Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis

Chang

Molecular & Cellular Proteomics

et al. 2013

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134

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

confidence: 99%

“…Proteins were extracted using the BPP protocol (19), separated via 2-DE, and visualized in the gels by means of GAP staining (21).…”

Section: Determination Of Salt-responsive Protein Spots Via 2-de-mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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

134

102

“…50000 Vh) followed by incubation in equilibration buffer and fractionation on SDS-PAGE as per the manufacturer's instruction (ReadyStrip IPG, Bio-Rad). Protein spots on 2D gels were visualized using Coomassie blue stain [8]. The experiment was carried out in triplicates.…”

Section: Two-dimensional Gel Electrophoresis (2de) Based Fractionatiomentioning

confidence: 99%

Analysis of Dynamics of Proteome in Resistant Cultivar of Pearl Millet Seedlings during Sclerospora graminicola Infection

Anup¹,

Kini

2016

J App Biol Biotech

The current study was performed to analyze the dynamics of proteome in resistant cultivar of pearl millet seedlings during early hours of downy mildew infection. This was achieved using two-dimensional gel electrophoresis (2DE)-nano LCMS/MS based approach. Total protein extract from two-day-old pearl millet seedlings harvested 6 h post inoculation with pathogen, was fractionated by 2DE on 5-8 pI range IPG strips. Among the 210 protein spots detected by the PD-Quest software, 21 proteins were found to be up-regulated and 14 proteins down-regulated. Differential regulation of these proteins during downy mildew infection was found to be significant at p ≤ 0.05. Majority of these proteins belonged to the following functional categories: energy and metabolism (32%) category as well as stress and defense (32%). Unlike the previous proteomic study in pearl millet, the present study showed differential accumulation of early expressing defense-related genes such as those involved in reactive oxygen species mediated pathways, pathogenesis-related proteins and other interconnecting pathways responsible for coordinating the resistance mechanism of the plant. The current findings shed light on the vital differentially regulated proteins of pearl millet during early phase of pathogen infection that can be targeted as future disease management strategy.

Herbaspirillum rubrisubalbicans, a mild pathogen impairs growth of rice by augmenting ethylene levels

et al. 2017

Herbaspirillum rubrisubalbicans decreases growth of rice. Inoculation of rice with H. rubrisubalbicans increased the ACCO mRNA levels and ethylene production. The H. rubrisubalbicans rice interactions were further characterized by proteomic approach. Herbaspirillum rubrisubalbicans is a well-known growth-promoting rhizobacteria that can also act as a mild phyto-pathogen. During colonisation of rice, RT-qPCR analyses showed that H. rubrisubalbicans up-regulates the methionine recycling pathway as well as phyto-siderophore synthesis genes. mRNA levels of ACC oxidase and ethylene levels also increased in rice roots but inoculation with H. rubrisubalbicans impaired growth of the rice plant. A proteomic approach was used to identify proteins specifically modulated by H. rubrisubalbicans in rice and amongst the differentially expressed proteins a V-ATPase and a 14-3-3 protein were down-regulated. Several proteins of H. rubrisubalbicans were identified, including the type VI secretion system effector Hcp1, suggesting that protein secretion play a role colonisation in rice. Finally, the alkyl hydroperoxide reductase, a primary scavenger of endogenous hydrogen peroxide was also identified. Monitoring the levels of reactive oxygen species in the epiphytic bacteria by flow cytometry revealed that H. rubrisubalbicans is subjected to oxidative stress, suggesting that the alkyl hydroperoxide reductase is an important regulator of redox homeostasis in plant-bacteria interactions.