New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.).

Xiaochun Wang,Lanxin Shi,Steven M Theg,Yihao Wei,Xinming Ma

doi:10.1093/jxb/erv388

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.

Highlights

Glutamine synthetase (GS; EC 6.3.1.2) assimilates ammonium into glutamine, which is used for the biosynthesis of all essential nitrogenous compounds (Miflin and Lea, 1977)
To elucidate the role of GS isoforms during wheat development, leaf extracts from three cultivars of wheat seedlings at different developmental stages were separated by nativePAGE and GS isoforms were detected using transferase activity staining
GSII had the highest mobility in native-PAGE, followed by GSIII and GSI

Summary

Introduction

Glutamine synthetase (GS; EC 6.3.1.2) assimilates ammonium into glutamine, which is used for the biosynthesis of all essential nitrogenous compounds (Miflin and Lea, 1977). GS1 functions in the assimilation and recycling of the ammonia generated from catabolic processes (Avila-Ospina et al, 2014; Bernard and Habash, 2009; Kamachi et al, 1992) This role, confirmed by quantitative trait locus analysis, or gene mutation or knockout, is important during grain development in cereals when nitrogen is remobilized to the reproductive sinks (Brestic et al, 2014; Guan et al, 2015; Martin et al, 2006; Tabuchi et al, 2005). Three developmentally regulated GS holoenzymes in wheat are reported that can be separated by native-PAGE in plants

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Results

Conclusion