The CLP and PREP protease systems coordinate maturation and degradation of the chloroplast proteome in Arabidopsis thaliana.

A network of peptidases governs proteostasis in plant chloroplasts and mitochondria. This study reveals strong genetic and functional interactions in Arabidopsis between the chloroplast stromal CLP chaperone-protease system and the PREP1,2 peptidases, which are dually localized to chloroplast stroma and the mitochondrial matrix. Higher order mutants defective in CLP or PREP proteins were generated and analyzed by quantitative proteomics and N-terminal proteomics (terminal amine isotopic labeling of substrates (TAILS)). Strong synergistic interactions were observed between the CLP protease system (clpr1-2, clpr2-1, clpc1-1, clpt1, clpt2) and both PREP homologs (prep1, prep2) resulting in embryo lethality or growth and developmental phenotypes. Synergistic interactions were observed even when only one of the PREP proteins was lacking, suggesting that PREP1 and PREP2 have divergent substrates. Proteome phenotypes were driven by the loss of CLP protease capacity, with little impact from the PREP peptidases. Chloroplast N-terminal proteomes showed that many nuclear encoded chloroplast proteins have alternatively processed N-termini in prep1prep2, clpt1clpt2 and prep1prep2clpt1clpt2. Loss of chloroplast protease capacity interferes with stromal processing peptidase (SPP) activity due to folding stress and low levels of accumulated cleaved cTP fragments. PREP1,2 proteolysis of cleaved cTPs is complemented by unknown proteases. A model for CLP and PREP activity within a hierarchical chloroplast proteolysis network is proposed.

Proteomics, phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP chaperone-protease system.

The chloroplast chaperone CLPC1 unfolds and delivers substrates to the stromal CLPPRT protease complex for degradation. We previously used an in vivo trapping approach to identify interactors with CLPC1 in Arabidopsis thaliana by expressing a STREPII-tagged copy of CLPC1 mutated in its Walker B domains (CLPC1-TRAP) followed by affinity purification and mass spectrometry. To create a larger pool of candidate substrates, adaptors, or regulators, we carried out a far more sensitive and comprehensive in vivo protein trapping analysis. We identified 59 highly enriched CLPC1 protein interactors, in particular proteins belonging to families of unknown functions (DUF760, DUF179, DUF3143, UVR-DUF151, HugZ/DUF2470), as well as the UVR domain proteins EXE1 and EXE2 implicated in singlet oxygen damage and signaling. Phylogenetic and functional domain analyses identified other members of these families that appear to localize (nearly) exclusively to plastids. In addition, several of these DUF proteins are of very low abundance as determined through the Arabidopsis PeptideAtlas http://www.peptideatlas.org/builds/arabidopsis/ showing that enrichment in the CLPC1-TRAP was extremely selective. Evolutionary rate covariation indicated that the HugZ/DUF2470 family coevolved with the plastid CLP machinery suggesting functional and/or physical interactions. Finally, mRNA-based coexpression networks showed that all 12 CLP protease subunits tightly coexpressed as a single cluster with deep connections to DUF760-3. Coexpression modules for other trapped proteins suggested specific functions in biological processes, e.g., UVR2 and UVR3 were associated with extraplastidic degradation, whereas DUF760-6 is likely involved in senescence. This study provides a strong foundation for discovery of substrate selection by the chloroplast CLP protease system.

Autocatalytic Processing and Substrate Specificity of Arabidopsis Chloroplast Glutamyl Peptidase.

Chloroplast proteostasis is governed by a network of peptidases. As a part of this network, we show that Arabidopsis (Arabidopsis thaliana) chloroplast glutamyl peptidase (CGEP) is a homo-oligomeric stromal Ser-type (S9D) peptidase with both exo- and endo-peptidase activity. Arabidopsis CGEP null mutant alleles (cgep) had no visible phenotype but showed strong genetic interactions with stromal CLP protease system mutants, resulting in reduced growth. Loss of CGEP upregulated the chloroplast protein chaperone machinery and 70S ribosomal proteins, but other parts of the proteostasis network were unaffected. Both comparative proteomics and mRNA-based coexpression analyses strongly suggested that the function of CGEP is at least partly involved in starch metabolism regulation. Recombinant CGEP degraded peptides and proteins smaller than ∼25 kD. CGEP specifically cleaved substrates on the C-terminal side of Glu irrespective of neighboring residues, as shown using peptide libraries incubated with recombinant CGEP and mass spectrometry. CGEP was shown to undergo autocatalytic C-terminal cleavage at E946, removing 15 residues, both in vitro and in vivo. A conserved motif (A[S/T]GGG[N/G]PE946) immediately upstream of E946 was identified in dicotyledons, but not monocotyledons. Structural modeling suggested that C-terminal processing increases the upper substrate size limit by improving catalytic cavity access. In vivo complementation with catalytically inactive CGEP-S781R or a CGEP variant with an unprocessed C-terminus in a cgep clpr2-1 background was used to demonstrate the physiological importance of both CGEP peptidase activity and its autocatalytic processing. CGEP homologs of photosynthetic and nonphotosynthetic bacteria lack the C-terminal prosequence, suggesting it is a recent functional adaptation in plants.

