Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant

Abstract

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.