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Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.)

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Abstract

We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2). Phylogenetic analysis showed that the wheat GS sub-families together with the GS genes from other monocotyledonous species form four distinct clades. Immunolocalisation studies in leaves, stems and rachis in plants at flowering showed GS protein to be present in parenchyma, phloem companion and perifascicular sheath cells. In situ localisation confirmed that GS1 transcripts were present in the perifascicular sheath cells whilst those for GSr were confined to the vascular cells. Studies of the expression and protein profiles showed that all GS sub-families were differentially expressed in the leaves, peduncle, glumes and roots. Expression of GS genes in leaves was developmentally regulated, with both GS2 and GS1 assimilating or recycling ammonia in leaves during the period of grain development and filling. During leaf senescence the cytosolic isozymes, GS1 and GSr, were the predominant forms, suggesting major roles in assimilating ammonia during the critical phases of remobilisation of nitrogen to the grain. A preliminary analysis of three different wheat genotypes showed that the ratio of leaf GS2 protein to GS1 protein was variable. Use of this genetic variation should inform future efforts to modulate this enzyme for pre-breeding efforts to improve nitrogen use in wheat.

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Abbreviations

GS:

Glutamine synthetase

QTL:

Quantitative trait loci

DH:

Doubled haploid

qRT-PCR:

Real-time reverse transcription polymerase chain reaction

RACE:

Rapid amplification of cDNA ends

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Acknowledgements

We thank Peter J. Lea for useful discussions. This work was funded by an EU-FPV project ‘SUSTAIN’ QLK5-CT-2001-01461. Rothamsted Research is grant aided by the Biotechnology and Biological Sciences Research Council of the UK.

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  1. Stéphanie M. Bernard

    Present address: Ecology Department, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley, CA, 94720, USA

Authors and Affiliations

  1. Plant Science Department, Centre for Crop Genetic Improvement, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK

    Stéphanie M. Bernard, Marcela Baudo, Christine H. Foyer, Martin A. J. Parry & Dimah Z. Habash

  2. Plant and Soil Science Laboratory, Department of Agricultural Science, Faculty of Life Sciences, The University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Copenhagen, Denmark

    Anders Laurell Blom Møller, Giuseppe Dionisio, Thomas P. Jahn & Jan K. Schjoerring

  3. Unité de Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie Jules Verne, 33, Rue saint-Leu, 80039, Amiens Cedex, France

    Thomas Kichey & Frederic Dubois

  4. Unitat de Fisiologia Vegetal, Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, 08028, Barcelona, Spain

    Marta S. Lopes & Jose Luis Araus

  5. Laboratoire de Nutrition Azotée des Plantes, INRA, Versailles, 78026, France

    Thérèse Tercé-Laforgue & Bertrand Hirel

  6. Lancaster Environment Centre, Department of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, UK

    Brian G. Forde

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Correspondence to Dimah Z. Habash.

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Table S1

Sequences of primers used in RACE I: primers for cloning experiments (DOC 76 kb)

Table S2

Sequences of primers and probes for TaqMan assays. Nucleotides in bold and underlined were Locked Nucleic Acid (LNA) bases. Primers were designed not to discriminate between possible homeoalleles (DOC 33 kb)

Fig. S1

Transmission electron microscopic immunolocalisation using pre immune serum for GS in leaf parenchyma cell (A). In situ localisation of sense probes for GS1 in leaf (B) and stem (C) tissue. M, mitochondria; pa, parenchyma; pl, plastid; vb, vascular bundle. Bars: 0.5 μm (A) and 100 μm (B, C) (TIF 3974 kb)

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Bernard, S.M., Møller, A.L.B., Dionisio, G. et al. Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.). Plant Mol Biol 67, 89–105 (2008). https://doi.org/10.1007/s11103-008-9303-y

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