Sequence and expression of embryogenesis-specific cDNAs encoding 2S seed storage proteins in Pseudotsuga menziesii...

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.