Chalcone synthase gene expression in transgenic subterranean clover correlates with localised accumulation of flavonoids

Expression patterns of two clover chalcone synthase promoters (TsCHS1 and TsCHS3) were examined in transgenic Trifolium subterraneum plants to investigate flavonoid gene regulation and to determine if there is differential regulation of these promoters from the CHS multigene family. The CHS1 and CHS3 genes occur as part of a tightly linked cluster of tandemly orientated genes in subterranean clover. The CHS promoters were fused to the beta-glucuronidase (GUS) reporter gene and reintroduced into subterranean clovers. Analysis of the expression of the reporter genes showed that near-identical expression patterns occurred in the transgenic plants containing either the CHS1:GUS or the CHS3:GUS fusions. Both promoters responded rapidly to wounding of the shoot. Distinct changes in the expression patterns of both promoters occurred during the development of leaves and flowers. In young shoots, expression occurred throughout the tissues and was restricted to the cambium and xylem parenchyma in mature tissue. Roots possessed the highest activity levels, which were primarily located in the meristematic tissues of the tap root, lateral root and nodule. Epifluorescent localisation of flavonoids using diphenylboric acid-2-aminoethylester correlated with the site of GUS activity in most tissues. These results indicate that histochemical detection of CHS expression in various root meristems and flower parts is correlated with localised flavonoid accumulation in these cells. Our studies suggest a physiological role for flavonoids at those sites.