Altering expression of the flavonoid 3'-hydroxylase gene modified flavonol ratios and pollen germination in transgenic Mitchell petunia plants

Abstract

Antisense technology was successfully used to reduce flavonoid 3'-hydroxylase (F3'H) gene expression and enzyme activity and to promote the accumulation of monohydroxylated flavonols in petunia flower tissue. The hydroxylation pattern of specific flavonoid groups is a target for modification because of the possible associated changes in a range of factors including colour, stress tolerance and reproductive viability. Petunia (cv. Mitchell) plants were transformed to express in the antisense orientation the sequences encoding the F3'H (asF3'H). Transformants showed a range of responses, in terms of the level of endogenous F3'H gene expression and the relative proportion of the monohydroxylated flavonol (kaempferol) glycosides that accumulated. Kaempferol glycosides increased from 7% of the total flavonols in flower limb tissue of the wild type plants, to 45% in the flower limb tissue of line 114, the transgenic line that also showed the greatest decrease in F3'H expression in flower tissue. In leaf tissue, the trend was for a decrease in total flavonol concentration, with the relative proportion of kaempferol glycosides varying from ~40 to 80% of the total flavonols. The changes in leaf tissue were not consistent with the changes observed in flower tissue of the same lines. Endogenous F3'H activity in flower limb tissue was not completely shut down, although an 80% decrease in enzyme activity was recorded for line 114. The residual F3'H activity was still sufficient that quercetin glycosides remained as the major flavonol form. Alteration of F3'H activity appears to have affected overall flavonoid biosynthesis. A decrease in total flavonol concentration was observed in leaf tissue and two other flavonoid biosynthetic genes were down-regulated. No morphological changes were observed in the transgenic plants; however, up to a 60% decrease in pollen germination was observed in line 13. Thus, the relatively small change in flavonoid biosynthesis induced by the asF3'H transgene, correlated with several other effects beyond just the specific biosynthetic step regulated by this enzyme.