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      The modulation of leaf metabolism plays a role in salt tolerance of Cymodocea nodosa exposed to hypersaline stress in mesocosms

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          Abstract

          Applying proteomics, we tested the physiological responses of the euryhaline seagrass Cymodocea nodosa to deliberate manipulation of salinity in a mesocosm system. Plants were subjected to a chronic hypersaline condition (43 psu) to compare protein expression and plant photochemistry responses after 15 and 30 days of exposure with those of plants cultured under normal/ambient saline conditions (37 psu). Results showed a general decline in the expression level of leaf proteins in hypersaline stressed plants, with more intense reductions after long-lasting exposure. Specifically, the carbon-fixing enzyme RuBisCo displayed a lower accumulation level in stressed plants relative to controls. In contrast, the key enzymes involved in the regulation of glycolysis, cytosolic glyceraldehyde-3-phosphate dehydrogenase, enolase 2 and triose-phosphate isomerase, showed significantly higher accumulation levels. These responses suggested a shift in carbon metabolism in stressed plants. Hypersaline stress also induced a significant alteration of the photosynthetic physiology of C. nodosa by means of a down-regulation in structural proteins and enzymes of both PSII and PSI. However we found an over-expression of the cytochrome b559 alpha subunit of the PSII initial complex, which is a receptor for the PSII core proteins involved in biogenesis or repair processes and therefore potentially involved in the absence of effects at the photochemical level of stressed plants. As expected hypersalinity also affects vacuolar metabolism by increasing the leaf cell turgor pressure and enhancing the up-take of Na + by over-accumulating the tonoplast specific intrinsic protein pyrophosphate-energized inorganic pyrophosphatase (H(+)-PPase) coupled to the Na +/H +-antiporter. The modulation of carbon metabolism and the enhancement of vacuole capacity in Na + sequestration and osmolarity changes are discussed in relation to salt tolerance of C. nodosa.

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          Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.

          Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.
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            Refinements to label free proteome quantitation: how to deal with peptides shared by multiple proteins.

            Quantitative shotgun proteomics is dependent on the detection, identification, and quantitative analysis of peptides. An issue arises with peptides that are shared between multiple proteins. What protein did they originate from and how should these shared peptides be used in a quantitative proteomics workflow? To systematically evaluate shared peptides in label-free quantitative proteomics, we devised a well-defined protein sample consisting of known concentrations of six albumins from different species, which we added to a highly complex yeast lysate. We used the spectral counts based normalized spectral abundance factor (NSAF) as the starting point for our analysis and compared an exhaustive list of possible combinations of parameters to determine what was the optimal approach for dealing with shared peptides and shared spectral counts. We showed that distributing shared spectral counts based on the number of unique spectral counts led to the most accurate and reproducible results.
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              A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis.

              A simple and universally applicable protocol for extracting high-quality proteins from recalcitrant plant tissues is described. We have used the protocol with no modification, for a wide range of leaves and fruits. In all cases, this protocol allows to obtain good electrophoretic separation of proteins. As the protocol is rapid, universal, and compatible with silver staining, it could be used for routine protein extraction from recalcitrant plant tissues for proteomic analysis.
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                Author and article information

                Contributors
                Journal
                Front Plant Sci
                Front Plant Sci
                Front. Plant Sci.
                Frontiers in Plant Science
                Frontiers Media S.A.
                1664-462X
                26 June 2015
                2015
                : 6
                : 464
                Affiliations
                [1] 1Laboratorio di Biologia e Proteomica Vegetale, Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria Rende, Italy
                [2] 2Spanish Institute of Oceanography, Oceanographic Centre of Murcia Murcia, Spain
                Author notes

                Edited by: Dominique Job, Centre National de la Recherche Scientifique, France

                Reviewed by: Tiago Santana Balbuena, São Paulo State University, Brazil; Ana Paulina Barba De La Rosa, Instituto Potosino de Investigación Científica y Tecnológica, Mexico

                *Correspondence: Silvia Mazzuca, Laboratorio di Biologia e Proteomica Vegetale, Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, Ponte Bucci 12C, 87036 Rende, Italy, silvia.mazzuca@ 123456unical.it

                This article was submitted to Plant Proteomics, a section of the journal Frontiers in Plant Science

                Article
                10.3389/fpls.2015.00464
                4482034
                cda27558-d811-49f6-b191-ebd81ee36f54
                Copyright © 2015 Piro, Marín-Guirao, Serra, Spadafora, Sandoval-Gil, Bernardeau-Esteller, Fernandez and Mazzuca.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 13 March 2015
                : 11 June 2015
                Page count
                Figures: 5, Tables: 1, Equations: 0, References: 56, Pages: 12, Words: 0
                Funding
                Funded by: Rocío García Muñoz of the Spanish Oceanography Institute, Oceanography Centre of Murcia (Spain)
                Award ID: COST-STSM-ES0906-06317
                Funded by: Ministerio de Medioambiente y Medio Rural y Marino
                Award ID: OSMOGRASS project no. 021/SGTB/2007/1.3
                Funded by: Ministerio de Ciencia e Innovación
                Award ID: OSMOGRASS II project no. CTM2009-08413MAR
                Funded by: European Commission
                Categories
                Plant Science
                Original Research

                Plant science & Botany
                seagrasses,leaf proteomics,hypersaline,mesocosm
                Plant science & Botany
                seagrasses, leaf proteomics, hypersaline, mesocosm

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