiotic stress responses.