Exploring the proteome associated with the mRNA encoding the D1 reaction center protein of Photosystem II in plant chloroplasts.

Synthesis of the D1 reaction center protein of Photosystem II is dynamically regulated in response to environmental and developmental cues. In chloroplasts, much of this regulation occurs at the post-transcriptional level, but the proteins responsible are largely unknown. To discover proteins that impact psbA expression, we identified proteins that associate with maize psbA mRNA by: (i) formaldehyde cross-linking of leaf tissue followed by antisense oligonucleotide affinity capture of psbA mRNA; and (ii) co-immunoprecipitation with HCF173, a psbA translational activator that is known to bind psbA mRNA. The S1 domain protein SRRP1 and two RNA Recognition Motif (RRM) domain proteins, CP33C and CP33B, were enriched with both approaches. Orthologous proteins were also among the enriched protein set in a previous study in Arabidopsis that employed a designer RNA-binding protein as a psbA RNA affinity tag. We show here that CP33B is bound to psbA mRNA in vivo, as was shown previously for CP33C and SRRP1. Immunoblot, pulse labeling, and ribosome profiling analyses of mutants lacking CP33B and/or CP33C detected some decreases in D1 protein levels under some conditions, but no change in psbA RNA abundance or translation. However, analogous experiments showed that SRRP1 represses psbA ribosome association in the dark, represses ycf1 ribosome association, and promotes accumulation of ndhC mRNA. As SRRP1 is known to harbor RNA chaperone activity, we postulate that SRRP1 mediates these effects by modulating RNA structures. The uncharacterized proteins that emerged from our analyses provide a resource for the discovery of proteins that impact the expression of psbA and other chloroplast genes.

In Vivo Trapping of Proteins Interacting with the Chloroplast CLPC1 Chaperone: Potential Substrates and Adaptors.

The chloroplast stromal CLP protease system is essential for growth and development. It consists of a proteolytic CLP core complex that likely dynamically interacts with oligomeric rings of CLPC1, CLPC2, or CLPD AAA+ chaperones. These ATP-dependent chaperones are predicted to bind and unfold CLP protease substrates, frequently aided by adaptors (recognins), and feed them into the proteolytic CLP core for degradation. To identify new substrates and possibly also new adaptors for the chloroplast CLP protease system, we generated an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity tag in Arabidopsis thaliana by mutating critical glutamate residues (E374A and E718A) in the two Walker B domains of CLPC1 required for the hydrolysis of ATP (CLPC1-TRAP). On the basis of homology to nonplant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released; that is, they are trapped. When expressed in the wild type, this CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared with affinity-purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1-WT). These enriched proteins likely represent CLP protease substrates or new adaptors. Several of these trapped proteins overaccumulated in clp mutants or were found as interactors for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, the affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR, and CLPT subunits, indicating the stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction.

Extreme variation in rates of evolution in the plastid Clp protease complex.

Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the level of multi-subunit complexes in mitochondria and plastids. One such complex in plants is the caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The proteolytic core of Clp comprises subunits from one plastid-encoded gene (clpP1) and multiple nuclear genes. TheclpP1 gene is highly conserved across most green plants, but it is by far the fastest evolving plastid-encoded gene in some angiosperms. To better understand these extreme and mysterious patterns of divergence, we investigated the history ofclpP1 molecular evolution across green plants by extracting sequences from 988 published plastid genomes. We find thatclpP1 has undergone remarkably frequent bouts of accelerated sequence evolution and architectural changes (e.g. a loss of introns andRNA-editing sites) within seed plants. AlthoughclpP1 is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that this is rarely true. We applied comparative native gel electrophoresis of chloroplast protein complexes followed by protein mass spectrometry in two species within the angiosperm genusSilene, which has highly elevated and heterogeneous rates ofclpP1 evolution. We confirmed thatclpP1 is expressed as a stable protein and forms oligomeric complexes with the nuclear-encoded Clp subunits, even in one of the most divergentSilene species. Additionally, there is a tight correlation between amino acid substitution rates inclpP1 and the nuclear-encoded Clp subunits across a broad sampling of angiosperms, suggesting continuing selection on interactions within this complex.

