A purification strategy for analysis of the DNA/RNA-associated sub-proteome from chloroplasts of mustard cotyledons.

Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleic acids binding sub-proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalog of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins.

Environmental control of plant nuclear gene expression by chloroplast redox signals.

Plant photosynthesis takes place in specialized cell organelles, the chloroplasts, which perform all essential steps of this process. The proteins involved in photosynthesis are encoded by genes located on the plastid and nuclear genomes. Proper function and regulation of light harvesting and energy fixation thus requires a tight coordination of the gene expression machineries in the two genetic compartments. This is achieved by a bi-directional exchange of information between nucleus and plastids. Signals emerging from plastids report the functional and developmental state of the organelle to the nucleus and initiate distinct nuclear gene expression profiles, which trigger responses that support or improve plastid functions. Recent research indicated that this signaling is absolutely essential for plant growth and development. Reduction/oxidation (redox) signals from photosynthesis are key players in this information network since they do report functional disturbances in photosynthesis, the primary energy source of plants. Such disturbances are caused by environmental fluctuations for instance in illumination, temperature, or water availability. These environmental changes affect the linear electron flow of photosynthesis and result in changes of the redox state of the components involved [e.g., the plastoquinone (PQ) pool] or coupled to it (e.g., the thioredoxin pool). Thus, the changes in redox state directly reflect the environmental impact and serve as immediate plastidial signals to the nucleus. The triggered responses range from counterbalancing reactions within the physiological range up to severe stress responses including cell death. This review focuses on physiological redox signals from photosynthetic electron transport (PET), their relation to the environment, potential transduction pathways to the nucleus and their impact on nuclear gene expression.

An Arabidopsis GluTR binding protein mediates spatial separation of 5-aminolevulinic acid synthesis in chloroplasts.

5-Aminolevulinic acid (ALA) is the universal precursor for tetrapyrrole biosynthesis and is synthesized in plants in three enzymatic steps: ligation of glutamate (Glu) to tRNA(Glu) by glutamyl-tRNA synthetase, reduction of activated Glu to Glu-1-semialdehyde by glutamyl-tRNA reductase (GluTR), and transamination to ALA by Glu 1-semialdehyde aminotransferase. ALA formation controls the metabolic flow into the tetrapyrrole biosynthetic pathway. GluTR is proposed to be the key regulatory enzyme that is tightly controlled at transcriptional and posttranslational levels. We identified a GluTR binding protein (GluTRBP; previously called PROTON GRADIENT REGULATION7) that is localized in chloroplasts and part of a 300-kD protein complex in the thylakoid membrane. Although the protein does not modulate activity of ALA synthesis, the knockout of GluTRBP is lethal in Arabidopsis thaliana, whereas mutants expressing reduced levels of GluTRBP contain less heme. GluTRBP expression correlates with a function in heme biosynthesis. It is postulated that GluTRBP contributes to subcompartmentalized ALA biosynthesis by maintaining a portion of GluTR at the plastid membrane that funnels ALA into the heme biosynthetic pathway. These results regarding GluTRBP support a model of plant ALA synthesis that is organized in two separate ALA pools in the chloroplast to provide appropriate substrate amounts for balanced synthesis of heme and chlorophyll.

Identification of essential subunits in the plastid-encoded RNA polymerase complex reveals building blocks for proper plastid development.

The major RNA polymerase activity in mature chloroplasts is a multisubunit, Escherichia coli-like protein complex called PEP (for plastid-encoded RNA polymerase). Its subunit structure has been extensively investigated by biochemical means. Beside the "prokaryotic" subunits encoded by the plastome-located RNA polymerase genes, a number of additional nucleus-encoded subunits of eukaryotic origin have been identified in the PEP complex. These subunits appear to provide additional functions and regulation modes necessary to adapt transcription to the varying functional situations in chloroplasts. However, despite the enormous progress in genomic data and mass spectrometry techniques, it is still under debate which of these subunits belong to the core complex of PEP and which ones represent rather transient or peripheral components. Here, we present a catalog of true PEP subunits that is based on comparative analyses from biochemical purifications, protein mass spectrometry, and phenotypic analyses. We regard reproducibly identified protein subunits of the basic PEP complex as essential when the corresponding knockout mutants reveal an albino or pale-green phenotype. Our study provides a clearly defined subunit catalog of the basic PEP complex, generating the basis for a better understanding of chloroplast transcription regulation. In addition, the data support a model that links PEP complex assembly and chloroplast buildup during early seedling development in vascular plants.

Analysis of oligomeric protein complexes in the chloroplast sub-proteome of nucleic acid-binding proteins from mustard reveals potential redox regulators of plastid gene expression.

Photosynthetic light quality acclimation in plants involves redox-controlled changes in plastid gene expression. To study proteins potentially involved in this regulation, we isolated low-abundant chloroplast nucleic acid-binding proteins from the crucifere mustard (Sinapis alba) and investigated if photosynthetic redox signals affect their composition and/or oligomeric structure. We purified chloroplasts from plants subjected to light quality shifts and applied organelle lysates to heparin-Sepharose chromatography followed by 2-D blue native PAGE. We studied accumulation and structure of oligomeric protein complexes and applied MS/MS to identify them. We found ten oligomeric protein complexes of higher order and eleven smaller protein complexes or spots including plastid-encoded RNA polymerase (PEP), plastid transcriptionally active chromosome proteins, RNA-binding proteins, ribosomal subunits and chaperones. A translation elongation factor was found to be the only protein displaying major differences in its amounts in response to the growth lights. Furthermore, we found a novel thioredoxin as a subunit of the PEP, a 2-Cys-peroxiredoxin complex and a (soluble) ferredoxin:NADP-oxido-reductase, which represent potential redox regulator of plastid gene expression. A T-DNA knock-out line of the thioredoxin from Arabidopsis exhibits a yellowish-pale phenotype, demonstrating that this novel PEP subunit is essential for proper plastid development.

The role of phosphorylation in redox regulation of photosynthesis genes psaA and psbA during photosynthetic acclimation of mustard.

The long-term response (LTR) to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded genes psaAB (encoding the P700 apoproteins of photosystem I) and psbA (encoding the D1 protein of photosystem II) and requires the action of plastid-localized kinases. To study the potential impact of phosphorylation events on plastid gene expression during the LTR, we analyzed mustard seedlings acclimated to light sources favoring either photosystem I or photosystem II. Primer extension analyses of psaA transcripts indicate that the redox regulation occurs at the principal bacterial promoters, suggesting that the plastid encoded RNA polymerase (PEP) is the target for redox signals. Chloroplast protein fractions containing PEP and other DNA-binding proteins were purified from mustard via heparin-Sepharose chromatography. The biochemical properties of these fractions were analyzed with special emphasis on promoter recognition and specificity, phosphorylation state, and kinase activity. The results demonstrate that the LTR involves the action of small DNA-binding proteins; three of them exhibit specific changes in the phosphorylation state. Auto-phosphorylation assays, in addition, exhibit large differences in the activity of endogenous kinase activities. Chloroplast run-on transcription experiments with the kinase inhibitor H7 and the reductant DTT indicate that phosphorylation events are essential for the mediation of redox signals toward psaA and psbA transcription initiation, but require the synergistic action of a thiol redox signal. The data support the idea that redox signals from the thylakoid membrane are linked to gene expression via phosphorylation events; however, this mediation appears to require a complex network of interacting proteins rather than a simple phosphorelay.