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iTRAQ-based quantitative proteome and phosphoprotein characterization reveals the central metabolism changes involved in wheat grain development.

Ma C, Zhou J, Chen G, Bian Y, Lv D, Li X, Wang Z, Yan Y - BMC Genomics (2014)

Bottom Line: Hierarchical clustering analysis indicated that the DEPs involved in starch biosynthesis, storage proteins, and defense/stress-related proteins significantly accumulated at the late grain development stages, while those related to protein synthesis/assembly/degradation and photosynthesis showed an opposite expression model during grain development.Numerous DEPs are involved in grain starch and protein syntheses as well as adverse defense, which set an important basis for wheat yield and quality.Particularly, some key DEPs involved in starch biosynthesis and stress/defense were phosphorylated, suggesting their roles in wheat grain development.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Capital Normal University, Beijing 100048, China. zhimin206@263.net.

ABSTRACT

Background: Wheat (Triticum aestivum L.) is an economically important grain crop. Two-dimensional gel-based approaches are limited by the low identification rate of proteins and lack of accurate protein quantitation. The recently developed isobaric tag for relative and absolute quantitation (iTRAQ) method allows sensitive and accurate protein quantification. Here, we performed the first iTRAQ-based quantitative proteome and phosphorylated proteins analyses during wheat grain development.

Results: The proteome profiles and phosphoprotein characterization of the metabolic proteins during grain development of the elite Chinese bread wheat cultivar Yanyou 361 were studied using the iTRAQ-based quantitative proteome approach, TiO2 microcolumns, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among 1,146 non-redundant proteins identified, 421 showed at least 2-fold differences in abundance, and they were identified as differentially expressed proteins (DEPs), including 256 upregulated and 165 downregulated proteins. Of the 421 DEPs, six protein expression patterns were identified, most of which were up, down, and up-down expression patterns. The 421 DEPs were classified into nine functional categories mainly involved in different metabolic processes and located in the membrane and cytoplasm. Hierarchical clustering analysis indicated that the DEPs involved in starch biosynthesis, storage proteins, and defense/stress-related proteins significantly accumulated at the late grain development stages, while those related to protein synthesis/assembly/degradation and photosynthesis showed an opposite expression model during grain development. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 12 representative genes encoding different metabolic proteins showed certain transcriptional and translational expression differences during grain development. Phosphorylated proteins analyses demonstrated that 23 DEPs such as AGPase, sucrose synthase, Hsp90, and serpins were phosphorylated in the developing grains and were mainly involved in starch biosynthesis and stress/defense.

Conclusions: Our results revealed a complex quantitative proteome and phosphorylation profile during wheat grain development. Numerous DEPs are involved in grain starch and protein syntheses as well as adverse defense, which set an important basis for wheat yield and quality. Particularly, some key DEPs involved in starch biosynthesis and stress/defense were phosphorylated, suggesting their roles in wheat grain development.

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An overview of the starch biosynthesis processes in wheat endosperm. The starch biosynthesis includes sucrose degradation pathway, photosynthesis pathway as the raw materials source and starch biosynthesis pathway, and consists of two distinct phases: the glucan initiation process and the starch amplification process. ADPG is mainly synthesized by the cytosolic AGPase SSU and LSU, or supplied by sucrose degradation. The subsequent mechanisms underlying the glucan initiation process remain to be established. Branched dextrins are putatively processed by the coordinated activities of SS, BE, and/or DBE to produce the prototype of an amylopectin cluster structure, which further develops into amylopectin to establish the basic structure. Amylose is mainly synthesized by GBSS. Two AGPase SSU, two AGPase LSU and one Susy were phosphorylated. The protein levels are shown in coloured squares, indicating the change of expression for each developmental stage.
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Fig8: An overview of the starch biosynthesis processes in wheat endosperm. The starch biosynthesis includes sucrose degradation pathway, photosynthesis pathway as the raw materials source and starch biosynthesis pathway, and consists of two distinct phases: the glucan initiation process and the starch amplification process. ADPG is mainly synthesized by the cytosolic AGPase SSU and LSU, or supplied by sucrose degradation. The subsequent mechanisms underlying the glucan initiation process remain to be established. Branched dextrins are putatively processed by the coordinated activities of SS, BE, and/or DBE to produce the prototype of an amylopectin cluster structure, which further develops into amylopectin to establish the basic structure. Amylose is mainly synthesized by GBSS. Two AGPase SSU, two AGPase LSU and one Susy were phosphorylated. The protein levels are shown in coloured squares, indicating the change of expression for each developmental stage.

Mentions: Our results provided dynamic changes and phosphorylated protein characterization of the DEPs involved in starch synthesis (Figure 8). Starch biosynthesis includes two main stages: glucan initiation and starch synthesis. Firstly, photosynthesis and sucrose hydrolysis provide the raw materials that are transformed into glucose-1-phosphate (G1P) by a series of enzymes; then, G1P is catalyzed into ADP-Glu by AGPase. ADP-Glu is carried to amyloplasts by BT1 and begins starch synthesis. ADP-Glu is elongated by GBSS and SS I-IV for amylase and amylopectin synthesis, respectively. SBE and DBE are involved in catalyzing the formation of α-(1, 6)-linkages within the polymers and hydrolyzing α-(1, 6)-linkages within a polyglucan to regularize the branching and maintain amylopectin crystallinity. Most of the enzymes involved in starch biosynthesis were identified in our work and showed a peak expression at 21 DPA, well consistent with the rapid increase of grain sizes and starch granules at 14–21 DPA (Figure 1). Some DEPs essential for starch biosynthesis were phosphorylated in the developing grains, which could increase the interaction between starch synthesis enzymes and enhance their activity to promote starch biosynthesis.Figure 8


iTRAQ-based quantitative proteome and phosphoprotein characterization reveals the central metabolism changes involved in wheat grain development.

