Characterization of transgenic aspen (Populus tremuloides Michx.) harboring a homologous cinnamate 4-hydroxylase (C4H) transgene and analysis of two aspen 4-coumarate: CoA ligase (4CL) gene promoters

To further our understanding of lignin biosynthesis in trees and to develop improved methods of genetic engineering for application in forest industry, two studies were conducted. The first study involved characterization of transgenic aspen with altered expression of cinnamate 4-hydroxylase (C4H), a phenylpropanoid pathway gene involved in lignin biosynthesis. The second study involved the use of nested deletions to examine the regulation of spatial gene expression by two aspen 4-coumarate: CoA ligase (4CL) promoters.; Transgenic aspen trees harboring a homologous C4H transgene in sense or antisense orientation were confirmed by PCR and Southern analysis. C4H mRNA levels were reduced in xylem of several antisense transgenic lines but no changes in lignin content or composition were found. Down-regulation of the C4H gene was also observed in xylem of several sense transgenic lines. Although lignin content remained unchanged, a slight reduction in S/G ratio was observed in several trees with co-suppression effects.; Two aspen 4CL promoters that directed gene expression in distinct tissues were analyzed by expressing promoter deletion::β-glucuronidase (GUS) fusions in transgenic tobacco. Histochemical GUS analysis indicated that a fragment between −243 and +77 of Pt4CL1 promoter was sufficient for xylem GUS expression in tobacco while further deletion down to −165 abolished GUS expression. Eletrophoretic mobility shift assay (EMSA) revealed that the region between −243 and −165 might not interact with xylem nuclear proteins directly. Instead, this region may contain DNA sequences that facilitate or stabilize DNA-protein interaction between −165/+1 and xylem nuclear exctract, and may be crucial for promoter activity in vivo. GUS analysis of the Pt4CL2 promoter and its deletions down to −797/−1 revealed its trichome-specific nature. The epidermal GUS expression reported earlier was not observed in greenhouse-grown tobacco plants in this study. GUS expression in trichomes and possibly in epidermis was detected in in vitro T ₁ seedlings with deletions down to −432/−1. Three-week-old T₁ greenhouse-grown plants had GUS expression in trichomes with deletions down to −657/−1 and in phloem of leaf veins and stems with deletions −797/−1 and −657/−1. These results suggested that the trichome- and epidermis-specific regulatory activities of the Pt4CL2 promoter may be developmentally regulated.