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

Chloroplast development is an integral part of plant survival and growth, and occurs in parallel with chlorophyll biosynthesis. However, little is known about the mechanisms underlying chloroplast development in hexaploid wheat. Here, we obtained a spaceflight-induced wheat albino mutant mta. Chloroplast ultra-structural observation showed that chloroplasts of mta exhibit abnormal morphology and distribution compared to wild type. Photosynthetic pigments content was also significantly decreased in mta. Transcriptome and chloroplast proteome profiling of mta and wild type were done to identify differentially expressed genes (DEGs) and proteins (DEPs), respectively. In total 4,588 DEGs including 1,980 up-and 2,608 down-regulated, and 48 chloroplast DEPs including 15 up-and 33 down-regulated were identified in mta. Classification of DEGs revealed that most were involved in chloroplast development, chlorophyll biosynthesis, or photosynthesis. Besides, transcription factors such as PIF3, GLK and MYB which might participate in those pathways were also identified. The correlation analysis between DEGs and DEPs revealed that the transcript-toprotein in abundance was functioned into photosynthesis and chloroplast relevant groups. Real time qPCR analysis validated that the expression level of genes encoding photosynthetic proteins was significantly decreased in mta. Together, our results suggest that the molecular mechanism for albino leaf color formation in mta is a thoroughly regulated and complicated process. The combined analysis of transcriptome and proteome afford comprehensive information for further research on chloroplast development mechanism in wheat. And spaceflight provides a potential means for mutagenesis in crop breeding.

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“…Chloroplasts were isolated from WT and mta following a modified protocol [34]. Each step was performed at 4°C or on ice.…”

Section: Methodsmentioning

confidence: 99%

Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta

Shi

Guo

et al. 2017

PLoS ONE

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“…The procedure for extracting proteins from chloroplasts was modified from the improved phenol (BPP) method [82]. Chloroplast pellets were re-suspended uniformly in ice-cold extraction buffer containing 100 mM Tris (pH 8.0), 100 mM EDTA, 50 mM borax, 50 mM vitamin C, 1% Triton X-100, 2% β-mercaptoethanol, and 30% sucrose.…”

Section: Methodsmentioning

confidence: 99%

Comparative Proteomic and Physiological Analysis Reveals the Variation Mechanisms of Leaf Coloration and Carbon Fixation in a Xantha Mutant of Ginkgo biloba L.

Liu

Wang

et al. 2016

IJMS

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Yellow-green leaf mutants are common in higher plants, and these non-lethal chlorophyll-deficient mutants are ideal materials for research on photosynthesis and plant development. A novel xantha mutant of Ginkgo biloba displaying yellow-colour leaves (YL) and green-colour leaves (GL) was identified in this study. The chlorophyll content of YL was remarkably lower than that in GL. The chloroplast ultrastructure revealed that YL had less dense thylakoid lamellae, a looser structure and fewer starch grains than GL. Analysis of the photosynthetic characteristics revealed that YL had decreased photosynthetic activity with significantly high nonphotochemical quenching. To explain these phenomena, we analysed the proteomic differences in leaves and chloroplasts between YL and GL of ginkgo using two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF MS. In total, 89 differential proteins were successfully identified, 82 of which were assigned functions in nine metabolic pathways and cellular processes. Among them, proteins involved in photosynthesis, carbon fixation in photosynthetic organisms, carbohydrate/energy metabolism, amino acid metabolism, and protein metabolism were greatly enriched, indicating a good correlation between differentially accumulated proteins and physiological changes in leaves. The identifications of these differentially accumulated proteins indicates the presence of a specific different metabolic network in YL and suggests that YL possess slower chloroplast development, weaker photosynthesis, and a less abundant energy supply than GL. These studies provide insights into the mechanism of molecular regulation of leaf colour variation in YL mutants.

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“…All selected spots were manually excised from the 2-DE gels. In-gel digestion and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) fingerprint spectra acquisition were performed according to Fan et al52. The peptide mass fingerprints were searched against the NCBInr (http://www.ncbi.nlm.nih.gov/) protein databases using MASCOT Version 2.1.…”

Section: Methodsmentioning

confidence: 99%

Stimulated Leaf Dark Respiration in Tomato in an Elevated Carbon Dioxide Atmosphere

Zhang

Sun

et al. 2013

Sci Rep

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It is widely accepted that leaf dark respiration is a determining factor for the growth and maintenance of plant tissues and the carbon cycle. However, the underlying effect and mechanism of elevated CO2 concentrations ([CO2]) on dark respiration remain unclear. In this study, tomato plants grown at elevated [CO2] showed consistently higher leaf dark respiratory rate, as compared with ambient control plants. The increased respiratory capacity was driven by a greater abundance of proteins, carbohydrates, and transcripts involved in pathways of glycolysis carbohydrate metabolism, the tricarboxylic acid cycle, and mitochondrial electron transport energy metabolism. This study provides substantial evidence in support of the concept that leaf dark respiration is increased by elevated [CO2] in tomato plants and suggests that the increased availability of carbohydrates and the increased energy status are involved in the increased rate of dark respiration in response to elevated [CO2].

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

Cited by 29 publications

References 63 publications

Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta

Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta

Comparative Proteomic and Physiological Analysis Reveals the Variation Mechanisms of Leaf Coloration and Carbon Fixation in a Xantha Mutant of Ginkgo biloba L.

Stimulated Leaf Dark Respiration in Tomato in an Elevated Carbon Dioxide Atmosphere

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