Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato(Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration)and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome

Organization and molecular evolution of a disease-resistance gene cluster in coffee trees

<p>Abstract</p> <p>Background</p> <p>Most disease-resistance (R) genes in plants encode NBS-LRR proteins and belong to one of the largest and most variable gene families among plant genomes. However, the specific evolutionary routes of NBS-LRR encoding genes remain elusive. Recently in coffee tree (<it>Coffea arabica</it>), a region spanning the <it>S</it>_<it>H</it><it>3 </it>locus that confers resistance to coffee leaf rust, one of the most serious coffee diseases, was identified and characterized. Using comparative sequence analysis, the purpose of the present study was to gain insight into the genomic organization and evolution of the <it>S</it>_<it>H</it><it>3 </it>locus.</p> <p>Results</p> <p>Sequence analysis of the <it>S</it>_<it>H</it><it>3 </it>region in three coffee genomes, E^aand C^asubgenomes from the allotetraploid <it>C. arabica </it>and C^cgenome from the diploid <it>C. canephora</it>, revealed the presence of 5, 3 and 4 R genes in E^a, C^a, and C^cgenomes, respectively. All these R-gene sequences appeared to be members of a CC-NBS-LRR (CNL) gene family that was only found at the <it>S</it>_<it>H</it><it>3 </it>locus in <it>C. arabica</it>. Furthermore, while homologs were found in several dicot species, comparative genomic analysis failed to find any CNL R-gene in the orthologous regions of other eudicot species. The orthology relationship among the <it>S</it>_<it>H</it><it>3</it>-CNL copies in the three analyzed genomes was determined and the duplication/deletion events that shaped the <it>S</it>_<it>H</it><it>3 </it>locus were traced back. Gene conversion events were detected between paralogs in all three genomes and also between the two sub-genomes of <it>C. arabica</it>. Significant positive selection was detected in the solvent-exposed residues of the <it>S</it>_<it>H</it><it>3</it>-CNL copies.</p> <p>Conclusion</p> <p>The ancestral <it>S</it>_<it>H</it><it>3</it>-CNL copy was inserted in the <it>S</it>_<it>H</it><it>3 </it>locus after the divergence between Solanales and Rubiales lineages. Moreover, the origin of most of the <it>S</it>_<it>H</it><it>3</it>-CNL copies predates the divergence between <it>Coffea </it>species. The <it>S</it>_<it>H</it><it>3</it>-CNL family appeared to evolve following the birth-and-death model, since duplications and deletions were inferred in the evolution of the <it>S</it>_<it>H</it><it>3 </it>locus. Gene conversion between paralog members, inter-subgenome sequence exchanges and positive selection appear to be the major forces acting on the evolution of <it>S</it>_<it>H</it><it>3</it>-CNL in coffee trees.</p

Carbon metabolism and regulation in C3 plants, isolation and characterization of high CO¦2 responsive mutants of Arabidopsis thaliana

grantor: University of TorontoGiven that atmospheric CO2 levels are expected to double during the 21st century and that crop and forest species account for two thirds of global photosynthesis, knowledge of mechanisms governing the acclimation response to high CO2 in plants is essential. The objective of this study was to better understand the mechanism of regulation affecting the response of higher plants to the availability of inorganic carbon. As genetic screens have proved useful in the understanding of physiology and signal transduction in several pathways, a genetic approach using the crucifer 'Arabidopsis thaliana' was taken. Initial characterization of conditions that elicited a response from wildtype 'Arabidopsis' plants was performed prior to screening mutagenized populations. Wildtype 'Arabidopsis' plants exhibit a stress response when exposed to elevated CO2 (0.1-0.3%). Aspects of this stress response include anthocyanin accumulation, curling of leaves, necrosis, hyper-accumulation of foliar carbohydrates and down-regulation of photosynthetic gene expression. Characterization of wildtype facilitated the isolation of mutants which showed a response differing from that of wildtype plants when exposed to elevated CO2. Mutants that resulted from this screen were placed in two general categories; CO2 non-responsive ('cnr') and CO2 hyper-responsive ('chr'). Initial characterization of representative mutants from the different groups was carried out. The largest group of mutants 'cnr' was chosen for further study. These 'cnr' mutants share varying degrees of a single combination of phenotypes which include reduced anthocyanin production, reduced or no curling of leaves, delayed senescence, and normal to vigorous growth under elevated CO2. Two non-allelic T-DNA tagged mutants, ' cnr' 1-1 and 'cnr' 2-1 were selected for genetic, molecular, biochemical and physiological analyses. 'cnr' 1-1 is a dominant mutation and the gene affected by the T-DNA insert in this mutant is a leucine rich repeat receptor kinase. The CO2 insensitive and glucose insensitive phenotype of 'cnr' 1-1 suggest a role for CNR1 in carbon metabolism and the molecular identity of CNR1 suggests a role in signal transduction. ' cnr' 2-1 is a recessive mutation and the T-DNA insert disrupts a P450 monoxygenase gene. Physiological and biochemical analyses suggest that the P450 monoxygenase is involved in the abscisic acid pathway.Ph.D