Expression of an Antirrhinum dihydroflavonol reductase gene results in changes in condensed tannin structure and accumulation in root cultures of Lotus corniculatus (bird's foot trefoil)

Condensed tannins (proanthocyanidins) are an important factor in the nutritive and dietary quality of many forage crops. We report here experiments aimed at altering the levels and monomer composition of condensed tannins (CTs) in hairy root cultures of Lotus corniculatus (bird's foot trefoil) using genetic manipulation. An Antirrhinum majus dihydroflavonol reductase (DFR) cDNA was expressed in sense in L. corniculatus and CT levels in transgenic root cultures were analysed. Two co-transformed lines were noted with decreased CT content relative to controls and these levels were comparable with antisense-DFR phenotypes. In ADFR10, a co-transformed line with the highest CT levels, CT structure was altered in a manner consistent with the substrate specificity of the introduced gene; that is an increase in pro-pelargonidin monomers noted after hydrolysis of CTs. RT-PCR confirmed the expression of endogenous DFR gene(s) in both putatively co-suppressed lines and also in ADFR10. Analysis of selected root culture lines indicated that the monomer composition of CTs did not change during growth and development but that levels of CTs varied in a regulated manner.     

A comparison of two strategies to modify the hydroxylation of condensed tannin polymers in Lotus corniculatus L

A full-length sense Antirrhinum majus dihydroflavonol reductase (DFR) sequence was introduced into birdsfoot trefoil (Lotus corniculatus L.) in experiments aimed at modifying condensed tannin content and polymer hydroxylation in a predictable manner. Analysis of transgenic plants indicated lines that showed enhanced tannin content in leaf and stem tissues. In contrast to previous data from root cultures, levels of propelargonidin units were not markedly elevated in lines with enhanced tannin content. RT-PCR analysis of four selected lines indicated a correlation between enhanced tannin content and expression of the introduced DFR transgene. Using a contrasting approach we introduced a flavonoid 3'5' hydroxylase (F3'5'H) sequence derived from Eustoma grandiflorum into Lotus root cultures. Expression of the transgene was associated with increased levels of condensed tannins and in this case there was also no alteration in polymer hydroxylation. These results suggest that additional mechanisms may exist that control the hydroxylation state of condensed tannins in this model species.     

Genetic Manipulation of Condensed Tannins in Higher Plants : 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.     

Molecular genetics of Pseudomonas syringae pathovar pisi: plasmid involvement in cultivar-specific incompatibility.

A mutant (PF24) of the race 1 strain, 299A, of Pseudomonas syringae pv. pisi has been characterized in terms of its interactions with pea (Pisum sativum) cultivars. The mutant showed a changed reaction (avirulence to virulence) with a group of pea cultivars, including cvs. Belinda and Puget, previously thought to contain resistance genes R1 and R3. Avirulence towards cv. Puget was restored by transfer of any one of five cosmid clones from a race 3 (strain 870A) gene library to a rifampicin-resistant derivative of PF24. These observations were in agreement with a revised race-specific resistance genotype for Belinda and similar cultivars comprising a single resistance gene, R3. An incompatible interaction was observed between strain PF24 and cvs. Vinco (postulated to harbour race-specific resistance genes R1, R2, R3 and R5) and Hurst's Greenshaft (R4 and possibly R1), indicating that the mutant retains at least one avirulence gene (A1 or A1 and A4). Mutant PF24 showed loss of a cryptic plasmid (pAV212) compared with its progenitor, strain 299A. A subclone (pAV233) of one of the race 3 restoration clones showed strong hybridization with similar-sized digestion fragments in race 3 plasmid DNA, confirming the A3 gene to be plasmid-borne. Strong cross-hybridization was also observed with a single 3.27 kb EcoRI fragment of plasmid DNA present in strain 299A but absent from strain PF24. This is consistent with the corresponding A3 determinant being located on pAV212 in the race 1 strain 299A. The novel avirulence gene corresponding to A3 in strain 870A is provisionally designated avrPpi3.(ABSTRACT TRUNCATED AT 250 WORDS)