Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase

A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS₂) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS₁). A sequence identicalto an N-terminal sequence determined from a purified preparation of the mature GS₂ polypeptide (NH₂-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46–61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS₂ subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS₂-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS₂ mRNA, and an anti-GS antiserum to assay for GS₂ protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS₂ mRNA; class II, which had normal or increased levels of GS₂ mRNA but very little GS₂ protein; and class III, which had significant amounts of GS₂ protein but little or no GS₂ activity.