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Initial characteristics of RbcX proteins from Arabidopsis thaliana.

ABSTRACT: Form I of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is composed of eight large (RbcL) and eight small (RbcS) subunits. Assembly of these subunits into a functional holoenzyme requires the assistance of additional assembly factors. One such factor is RbcX, which has been demonstrated to act as a chaperone in the assembly of most cyanobacterial Rubisco complexes expressed in heterologous system established in Escherichia coli cells. Analysis of Arabidopsis thaliana genomic sequence revealed the presence of two genes encoding putative homologues of cyanobacterial RbcX protein: AtRbcX1 (At4G04330) and AtRbcX2 (At5G19855). In general, both RbcX homologues seem to have the same function which is chaperone activity during Rubisco biogenesis. However, detailed analysis revealed slight differences between them. AtRbcX2 is localized in the stromal fraction of chloroplasts whereas AtRbcX1 was found in the insoluble fraction corresponding with thylakoid membranes. Search for putative "partners" using mass spectrometry analysis suggested that apart from binding to RbcL, AtRbcX1 may also interact with ? subunit of chloroplast ATP synthase. Quantitative RT-PCR analysis of AtRbcX1 and AtRbcX2 expression under various stress conditions indicated that AtRbcX2 is transcribed at a relatively stable level, while the transcription level of AtRbcX1 varies significantly. In addition, we present the attempts to elucidate the secondary structure of AtRbcX proteins using CD spectroscopy. Presented results are the first known approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.

SUBMITTER: Kolesinski P

PROVIDER: S-EPMC3195260 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Initial characteristics of RbcX proteins from Arabidopsis thaliana.

Kolesiński Piotr P Piechota Janusz J Szczepaniak Andrzej A

Plant molecular biology 20110916 4-5

Form I of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is composed of eight large (RbcL) and eight small (RbcS) subunits. Assembly of these subunits into a functional holoenzyme requires the assistance of additional assembly factors. One such factor is RbcX, which has been demonstrated to act as a chaperone in the assembly of most cyanobacterial Rubisco complexes expressed in heterologous system established in Escherichia coli cells. Analysis of Arabidopsis thaliana genomic sequence ...[more]

PMID: 21922322

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Project description:Organellar gene expression (OGE) is crucial for plant development, photosynthesis, and respiration, but our understanding of the mechanisms that control it is still relatively poor. Thus, OGE requires various nucleus-encoded proteins that promote transcription, splicing, trimming, and editing of organellar RNAs, and regulate translation. In metazoans, proteins of the mitochondrial Transcription tERmination Factor (mTERF) family interact with the mitochondrial chromosome and regulate transcriptional initiation and termination. Sequencing of the Arabidopsis thaliana genome led to the identification of a diversified MTERF gene family but, in contrast to mammalian mTERFs, knowledge about the function of these proteins in photosynthetic organisms is scarce. In this hypothesis article, I show that tandem duplications and one block duplication contributed to the large number of MTERF genes in A. thaliana, and propose that the expansion of the family is related to the evolution of land plants. The MTERF genes-especially the duplicated genes-display a number of distinct mRNA accumulation patterns, suggesting functional diversification of mTERF proteins to increase adaptability to environmental changes. Indeed, hypothetical functions for the different mTERF proteins can be predicted using co-expression analysis and gene ontology (GO) annotations. On this basis, mTERF proteins can be sorted into five groups. Members of the "chloroplast" and "chloroplast-associated" clusters are principally involved in chloroplast gene expression, embryogenesis, and protein catabolism, while representatives of the "mitochondrial" cluster seem to participate in DNA and RNA metabolism in that organelle. Moreover, members of the "mitochondrion-associated" cluster and the "low expression" group may act in the nucleus and/or the cytosol. As proteins involved in OGE and presumably nuclear gene expression (NGE), mTERFs are ideal candidates for the coordination of the expression of organelle and nuclear genomes.

Project description:Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30-45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and properties of AtNSPs and related proteins and the biological implications.

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Initial characteristics of RbcX proteins from Arabidopsis thaliana.

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Initial characteristics of RbcX proteins from Arabidopsis thaliana.

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