A protein extraction method compatible with proteomic analysis for the euhalophyte Salicornia europaea

Chang

Molecular & Cellular Proteomics

et al. 2013

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136

102

Thellungiella halophila, a close relative of Arabidopsis, is a model halophyte used to study plant salt tolerance. The proteomic/physiological/transcriptomic analyses of Thellungiella plant leaves subjected to different salinity levels, reported herein, indicate an extraordinary ability of Thellungiella to adapt to large concentrations of exogenous saline by compartmentalizing Na ؉ into cell vacuoles and accumulating proline and soluble sugars as organic osmolytes. Salinity stress stimulated the accumulation of starch in chloroplasts, which resulted in a greatly increased content of starch and total sugars in leaves. Comparative proteomics of Thellungiella leaves identified 209 salt-responsive proteins. Among these, the sequences of 108 proteins were strongly homologous to Arabidopsis protein sequences, and 30 had previously been identified as Thellungiella proteins. Functional classification of these proteins into 16 categories indicated that the majority are involved in carbohydrate metabolism, followed by those involved in energy production and conversion, and then those involved in the transport of inorganic ions. Pathway analysis revealed that most of the proteins are involved in starch and sucrose metabolism, carbon fixation, photosynthesis, and glycolysis. Of these processes, the most affected were starch and sucrose metabolism, which might be pivotal for salt tolerance. The gene expression patterns of the 209 salt-responsive proteins revealed through hierarchical clustering of microarray data and the expression patterns of 29 Thellungiella genes evaluated via quantitative RT-PCR were similar to those deduced via proteomic analysis, which underscored the possibility that starch and sucrose metabolism might play pivotal roles in determining the salt tolerance ability of Thellungiella. Our observations enabled us to propose a schematic representation of the systematic salt-tolerance phenotype in Thellungiella and suggested that the increased accumulation of starch, soluble sugars, and proline, as well as subcellular compartmentalization of sodium, might collectively denote important mechanisms for halophyte salt tolerance. Molecular & Cellular

Section: -De and Gelmentioning

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Comparative Proteomics of Thellungiella halophila Leaves from Plants Subjected to Salinity Reveals the Importance of Chloroplastic Starch and Soluble Sugars in Halophyte Salt Tolerance

Chang

Molecular & Cellular Proteomics

et al. 2013

Self Cite

136

102

An efficient method for the extraction of chloroplast proteins compatible for 2‐DE and MS analysis

et al. 2009

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Comparative proteomic analysis of chloroplast by 2-DE has received significant attention in recent years. However, the complication of membrane systems in chloroplast made it challenging to elucidate entire chloroplast proteome by 2-DE. Here, we developed an efficient method for extracting chloroplast proteins, and produced excellent 2-DE profiles from both Arabidopsis thaliana and Salicornia europaea. Comparison of this method with another two protocols for the extraction of A. thaliana chloroplast proteins showed that our method obtained higher protein yields and produced more protein spots on both pH 3-10 and 4-7 2-DE gels. Moreover, this method recovered more proteins in the basic and high M(r) regions, thereby offering the best extraction of chloroplast proteins. Identification of 15 specific chloroplast-targeted proteins on our gels by MALDI-TOF MS revealed that this method was compatible with MS, and recovered more chloroplast membrane proteins than the commonly used methods. This protocol is expected to have a wide application in future chloroplast proteomic analysis.

Simultaneous isolation of DNA, RNA, and protein from Medicago truncatula L.

Xiong¹,

Yang²,

Sun³

et al. 2010

Electrophoresis

We describe a method for the simultaneous extraction of proteins and nucleic acids from Medicago truncatula tissues. Using a modified TRIzol reagent method, we developed a simple and an effective way to simultaneously extract proteins and nucleic acids from a single sample. We verified that this method does not affect the quality or quantitation of the isolated DNA and RNA. Furthermore, we used 2-DE to compare M. truncatula leaf, stem, and root samples processed using this new method with two commonly used methods: phenol extraction/methanol-ammonium acetate precipitation and trichloroacetic acid/acetone precipitation. The results showed that our method was superior to the other methods, based on 2-DE patterns. We also demonstrated that our protocol is compatible with proteomic analysis, as 10 out of 14 selected proteins isolated by the method were identified by MALDI-TOF-MS/MS. The protocol described can be used with sample preparation protocols for proteomic, transcriptomic, and genomic studies.