Dissecting the molecular puzzle of the editosome core in Arabidopsis organelles.

Over the last decade, the composition of the C-to-U RNA editing complex in embryophyte organelles has turned out to be much more complex than first expected. While PPR proteins were initially thought to act alone, significant evidences have clearly depicted a sophisticated mechanism with numerous protein-protein interaction involving PPR and non-PPR proteins. Moreover, the identification of specific functional partnership between PPRs also suggests that, in addition to the highly specific PPRs directly involved in the RNA target recognition, non-RNA-specific ones are required.

Adenylates regulate Arabidopsis plastidial thioredoxin activities through the binding of a CBS domain protein.

Cystathionine-β-synthase (CBS) domains are found in proteins of all living organisms and have been proposed to play a role as energy sensors regulating protein activities through their adenosyl ligand binding capacity. In plants, members of the CBSX protein family carry a stand-alone pair of CBS domains. In Arabidopsis (Arabidopsis thaliana), CBSX1 and CBSX2 are targeted to plastids where they have been proposed to regulate thioredoxins (TRXs).

Role of ClpP in the Biogenesis and Degradation of RuBisCO and ATP Synthase in .

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) associates a chloroplast- and a nucleus-encoded subunit (LSU and SSU). It constitutes the major entry point of inorganic carbon into the biosphere as it catalyzes photosynthetic CO fixation. Its abundance and richness in sulfur-containing amino acids make it a prime source of N and S during nutrient starvation, when photosynthesis is downregulated and a high RuBisCO level is no longer needed.

Targeted Profiling of Subproteomes Illuminates Co- and Posttranslationally N-Terminal Myristoylated Proteins.

N-terminal myristoylation, a major eukaryotic protein lipid modification, is difficult to detect in vivo and challenging to predict in silico. We developed a proteomics strategy involving subfractionation of cellular membranes, combined with separation of hydrophobic peptides by mass spectrometry-coupled liquid chromatography to identify the myristoylated proteome. This approach identified a starting pool of 8837 proteins in all analyzed cellular fractions, comprising 32% of the Arabidopsis proteome.

Golgi traffic and integrity depend on N-myristoyl transferase-1 in Arabidopsis.

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified.

Construction of plastid reference proteomes for maize and Arabidopsis and evaluation of their orthologous relationships; the concept of orthoproteomics.

Plastids are essential organelles because they contribute to primary and secondary metabolism and plant signaling networks. A high-quality inventory of the plastid proteome is therefore a critical tool in plant research. We present reference plastid proteomes for maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) with, respectively, 1564 and 1559 proteins.

The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features.

The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization.

Nucleoid-enriched proteomes in developing plastids and chloroplasts from maize leaves: a new conceptual framework for nucleoid functions.

Plastids contain multiple copies of the plastid chromosome, folded together with proteins and RNA into nucleoids. The degree to which components of the plastid gene expression and protein biogenesis machineries are nucleoid associated, and the factors involved in plastid DNA organization, repair, and replication, are poorly understood. To provide a conceptual framework for nucleoid function, we characterized the proteomes of highly enriched nucleoid fractions of proplastids and mature chloroplasts isolated from the maize (Zea mays) leaf base and tip, respectively, using mass spectrometry.

Structural and metabolic transitions of C4 leaf development and differentiation defined by microscopy and quantitative proteomics in maize.

C(4) grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C(4) photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C(4) differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry.

Reconstruction of metabolic pathways, protein expression, and homeostasis machineries across maize bundle sheath and mesophyll chloroplasts: large-scale quantitative proteomics using the first maize genome assembly.

Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C(4) photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization.

Multistep processing of an insertion sequence in an essential subunit of the chloroplast ClpP complex.

In Chlamydomonas reinhardtii, the clpP1 chloroplast gene encoding one of the catalytic subunits of the ClpP protease complex contains a large in-frame insertion sequence (IS1). Based on the Escherichia coli ClpP structure, IS1 is predicted to protrude at the apical surface of the complex, likely influencing the interaction of the catalytic core with ClpC/HSP100 chaperones. Immunoblotting with an anti-ClpP1 antibody detected two immunoreactive forms of ClpP1: ClpP1(H) (59 kDa) and ClpP1(L) (25 kDa).

Cell-type-specific differentiation of chloroplasts in C4 plants.

In leaves of C4 grasses such as maize, photosynthetic activities are partitioned between bundle-sheath and mesophyll cells, leading to increased photosynthetic yield, particularly under stress conditions. As we discuss here, recent comparative chloroplast proteome analyses in maize have shown specific adaptation to C4-cell-specific differentiation of the photosynthetic apparatus, primary and secondary metabolism and metabolite transporters, as well as the chloroplast protein homeostasis machinery. Furthermore, a novel bundle-sheath-enriched 1000-kDa NADPH dehydrogenase 'supercomplex' has been identified, and we discuss its possible role in inorganic carbon concentration.

PPDB, the Plant Proteomics Database at Cornell.

The Plant Proteomics Database (PPDB; http://ppdb.tc.cornell.

Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells.

Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g.

Deregulation of maize C4 photosynthetic development in a mesophyll cell-defective mutant.

During maize (Zea mays) C(4) differentiation, mesophyll (M) and bundle sheath (BS) cells accumulate distinct sets of photosynthetic enzymes, with very low photosystem II (PSII) content in BS chloroplasts. Consequently, there is little linear electron transport in the BS and ATP is generated by cyclic electron flow. In contrast, M thylakoids are very similar to those of C(3) plants and produce the ATP and NADPH that drive metabolic activities.

The chloroplast ClpP complex in Chlamydomonas reinhardtii contains an unusual high molecular mass subunit with a large apical domain.

The composition of the chloroplast-localized protease complex, ClpP, from the green alga Chlamydomonas reinhardtii was characterized by nondenaturing electrophoresis, immunoblotting and MS. The detected ClpP complex has a native mass of approximately 540 kDa, which is approximately 200 kDa higher than ClpP complexes in higher plant chloroplasts, mitochondria or bacteria. The 540-kDa ClpP complex contains two nuclear-encoded ClpP proteins (ClpP3 and P5) and five ClpR (R1, R2, R3, R4 and R6) proteins, as well two proteins, ClpP1L and ClpP1H, both probably derived from the plastid clpP1 gene.

Functional differentiation of bundle sheath and mesophyll maize chloroplasts determined by comparative proteomics.

Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags.