Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics.

Posidonia oceanica is a marine angiosperm, or seagrass, adapted to grow to the underwater life from shallow waters to 50 m depth. This raises questions of how their photosynthesis adapted to the attenuation of light through the water column and leads to the assumption that biochemistry and metabolism of the chloroplast are the basis of adaptive capacity. In the present study, we described a protocol that was adapted from those optimized for terrestrial plants, to extract chloroplasts from as minimal tissue as possible. We obtained the best balance between tissue amount/intact chloroplasts yield using one leaf from one plant. After isopynic separations, the chloroplasts purity and integrity were evaluated by biochemical assay and using a proteomic approach. Chloroplast proteins were extracted from highly purified organelles and resolved by 1DE SDS-PAGE. Proteins were sequenced by nLC-ESI-IT-MS/MS of 1DE gel bands and identified against NCBInr green plant databases, Dr. Zompo database for seagrasses in a local customized dataset. The curated localization of proteins in sub-plastidial compartments (i.e. envelope, stroma and thylakoids) was retrieved in the AT_CHLORO database. This purification protocol and the validation of compartment markers may serve as basis for sub-cellular proteomics in P. oceanica and other seagrasses.

Leaf proteome modulation and cytological features of seagrass Cymodocea nodosa in response to long-term high CO2 exposure in volcanic vents.

Seagrass Cymodocea nodosa was sampled off the Vulcano island, in the vicinity of a submarine volcanic vent. Leaf samples were collected from plants growing in a naturally acidified site, influenced by the long-term exposure to high CO2 emissions, and compared with others collected in a nearby meadow living at normal pCO2 conditions. The differential accumulated proteins in leaves growing in the two contrasting pCO2 environments was investigated. Acidified leaf tissues had less total protein content and the semi-quantitative proteomic comparison revealed a strong general depletion of proteins belonging to the carbon metabolism and protein metabolism. A very large accumulation of proteins related to the cell respiration and to light harvesting process was found in acidified leaves in comparison with those growing in the normal pCO2 site. The metabolic pathways linked to cytoskeleton turnover also seemed affected by the acidified condition, since a strong reduction in the concentration of cytoskeleton structural proteins was found in comparison with the normal pCO2 leaves. Results coming from the comparative proteomics were validated by the histological and cytological measurements, suggesting that the long lasting exposure and acclimation of C. nodosa to the vents involved phenotypic adjustments that can offer physiological and structural tools to survive the suboptimal conditions at the vents vicinity.

Depth-specific fluctuations of gene expression and protein abundance modulate the photophysiology in the seagrass Posidonia oceanica.

Here we present the results of a multiple organizational level analysis conceived to identify acclimative/adaptive strategies exhibited by the seagrass Posidonia oceanica to the daily fluctuations in the light environment, at contrasting depths. We assessed changes in photophysiological parameters, leaf respiration, pigments, and protein and mRNA expression levels. The results show that the diel oscillations of P. oceanica photophysiological and respiratory responses were related to transcripts and proteins expression of the genes involved in those processes and that there was a response asynchrony between shallow and deep plants probably caused by the strong differences in the light environment. The photochemical pathway of energy use was more effective in shallow plants due to higher light availability, but these plants needed more investment in photoprotection and photorepair, requiring higher translation and protein synthesis than deep plants. The genetic differentiation between deep and shallow stands suggests the existence of locally adapted genotypes to contrasting light environments. The depth-specific diel rhythms of photosynthetic and respiratory processes, from molecular to physiological levels, must be considered in the management and conservation of these key coastal ecosystems.

The Citrus clementina putative allergens: from proteomic analysis to structural features.

Several allergens have been identified and characterized in the genus Citrus, which belongs to the germin-like proteins (GPLs), profilins, and non-specific lipid transfer proteins (nsLTPs). In this work, in silico sequence analysis, protein purification, mass spectrometry identification, and the spectral counting method were integrated to identify new putative allergens of Citrus clementina and their expression level in the fruit peel. The in silico analysis revealed fifteen new sequences belonging to GLPs (Cit cl 1), and two more belonging to nsLTPs (Cit cl 3). No other new sequences were found as regards profilins (Cit cl 2). Each putative allergen from fruit peel was obtained using different protein extraction methods, and the protein sequences of the putative allergens were identified by means of LTQ-Orbitrap XL mass spectrometer. The spectral counting strategy revealed that Cit cl 1 had a higher expression level than Cit cl 2 and Cit cl 3. To predict the quaternary structure and deduced function of Cit cl 1, its primary sequence was used as a template to search a homologous protein structure in the RCSB PDB Database, getting high correspondence with the oxalate oxidase protein in barley.