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Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme

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Abstract

The NH2-terminal sequences of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) purified from barley (Hordeum vulgare L.) and Chlamydomonas reinhardtii (Dangeard), and of a barley peptide, were determined and the barley sequences were used to design oligonucleotide primers for the polymerase chain reaction. A specific 1.3-kilobase (kb) cDNA fragment specifying the NH2-terminal one-third of the mature barley polypeptide, was amplified, cloned and sequenced. The NH2-terminus of plant Fd-GOGAT is highly conserved and homologous to the NH2-terminus of the heavy subunit of Escherichia coli NADPH-GOGAT. Based on sequence homologies, we tentatively identified the NH2-terminal region of Fd-GOGAT as the glutamine-amidotransferase domain, which is related to the corresponding domain of the purF-type amidotransferases. The Fd-GOGAT cDNA clone, and polyclonal antibodies raised against the barley enzyme, were used to analyse four Fd-GOGAT-deficient photorespiratory mutants. Three mutants (RPr 82/1, RPr 82/9 and RPr 84/82) had no detectable Fd-GOGAT protein in leaves, while the fourth (RPr 84/42) had a small amount of cross-reacting material. Hybridization to Northern blots of total leaf RNA revealed that both RPr 82/9 and RPr 84/82 were indistinguishable from the parental line (Maris Mink), having normal amounts of a 5.7-kb mRNA species. On the other hand, RPr 82/2 and RPr 84/42 each contained two distinct hybridizing RNA species, one of which was larger than 5.7 kb, the other smaller. Using a set of wheat-barley telosomic addition lines we have assigned the Fd-GOGAT structural locus to the short arm of chromosome 2.

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Abbreviations

bp:

kbase pairs

cDNA:

copy DNA

Fd:

ferredoxin

GOGAT:

glutamate synthase

GAT:

glutamine amidotransferase

kb:

kilobase

PCR:

polymerase chain reaction

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Authors and Affiliations

  1. Biochemistry and Physiology Department, Rothamsted Experimental Station, AFRC Institute of Arable Crops Research, AL5 2JQ, Harpenden, Herts., UK

    Concepción Avila, Martin E. Cannell, Roger M. Wallsgrove & Brian G. Forde

  2. Departamento de Bioquímica Vegetal y Biología Molecular, Universidad de Sevilla, Apartado 553, 41080, Sevilla, Spain

    Antonio J. Márquez & Purificación Pajuelo

Authors
  1. Concepción Avila
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  2. Antonio J. Márquez
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  3. Purificación Pajuelo
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  4. Martin E. Cannell
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  5. Roger M. Wallsgrove
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  6. Brian G. Forde
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Additional information

C.A. was the holder of a Fleming award from the British Council and the Spanish Ministry of Education and Science. A.J.M. was for part of the work the recipient of a European Molecular Biology Organization postdoctoral fellowship. The research was also partly supported by contract no. BAP/O354/E of the Biotechnology Action Programme of the E.C., by an Acciones Integradas award (no. 40/125) from the British Council and the Spanish Ministry of Education and Science, by the Junta de Andalucia (to Group 3263) and by project PB91-0613 from DGICYT (Spain). We thank Daryl Pappin (Department of Biochemistry, University of Leeds) for amino-acid sequencing, and Martin Cornelius (Rothamsted Experimental Station, Harpenden, Herts., UK) for synthesis of oligonuleotides.

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Avila, C., Márquez, A.J., Pajuelo, P. et al. Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme. Planta 189, 475–483 (1993). https://doi.org/10.1007/BF00198209

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