Consequences of the loss of catalytic triads in chloroplast CLPPR protease core complexes in vivo.

The essential chloroplast CLP protease system consists of a tetradecameric proteolytic core with catalytic P (P1, 3-6) and non-catalytic R (R1-4) subunits, CLP chaperones and adaptors. The chloroplast CLP complex has a total of ten catalytic sites,but it is not known how many of these catalytic sites can be inactivated before plants lose viability. Here we show that CLPP3 and the catalytically inactive variant CLPP3S164A fully complement the developmental arrest of the clpp3-1 null mutant, even under environmental stress. In contrast, whereas the inactive variant CLPP5S193A assembled into the CLP core, it cannot rescue the embryo lethal phenotype of the clpp5-1 null mutant. This shows that CLPP3 makes a unique structural contribution but its catalytic site is dispensable, whereas the catalytic activity of CLPP5 is essential. Mass spectrometry of affinity-purified CLP cores of the complemented lines showed highly enriched CLP cores. Other chloroplast proteins were co-purified with the CLP cores and are candidate substrates. A strong overlap of co-purified proteins between the CLP core complexes with active and inactive subunits indicates that CLP cores with reduced number of catalytic sites do not over-accumulate substrates, suggesting that the bottle-neck for degradation is likely substrate recognition and unfolding by CLP adaptors and chaperones, upstream of the CLP core.

The Plastoglobule-Localized Metallopeptidase PGM48 Is a Positive Regulator of Senescence in Arabidopsis thaliana.

Plastoglobuli (PG) are thylakoid-associated monolayer lipid particles with a specific proteome of ∼30 PG core proteins and isoprenoid and neutral lipids. During senescence, PGs increase in size, reflecting their role in dismantling thylakoid membranes. Here, we show that the only PG-localized peptidase PGM48 positively regulates leaf senescence. We discovered that PGM48 is a member of the M48 peptidase family with PGM48 homologs, forming a clade (M48D) only found in photosynthetic organisms. Unlike the M48A, B, and C clades, members of M48D have no transmembrane domains, consistent with their unique subcellular location in the PG. In vitro assays showed Zn-dependent proteolytic activity and substrate cleavage upstream of hydrophobic residues. Overexpression of PGM48 accelerated natural leaf senescence, whereas suppression delayed senescence. Quantitative proteomics of PG from senescing rosettes of PGM48 overexpression lines showed a dramatically reduced level of CAROTENOID CLEAVAGE ENZYME4 (CCD4) and significantly increased levels of the senescence-induced ABC1 KINASE7 (ABC1K7) and PHYTYL ESTER SYNTHASE1 (PES1). Yeast two-hybrid experiments identified PG core proteins ABC1K3, PES1, and CCD4 as PGM48 interactors, whereas several other PG-localized proteins and chlorophyll degradation enzymes did not interact. We discuss mechanisms through which PGM48 could possibly accelerate the senescence process.

Phosphorylation of plastoglobular proteins in Arabidopsis thaliana.

Plastoglobules (PGs) are plastid lipid-protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles.

Discovery of a Unique Clp Component, ClpF, in Chloroplasts: A Proposed Binary ClpF-ClpS1 Adaptor Complex Functions in Substrate Recognition and Delivery.

Clp proteases are found in prokaryotes, mitochondria, and plastids where they play crucial roles in maintaining protein homeostasis (proteostasis). The plant plastid Clp machinery comprises a hetero-oligomeric ClpPRT proteolytic core, ATP-dependent chaperones ClpC and ClpD, and an adaptor protein, ClpS1. ClpS1 selects substrates to the ClpPR protease-ClpC chaperone complex for degradation, but the underlying substrate recognition and delivery mechanisms are currently unclear. Here, we characterize a ClpS1-interacting protein in Arabidopsis thaliana, ClpF, which can interact with the Clp substrate glutamyl-tRNA reductase. ClpF and ClpS1 mutually stimulate their association with ClpC. ClpF, which is only found in photosynthetic eukaryotes, contains bacterial uvrB/C and YccV protein domains and a unique N-terminal domain. We propose a testable model in which ClpS1 and ClpF form a binary adaptor for selective substrate recognition and delivery to ClpC, reflecting an evolutionary adaptation of the Clp system to the plastid proteome.

Posttranslational Protein Modifications in Plant Metabolism.

