Dirigent proteins

There are some 16 isovariants of dirigent proteins harboring consensus regions believed involved in monolignol radical (and possibly other phenylpropanoid radical) binding sites (Table 21). The dirigent proteins (DPs) were discovered in this laboratory (34) with the first, a (+)-pinoresinol-forming DP being cloned, and its (glycosylated) recombinant functional protein obtained (35). Its detailed kinetic study revealed an unique biochemical mechanism involving trapping by the DP of preformed coniferyl alcohol radical intermediates, these undergoing stereoselective coupling to give (+)-pinoresinol (36-38). Interestingly, DPs appear to have emerged with vascular land plant evolution, given that both their gene sequences are truly unique (not found in other life forms, including simple aquatic plants) as are their biochemical modes of action. We have proposed that different forms of DPs, or proteins harboring arrays of monolignol radical (or related phenylpropanoid) DP binding sites, control both lignin and lignan formation (9,34,35,39,40), and presumably assembly of the phenolic components of suberin and sporopollenin as well. Furthermore, evidence has now been obtained for both stereoselective (34-38,41,42) and regiospecific coupling (43), with the latter only giving 8–8´ linked racemic products.

All 16 Arabidopsis isovariants were cloned, with corresponding promoters isolated (44,45). These were individually employed to obtain both GUS-DIR/GFP-DIR promoter constructs (see Figures 1 and 2) with each introduced into Arabidopsis via the transformation procedures previously described. The results obtained revealed a complex pattern of partially overlapping/partially unique gene expression in all vascular components (e.g. stem vascular apparatus, leaf tissue veins [primary and secondary], roots, hydathodes, base of trichomes, meristematic regions, petals, stamen filament vasculature, stigma, papillar cells, style, gynoecium and silique abscission zones, etc.); this finding is in general agreement with an earlier report with a GUS-DIR study (46) for the western red cedar multigene family (47), which were also expressed differentially (albeit in overlapping patterns) in distinct components of the vascular apparatus in different tissues and organs (46). Additionally, both sense and antisense lines unique for each DIR gene have been prepared (Table 22) and these, together with 12 homozygous knockout lines, are being comprehensively examined.