The isolation of a novel metallothionein-related cDNA expressed in somatic and zygotic embryos of Douglas-fir: Regulation by ABA, osmoticum, and metal ions

To isolate genes which are expressed preferentially during embryogenesis, a Douglas-fir embryogenesis cDNA library was constructed and differentially screened with cDNA probes made with mRNA from developing and mature embryos, respectively. The cDNA clone PM 2.1 was isolated based on its abundance in developing seeds and absence in mature seeds, and its predicted amino acid sequence was shown to have structural features characteristic of plant MT-like proteins. Alignment of the PM 2.1 predicted amino acid sequence with other plant MT-like protein sequences revealed a general paucity of Cys and Cys-Xaa-Cys sequences and the presence of novel serine residues within the conserved Cys-Xaa-Cys motifs in the C-terminal domain. The consensus sequence following the Cys-poor spacer in type 2 MT-like proteins, CXCXXXCXCXXCXCX, was modified in PM 2.1 to CXSXXXSXYXX-XCX. Phylogenetic analysis supported PM 2.1 was distinct from other MT and grouped with MT-like proteins from Arabidopsis (OEST), rice (AEST) and kiwifruit (AD1), which do not belong to type 1 or 2. The PM 2.1 gene was expressed in somatic and zygotic embryos, in haploid maternal tissue, as well as in hormone- and metal-treated seeds and seedlings. The PM 2.1 transcripts were detected in the needles of 14-week-old seedlings, but not the root tissue or mature pollen. The expression of the PM 2.1 gene in embryos was dependent upon ABA and osmoticum and in seedlings was differentially modulated by metals, suggesting a role of the PM 2.1 gene product in the control of microelement availability during Douglas-fir seed development and germination. The novel structural features, and the developmental, hormonal and metal modulation of PM 2.1 expression, are evidence for a new type of MT-related protein in plants.