Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme.
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.
Avila
C
C
Biochemistry and Physiology Department, Rothamsted Experimental Station, AFRC Institute of Arable Crops Research, Harpenden, Herts, UK.
Márquez
A J
AJ
Pajuelo
P
P
Cannell
M E
ME
Wallsgrove
R M
RM
Forde
B G
BG
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000878:Anthranilate Synthase; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004247:DNA; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019884:Nitrogenous Group Transferases; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000235:genetics
D004247:DNA; D000594:Amino Acid Oxidoreductases; C042233:glutamate synthase (ferredoxin); D014166:Transferases; D019884:Nitrogenous Group Transferases; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit