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Molecular analysis of two mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes

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Abstract

Two photorespiratory mutants from Lotus japonicus, namely Ljgln2-1 and Ljgln2-2, deficient in plastidic glutamine synthetase (GLN2), were analysed at the molecular level. Both mutants showed normal levels of Gln2 mRNA, indicating that they were affected post-transcriptionally. Complete sequencing of full-length Gln2 cDNAs revealed the presence of a single point mutation on each mutant, leading to G85R and L278H amino acid replacements, respectively. Different types of experimental approaches, including heterologous expression and complementation tests in Escherichia coli, showed that both GLN2 mutant proteins completely lacked of biosynthetic and transferase enzyme activities. Moreover, it was also shown that while GLN2-1 mutant protein was assembled into a less stable inactive octamer, GLN2-2 mutant protein was unable to acquire a proper quaternary structure and was rapidly degraded. Therefore, the mutations analysed are the first of their type affecting the stability and/or the quaternary structure of the GLN2 enzyme. The kinetic parameters of purified recombinant GLN2 were determined. The enzyme showed positive cooperativity towards ammonium and Mg2+. Thiol compounds stimulated by twofold the biosynthetic activity but not the transferase activity of recombinant GLN2 and were able to alter the kinetics towards glutamate of the enzyme. Moreover, the biosynthetic activity of recombinant GLN2 was stimulated by more than tenfold by the presence of free Mg2+.

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Abbreviations

GS:

Glutamine synthetase

GOGAT:

Glutamate synthase

GLN1:

Cytosolic glutamine synthetase (also called GS1)

GLN2:

Plastidic glutamine synthetase (also called GS2)

CTAB:

Hexadecyltrimethylammonium bromide

DTE:

Dithioerythritol

DTT:

Dithiothreitol

EMS:

Ethyl methanesulphonate

EST:

Expressed sequenced tag

γ-GHA:

γ-Glutamylhydroxamate

GSH:

Reduced glutathione

h:

Hill number

IPTG:

Isopropyl-beta-d-thiogalactopyranoside

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Acknowledgements

The authors wish to thank funding from research projects BMC2001-3162/BFU2004-02753/BFU2005-03120 from MCYT/MEC-FEDER (Spain), HPRN-CT2000-00086, MRTN-CT-2003-505227 and INCO-Dev 517617 from the European Union, as well as support given by Junta de Andalucía to group CVI-163. The authors also wish to thank Prof. Peter J. Lea (Lancaster University) and Prof. Brian G. Forde (Lancaster University) for a critical reading of this manuscript. Analytical ultracentrifugation facilities were provided by Drs. G. Rivas and C. Alfonso from the service of analytical ultracentrifugation (CIB, Madrid, Spain). Technical assistance of MJ Cubas (Universidad de Sevilla) is also gratefully acknowledged.

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  1. Tania Arcondéguy

    Present address: Laboratoire de Chimie Bacterienne, 31 Chem Joseph Aiguier, 13402, Marseille cedex 20, France

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  1. Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química, Universidad de Sevilla, Apartado 553, 41080, Sevilla, Spain

    Marco Betti, Tania Arcondéguy & Antonio J. Márquez

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Correspondence to Antonio J. Márquez.

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Betti, M., Arcondéguy, T. & Márquez, A.J. Molecular analysis of two mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes. Planta 224, 1068–1079 (2006). https://doi.org/10.1007/s00425-006-0279-z

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