Posttranslational modifications (PTMs) of proteins greatly expand proteome diversity, increase functionality, and allow for rapid responses, all at relatively low costs for the cell. PTMs play key roles in plants through their impact on signaling, gene expression, protein stability and interactions, and enzyme kinetics. Following a brief discussion of the experimental and bioinformatics challenges of PTM identification, localization, and quantification (occupancy), a concise overview is provided of the major PTMs and their (potential) functional consequences in plants, with emphasis on plant metabolism. Classic examples that illustrate the regulation of plant metabolic enzymes and pathways by PTMs and their cross talk are summarized. Recent large-scale proteomics studies mapped many PTMs to a wide range of metabolic functions. Unraveling of the PTM code, i.e. a predictive understanding of the (combinatorial) consequences of PTMs, is needed to convert this growing wealth of data into an understanding of plant metabolic regulation.

Structures, Functions, and Interactions of ClpT1 and ClpT2 in the Clp Protease System of Arabidopsis Chloroplasts.

Plastid ClpT1 and ClpT2 are plant-specific proteins that associate with the ClpPR protease. However, their physiological significance and structures are not understood. Arabidopsis thaliana loss-of-function single clpt1 and clpt2 mutants showed no visible phenotypes, whereas clpt1 clpt2 double mutants showed delayed development, reduced plant growth, and virescent, serrated leaves but were viable and produced seed. The clpt1 and clpt1 clpt2 mutants showed partial destabilization of the ClpPR complex, whereas clpt2 null mutants showed only marginal destabilization. Comparative proteomics of clpt1 clpt2 plants showed a proteostasis phenotype similar to viable mutants in ClpPR core subunits, indicating reduced Clp protease capacity. In vivo and in vitro assays showed that ClpT1 and ClpT2 can independently interact with the single ClpP ring and ClpPR core, but not with the single ClpR ring. We determined ClpT1 and ClpT2 structures (2.4- and 2.0-Å resolution) and detailed the similarities to the N-domains of bacterial ClpA/C chaperones. The ClpT structures suggested a conserved MYFF motif for interaction with the ClpPR core near the interface between the P- and R-rings. In vivo complementation showed that ClpT function and ClpPR core stabilization require the MYFF motif. Several models are presented that may explain how ClpT1,2 contribute to ClpPR protease activity.

A zinc finger motif-containing protein is essential for chloroplast RNA editing.

C-to-U editing of transcripts in plant organelles is carried out by small (<400 kD) protein complexes called editosomes. Recognition of the proper C target for editing is mediated by pentatricopeptide repeat (PPR) containing proteins that recognize cis-elements. Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts. By co-immunoprecipitation of the chloroplast editing factor ORRM1, followed by mass spectrometry, we have now identified a member of the RanBP2 type zinc fingers (pFAM00641) protein family that is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. In yeast two-hybrid assays, OZ1 (Organelle Zinc finger 1) interacts with PPR site recognition factors whose cognate sites are affected when OZ1 is mutated. No interaction of OZ1 with the chloroplast editing factors RIP2 and RIP9 was detected; however, OZ1 interacts with ORRM1, which binds to RIP proteins, allowing us to build a model for the chloroplast RNA editosome. The RNA editosomes that act upon most chloroplast C targets are likely to contain a PPR protein recognition factor, either RIP2 or RIP9, ORRM1, and OZ1. The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria. With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing.

Developmental and Subcellular Organization of Single-Cell C4 Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing.

Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

MET1 is a thylakoid-associated TPR protein involved in photosystem II supercomplex formation and repair in Arabidopsis.

Photosystem II (PSII) requires constant disassembly and reassembly to accommodate replacement of the D1 protein. Here, we characterize Arabidopsis thaliana MET1, a PSII assembly factor with PDZ and TPR domains. The maize (Zea mays) MET1 homolog is enriched in mesophyll chloroplasts compared with bundle sheath chloroplasts, and MET1 mRNA and protein levels increase during leaf development concomitant with the thylakoid machinery. MET1 is conserved in C3 and C4 plants and green algae but is not found in prokaryotes. Arabidopsis MET1 is a peripheral thylakoid protein enriched in stroma lamellae and is also present in grana. Split-ubiquitin assays and coimmunoprecipitations showed interaction of MET1 with stromal loops of PSII core components CP43 and CP47. From native gels, we inferred that MET1 associates with PSII subcomplexes formed during the PSII repair cycle. When grown under fluctuating light intensities, the Arabidopsis MET1 null mutant (met1) showed conditional reduced growth, near complete blockage in PSII supercomplex formation, and concomitant increase of unassembled CP43. Growth of met1 in high light resulted in loss of PSII supercomplexes and accelerated D1 degradation. We propose that MET1 functions as a CP43/CP47 chaperone on the stromal side of the membrane during PSII assembly and repair. This function is consistent with the observed differential MET1 accumulation across dimorphic maize chloroplasts.

