Molecular characterization and regulation of embryogenesis-associated genes in Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco)




Chatthai, Malinee

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As a direct approach to investigate the molecular basis of embryogenesis in Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco), a cDNA library made from poly(A)⁺ RNA of developing seeds was differentially screened for clones representing transcripts abundant in the developing seeds but absent in mature seeds. Of a number of clones isolated, two groups were selected for further sequence and gene expression analysis. A group of four cDNA clones (PM2S1, PM2S2, PM2S3 and PM2S4) shared a significant nucleotide and deduced amino acid sequence similarity with each other and with gymnosperm 2S seed storage protein cDNAs. The deduced amino acid sequences had low similarity with angiosperm 2S storage proteins but contained all conserved cysteine residues in an arrangement suggestive of a structural similarity between the 2S seed storage proteins from gymnosperms and angiosperms. Northern blot analysis revealed PM2S mRNAs were present specifically in seeds and temporally during seed development. However, the relatively low abundance of PM2S3 mRNAs and the decline of PM2S2 mRNAs in megagametophyte which occurred before that of the other mRNAs suggested that their expression was regulated differentially. The accumulation of PM2S transcripts in megagametophyte started during the early embryogenesis and reached a peak before that in zygotic embryos. PM2S mRNAs were present in Douglas-fir somatic embryos at the same developmental stages as those in zygotic embryos, and ABA and osmoticum stress were necessary for the expression of PM2S genes in somatic embryos. 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. A gene designated gPm2Sl was isolated and sequenced. A comparison of the upstream sequence of gPm2Sl with the promoters of known 2S storage protein genes did not reveal significant sequence similarity except the presence of RY-repeated element (GCATGC), and the frequent occurrence of ACGT-containing motifs and E-box motifs (CANNTG). The 1.2-kb gPm2Sl promoter was fused to a P-glucuronidase (uidA) reporter gene and transformed into developing Douglas-fir seeds using particle bombardment and into tobacco via Agrobacterium tumefaciens. Histochemical analysis showed that the promoter was active in both systems and the gene expression was confined to endosperm and embryos of transgenic tobacco, indicating a common seed-specific regulatory mechanism between angiosperms and conifers. Another cDNA clone, PM2.1, hybridized to a 0.5 kb transcript and was predicted to encode a metallothionein (MT)-like protein. Alignment of the PM2.1 predicted amino acid sequence with other plant MT-like gene products revealed a general paucity of Cys and Cys-Xaa-Cys sequences and the presence of serine residues within the conserved Cys-Xaa-Cys motifs in the C-terminal domain. Phylogenetic analysis showed that PM2.1 grouped with class I/type 3 MT-like genes. The PM2.1 was expressed in somatic and zygotic embryos, in megagametophyte, as well as in hormone- and metal-treated seeds and seedlings. The PM2.1 transcripts were detected in the needles of 10-week-old seedlings, but not the root tissue or mature pollen. The expression of the PM2.1 gene in embryos was dependent upon ABA and osmoticum and was differentially modulated by metals, suggesting that the PM2.1 gene product may play a role in the control of microelement availability during Douglas-fir seed development and germination. Southern blot analysis of genomic DNA suggested that the PM2.1 was encoded by a multigene family. Three genomic clones were isolated and one of these clones (gPmMTa) was cloned and sequenced. The sequence analysis of its 5'-flanking region identified a number of putative regulatory elements such as ACGT-containing motifs, metal-responsive element (TGCGCC) and ethylene-responsive elements (ATTTCAAA) which may be responsible for gene transcription. DNase I-footprinting experiments with nuclear extracts isolated from Douglas-fir megagametophyte identified two protein-protected sites, a 31-bp sequence locating in the -176/-146 region that contained two ACGT-core motifs, and a 12-bp sequence, 5'-TGCCACGGAAGG-3', of unknown function. To identify promoter regions responsible for the regulation of gPmMTa gene expression, a series of deletions in the 0.9-kb fragment of the gPmMTa promoter was fused to the uidA reporter gene and the chimeric gene constructs were assayed in Douglas-fir and transgenic tobacco. Transient expression assays in megagametophyte and zygotic embryos indicated that the sequence lying between -190 and +88 of gPmMTa was sufficient to drive the expression of the reporter gene and the 225-bp fragment (-677 to -453) contained sequences necessary for high level expression. The gPmMTa promoter was not active in the seeds of transgenic tobacco.



Plant embryology, Forest genetics, Douglas fir, Seedlings