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

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.

Sequence analysis, transcriptional and posttranscriptional regulation of the rice vdac family.

The voltage-dependent anion-selective channel (VDAC) is a mitochondrial outer membrane ion channel. Different isoforms exist in plants but information about their specific role remains to be established. Our purpose is to find out the structural features common to three rice VDAC isoforms and to investigate their (post)transcriptional regulation in response to an osmotic stress. Two new cDNAs encoding mitochondrial VDAC from rice (Oryza sativa) were isolated, sequenced and characterized: a phylogenetic reconstruction permitted identification of orthologues in Poaceae and computer-based analyses predicted 18 transmembrane beta-strands, one amphipathic alpha-helix and two different phosphorylation motifs. The expression of three rice vdac genes was investigated. Northern blot analyses indicated that they were expressed in all plant tissues. There was a differential expression of osvdac1 and osvdac3, whereas osvdac2 was homogeneously expressed in all tissues. No change in vdac expression was observed under an osmotic stress. However, a fast-enhanced expression of vdac was observed in roots during the recovery period after stress release. This enhanced expression is not correlated to the amount of VDAC protein detected in roots suggesting a posttranscriptional regulation.

Expression of the rice vdac isoform2: histochemical localization and expression level.

The voltage-dependent anion-selective channel (VDAC) is a mitochondrial outer membrane ion channel. The putative promoter of the rice vdac isoform2 (osvdac2) was isolated by screening a rice genomic library. Computer-based analyses predicted a TATA box, a putative transcription start and several transcription factor-binding sites including pollen specific elements. The promoter region was fused to the gus reporter gene and introduced into rice by Agrobacterium-mediated transformation. Histochemical and cell-type localizations indicated an overall expression of this promoter with a strong expression in actively growing lateral roots and in the pollen grains. Quantitative experiments showed that the osvdac2 promoter has a strong specific activity in both root and shoot. Thus, the osvdac2 promoter could be a good alternative to viral promoters (e.g. CaMV 35S) to overexpress genes in transgenic Poaceae.