Ma C, Zhou J, Chen G, Bian Y, Lv D, Li X, Wang Z, Yan Y - BMC Genomics (2014)

An overview of the starch biosynthesis processes in wheat endosperm. The starch biosynthesis includes sucrose degradation pathway, photosynthesis pathway as the raw materials source and starch biosynthesis pathway, and consists of two distinct phases: the glucan initiation process and the starch amplification process. ADPG is mainly synthesized by the cytosolic AGPase SSU and LSU, or supplied by sucrose degradation. The subsequent mechanisms underlying the glucan initiation process remain to be established. Branched dextrins are putatively processed by the coordinated activities of SS, BE, and/or DBE to produce the prototype of an amylopectin cluster structure, which further develops into amylopectin to establish the basic structure. Amylose is mainly synthesized by GBSS. Two AGPase SSU, two AGPase LSU and one Susy were phosphorylated. The protein levels are shown in coloured squares, indicating the change of expression for each developmental stage.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4301063&req=5

Fig8: An overview of the starch biosynthesis processes in wheat endosperm. The starch biosynthesis includes sucrose degradation pathway, photosynthesis pathway as the raw materials source and starch biosynthesis pathway, and consists of two distinct phases: the glucan initiation process and the starch amplification process. ADPG is mainly synthesized by the cytosolic AGPase SSU and LSU, or supplied by sucrose degradation. The subsequent mechanisms underlying the glucan initiation process remain to be established. Branched dextrins are putatively processed by the coordinated activities of SS, BE, and/or DBE to produce the prototype of an amylopectin cluster structure, which further develops into amylopectin to establish the basic structure. Amylose is mainly synthesized by GBSS. Two AGPase SSU, two AGPase LSU and one Susy were phosphorylated. The protein levels are shown in coloured squares, indicating the change of expression for each developmental stage.
Mentions: Our results provided dynamic changes and phosphorylated protein characterization of the DEPs involved in starch synthesis (Figure 8). Starch biosynthesis includes two main stages: glucan initiation and starch synthesis. Firstly, photosynthesis and sucrose hydrolysis provide the raw materials that are transformed into glucose-1-phosphate (G1P) by a series of enzymes; then, G1P is catalyzed into ADP-Glu by AGPase. ADP-Glu is carried to amyloplasts by BT1 and begins starch synthesis. ADP-Glu is elongated by GBSS and SS I-IV for amylase and amylopectin synthesis, respectively. SBE and DBE are involved in catalyzing the formation of α-(1, 6)-linkages within the polymers and hydrolyzing α-(1, 6)-linkages within a polyglucan to regularize the branching and maintain amylopectin crystallinity. Most of the enzymes involved in starch biosynthesis were identified in our work and showed a peak expression at 21 DPA, well consistent with the rapid increase of grain sizes and starch granules at 14–21 DPA (Figure 1). Some DEPs essential for starch biosynthesis were phosphorylated in the developing grains, which could increase the interaction between starch synthesis enzymes and enhance their activity to promote starch biosynthesis.Figure 8

Bottom Line: Hierarchical clustering analysis indicated that the DEPs involved in starch biosynthesis, storage proteins, and defense/stress-related proteins significantly accumulated at the late grain development stages, while those related to protein synthesis/assembly/degradation and photosynthesis showed an opposite expression model during grain development.Numerous DEPs are involved in grain starch and protein syntheses as well as adverse defense, which set an important basis for wheat yield and quality.Particularly, some key DEPs involved in starch biosynthesis and stress/defense were phosphorylated, suggesting their roles in wheat grain development.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Capital Normal University, Beijing 100048, China. zhimin206@263.net.

ABSTRACT

Background: Wheat (Triticum aestivum L.) is an economically important grain crop. Two-dimensional gel-based approaches are limited by the low identification rate of proteins and lack of accurate protein quantitation. The recently developed isobaric tag for relative and absolute quantitation (iTRAQ) method allows sensitive and accurate protein quantification. Here, we performed the first iTRAQ-based quantitative proteome and phosphorylated proteins analyses during wheat grain development.

Results: The proteome profiles and phosphoprotein characterization of the metabolic proteins during grain development of the elite Chinese bread wheat cultivar Yanyou 361 were studied using the iTRAQ-based quantitative proteome approach, TiO2 microcolumns, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among 1,146 non-redundant proteins identified, 421 showed at least 2-fold differences in abundance, and they were identified as differentially expressed proteins (DEPs), including 256 upregulated and 165 downregulated proteins. Of the 421 DEPs, six protein expression patterns were identified, most of which were up, down, and up-down expression patterns. The 421 DEPs were classified into nine functional categories mainly involved in different metabolic processes and located in the membrane and cytoplasm. Hierarchical clustering analysis indicated that the DEPs involved in starch biosynthesis, storage proteins, and defense/stress-related proteins significantly accumulated at the late grain development stages, while those related to protein synthesis/assembly/degradation and photosynthesis showed an opposite expression model during grain development. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 12 representative genes encoding different metabolic proteins showed certain transcriptional and translational expression differences during grain development. Phosphorylated proteins analyses demonstrated that 23 DEPs such as AGPase, sucrose synthase, Hsp90, and serpins were phosphorylated in the developing grains and were mainly involved in starch biosynthesis and stress/defense.

Conclusions: Our results revealed a complex quantitative proteome and phosphorylation profile during wheat grain development. Numerous DEPs are involved in grain starch and protein syntheses as well as adverse defense, which set an important basis for wheat yield and quality. Particularly, some key DEPs involved in starch biosynthesis and stress/defense were phosphorylated, suggesting their roles in wheat grain development.

Show MeSH