Genetic Manipulation of Condensed Tannins in Higher Plants1 : II. Analysis of Birdsfoot Trefoil Plants Harboring Antisense Dihydroflavonol Reductase Constructs

We have produced and analyzed transgenic birdsfoot trefoil (Lotus corniculatus L.) plants harboring antisense dihydroflavonol reductase (AS-DFR) sequences. In initial experiments the effect of introducing three different antisense Antirrhinum majus L. DFR constructs into a single recipient genotype (S50) was assessed. There were no obvious effects on plant biomass, but levels of condensed tannins showed a statistical reduction in leaf, stem, and root tissues of some of the antisense lines. Transformation events were also found, which resulted in increased levels of condensed tannins. In subsequent experiments a detailed study of AS-DFR phenotypes was carried out in genotype S33 using pMAJ2 (an antisense construct comprising the 5′ half of the A. majus cDNA). In this case, reduced tannin levels were found in leaf and stem tissues and in juvenile shoot tissues. Analysis of soluble flavonoids and isoflavonoids in tannin down-regulated shoot tissues indicated few obvious default products. When two S33 AS-DFR lines were outcrossed, there was an underrepresentation of transgene sequences in progeny plants and no examples of inheritance of an antisense phenotype were observed. To our knowledge, this is the first report of the genetic manipulation of condensed tannin biosynthesis in higher plants.

A Fruit-Specific Putative Dihydroflavonol 4-Reductase Gene Is Differentially Expressed in Strawberry during the Ripening Process1

A cDNA clone encoding a putative dihydroflavonol 4-reductase gene has been isolated from a strawberry (Fragaria × ananassa cv Chandler) DNA subtractive library. Northern analysis showed that the corresponding gene is predominantly expressed in fruit, where it is first detected during elongation (green stages) and then declines and sharply increases when the initial fruit ripening events occur, at the time of initiation of anthocyanin accumulation. The transcript can be induced in unripe green fruit by removing the achenes, and this induction can be partially inhibited by treatment of de-achened fruit with naphthylacetic acid, indicating that the expression of this gene is under hormonal control. We propose that the putative dihydroflavonol 4-reductase gene in strawberry plays a main role in the biosynthesis of anthocyanin during color development at the late stages of fruit ripening; during the first stages the expression of this gene could be related to the accumulation of condensed tannins.