RESUMEN
Differential screening of a cDNA library constructed from polyadenylated RNA from developing Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) seeds resulted in the isolation of four full-length cDNA clones (PM2S1, PM2S2, PM2S3 and PM2S4) encoding isoforms of 2S seed storage proteins. The deduced amino acid sequences had low similarity to the angiosperm 2S storage proteins such as 2S albumin, napin and alpha-amylase/trypsin inhibitor yet contained all conserved cysteines in an arrangement suggestive of a structural similarity between the 2S seed storage proteins from gymnosperms and angiosperms. Southern blot analysis of genomic DNA suggested that the Douglas-fir 2S seed protein genes consisted of at least two sub-families, each including several gene members. Northern blot analysis revealed that all four PM2S mRNAs were present specifically in seeds and temporally during seed development. However, the decline in PM2S2 mRNAs in the megagametophytes occurred before that of the other mRNAs, suggesting that their gene expression was regulated differentially. The accumulation of PM2S transcripts in the haploid megagametophytes started during early embryogenesis and reached a peak before that in diploid zygotic embryos. The mRNAs for PM2S were present in Douglas-fir somatic embryos at the same developmental stages as those in the zygotic embryos, and abscisic acid and osmoticum stress were necessary for the expression of PM2S genes in somatic embryos.
Asunto(s)
Proteínas de Plantas/genética , Ácido Abscísico/metabolismo , Albúminas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes de Plantas , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Proteínas de Almacenamiento de Semillas , Análisis de Secuencia de ADN , Árboles/genéticaRESUMEN
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.