title: New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.).
abstract: Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development.
authors: Collaborative Innovation Center of Henan Grain Crops, Henan Agriculture University, Zhengzhou 450002, China State Key Laboratory of Wheat and Maize Crop Science in China, Henan Agriculture University, Zhengzhou 450002, China Department of Biochemistry, College of Life Science, Henan Agriculture University, Zhengzhou 450002, China.
authors: Xiaochun
authors: X
authors: Wang
authors: Department of Biochemistry, College of Life Science, Henan Agriculture University, Zhengzhou 450002, China.
authors: Yihao
authors: Y
authors: Wei
authors: Department of Plant Biology, University of California-Davis, One Shields Avenue, Davis, CA 95616, USA.
authors: Lanxin
authors: L
authors: Shi
authors: Collaborative Innovation Center of Henan Grain Crops, Henan Agriculture University, Zhengzhou 450002, China xinmingma@126.com smtheg@ucdavis.edu.
authors: Xinming
authors: X
authors: Ma
authors: Department of Plant Biology, University of California-Davis, One Shields Avenue, Davis, CA 95616, USA xinmingma@126.com smtheg@ucdavis.edu.
authors: Steven M
authors: SM
authors: Theg
mesh_terms: D005974:Glutamate-Ammonia Ligase / Q000737:chemistry*; D008970:Molecular Weight; D063347:Native Polyacrylamide Gel Electrophoresis; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry; D012394:Rosaniline Dyes; D014908:Triticum / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; D020033:Protein Isoforms; D012394:Rosaniline Dyes; C048139:Coomassie blue; D005974:Glutamate-Ammonia Ligase
keywords: Assembly; enzyme isoform; glutamine synthetase; nitrogen; protein modification; wheat.