Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase

Abstract

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 identical to 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.

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

“…For NHz-terminal sequencing of the intact enzyme, the protein was purified by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS. Amino-acid sequencing was performed by Dr. D. Pappin at the University of Leeds (UK) as described by Freeman et al (1990).…”

“…Total RNA was prepared from leaves of 5-d-old barley seedlings as described by Freeman et al (1990), and the poly(A)+RNA fraction was purified by two cycles of oligo(dT) chromatography.…”

“…A similar analysis of a set of GSz-deficient barley photorespiratory mutants led Freeman et al (1990) to identify three distinct classes of mutant: class I, deficient in both GS2 m R N A and protein; class II, with normal or increased amounts of m R N A but little or no protein, and class III, with normal amounts of both m R N A and protein but little or no enzyme activity. Two of the F d -G O G A T mutants (RPr 82/9 and RPr 84/82) clearly belong to class II.…”

“…As pointed out by Freeman et al (1990), because translational defects lead to m R N A instability (Scallon et al 1987;Jofuku et al 1989), it is likely that class II mutants are affected at a post-translational level. Thus RPr 82/9 and RPr 84/82 may produce an aberrant Fd-G O G A T precursor that is for some reason unstable, perhaps because of incorrect folding or because of defective targeting to the chloroplast.…”

Arabidopsis thaliana GLN2-Encoded Glutamine Synthetase Is Dual Targeted to Leaf Mitochondria and Chloroplasts

“…The GS-2 precursor is synthesized on cytosolic polysomes, taken up by chloroplasts, and is active in stromal fractions (Tingey et al, 1988;Tjaden et al, 1995). In corroborative work, light-inducible, plastidic GS-2 activity was confirmed in various plants, including bean (Phaseolus vulgaris) (Lightfoot et al, 1988) and barley (Hordeum vulgare) (Freeman et al, 1990).…”