Salicylic Acid Inhibits the Replication of Tomato bushy stunt virus by Directly Targeting a Host Component in the Replication Complex.

Although the plant hormone salicylic acid (SA) plays a central role in signaling resistance to viral infection, the underlying mechanisms are only partially understood. Identification and characterization of SA's direct targets have been shown to be an effective strategy for dissecting the complex SA-mediated defense signaling network. In search of additional SA targets, we previously developed two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SA-binding proteins (SABPs) from Arabidopsis. Using these approaches, we have now identified several members of the Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein family, including two chloroplast-localized and two cytosolic isoforms, as SABPs. Cytosolic GAPDH is a well-known glycolytic enzyme; it also is an important host factor involved in the replication of Tomato bushy stunt virus (TBSV), a single-stranded RNA virus. Using a yeast cell-free extract, an in vivo yeast replication system, and plant protoplasts, we demonstrate that SA inhibits TBSV replication. SA does so by inhibiting the binding of cytosolic GAPDH to the negative (-)RNA strand of TBSV. Thus, this study reveals a novel molecular mechanism through which SA regulates virus replication.

Meta-Analysis of Arabidopsis thaliana Phospho-Proteomics Data Reveals Compartmentalization of Phosphorylation Motifs.

Protein (de)phosphorylation plays an important role in plants. To provide a robust foundation for subcellular phosphorylation signaling network analysis and kinase-substrate relationships, we performed a meta-analysis of 27 published and unpublished in-house mass spectrometry-based phospho-proteome data sets for Arabidopsis thaliana covering a range of processes, (non)photosynthetic tissue types, and cell cultures. This resulted in an assembly of 60,366 phospho-peptides matching to 8141 nonredundant proteins. Filtering the data for quality and consistency generated a set of medium and a set of high confidence phospho-proteins and their assigned phospho-sites. The relation between single and multiphosphorylated peptides is discussed. The distribution of p-proteins across cellular functions and subcellular compartments was determined and showed overrepresentation of protein kinases. Extensive differences in frequency of pY were found between individual studies due to proteomics and mass spectrometry workflows. Interestingly, pY was underrepresented in peroxisomes but overrepresented in mitochondria. Using motif-finding algorithms motif-x and MMFPh at high stringency, we identified compartmentalization of phosphorylation motifs likely reflecting localized kinase activity. The filtering of the data assembly improved signal/noise ratio for such motifs. Identified motifs were linked to kinases through (bioinformatic) enrichment analysis. This study also provides insight into the challenges/pitfalls of using large-scale phospho-proteomic data sets to nonexperts.

Identification of multiple salicylic acid-binding proteins using two high throughput screens.

Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [(3)H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays.

ClpS1 is a conserved substrate selector for the chloroplast Clp protease system in Arabidopsis.

Whereas the plastid caseinolytic peptidase (Clp) P protease system is essential for plant development, substrates and substrate selection mechanisms are unknown. Bacterial ClpS is involved in N-degron substrate selection and delivery to the ClpAP protease. Through phylogenetic analysis, we show that all angiosperms contain ClpS1 and some species also contain ClpS1-like protein(s). In silico analysis suggests that ClpS1 is the functional homolog of bacterial ClpS. We show that Arabidopsis thaliana ClpS1 interacts with plastid ClpC1,2 chaperones. The Arabidopsis ClpS1 null mutant (clps1) lacks a visible phenotype, and no genetic interactions with ClpC/D chaperone or ClpPR core mutants were observed. However, clps1, but not clpc1-1, has increased sensitivity to the translational elongation inhibitor chloramphenicol suggesting a link between translational capacity and ClpS1. Moreover, ClpS1 was upregulated in clpc1-1, and quantitative proteomics of clps1, clpc1, and clps1 clpc1 showed specific molecular phenotypes attributed to loss of ClpC1 or ClpS1. In particular, clps1 showed alteration of the tetrapyrrole pathway. Affinity purification identified eight candidate ClpS1 substrates, including plastid DNA repair proteins and Glu tRNA reductase, which is a control point for tetrapyrrole synthesis. ClpS1 interaction with five substrates strictly depended on two conserved ClpS1 residues involved in N-degron recognition. ClpS1 function, substrates, and substrate recognition mechanisms are discussed.

Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway.

Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen-derived carotenoid ß-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyl-lipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wild-type PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.