Identification and Biochemical Characterization of Mutants in the Proanthocyanidin Pathway in Arabidopsis

Abstract

Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO 4 ) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds (tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3, tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutants tds1, tds2, tds3, and tds5 (which do not produce PA) and tds6 (6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 and tt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation.Flavonoids are a diverse group of plant secondary metabolites that accumulate in a wide variety of plant tissues and include anthocyanins, flavonols, and the polymeric flavanols known as proanthocyanidins (PAs; Fig. 1). Like their flavanol constituents, PAs are rich in hydrophobic aromatic rings and hydroxyl groups that can interact with biological molecules, particularly proteins, by hydrogen bonds and hydrophobic interactions. Because PAs are polymeric, their interaction with proteins is much stronger than that of monomeric flavanols, presumably because of a "chelate" effect where polymeric PAs can interact with large proteins at multiple sites, increasing the strength of overall molecular interaction as well as minimizing dissociation of the complex once it is formed (Fersht, 1985). This strong interaction of PAs with proteins is probably the basis of their main role in plants and their uses by man.Because PA content of dietary plants can have both positive and negative effects on animal nutrition (Waghorn and Jones, 1989), an important agricultural objective is to manipulate the level of PA in pasture legumes (Morris and Robbins, 1997). When present in bloat-safe forage legumes, PA can bind to and cause the precipitation of dietary proteins, inhibiting the formation of stable proteinaceous foams, thereby preventing bloat (Tanner et al., 1995). Increasing the PA content of pastures such as alfalfa (Medicago s...

Proanthocyanidin Synthesis and Expression of Genes Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase in Developing Grape Berries and Grapevine Leaves

“…In Arabidopsis (Arabidopsis thaliana), the transparent testa (tt) mutants that have an altered seed coat color define most reactions in flavonoid biosynthesis as well as several specifically involved in PA synthesis (Shirley et al, 1992;Abrahams et al, 2002). The tt genes necessary for PA accumulation include the transcription factors TT8, TT2, and TTG1 (Nesi et al, 2000(Nesi et al, , 2001Baudry et al, 2004); three genes, TT19, TT12, and AHA10, involved in transport processes Kitamura et al, 2004;Baxter et al, 2005); and the BANYULS gene that catalyzes the conversion of anthocyanidins to the corresponding 2,3-cis-flavan-3-ols, (2)-epiafzelechin, (2)-epicatechin, or (2)-epigallocatechin (Xie et al, 2003).…”

Use of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique to analyse the Anthocyanidin Synthase (ANS) gene Locus in Zimbabwean sorghum landraces with different seed proanthocyanidin profiles

“…The flavonoid pathway is a complicated pathway with over 10 known and unknown structural and regulatory genes (WinkelShirley, 2001). Studies on mutations at these different loci are yet to be carried out in sorghum, even though a lot of such work has been carried out in other plants (von Wettstein, 2007;Abrahams et al, 2002;Debeaujon et al, 2001). Due to the complex nature of the flavonoid pathway, it is difficult to accurately predict how a mutation in a given flavonoid pathway gene would impact on the flavonoid pathway in sorghum.…”

“…This gene, also called leucoanthocyanidin dioxygenase (LDOX) catalyses the conversion of leucoanthocyanidins into anthocyanidins and sits at the branch-point between the PAspecific branch and the anthocyanin-specific branch of the flavonoid pathway (Abrahams et al, 2002). Our interest is to find out if any sequence polymorphisms at this locus may explain observed differences in grain proanthocyanidin profiles.…”

“…Our interest is to find out if any sequence polymorphisms at this locus may explain observed differences in grain proanthocyanidin profiles. A number of flavonoid ANS gene mutants in Arabidopsis, namely tt 11, tt17 and tt 18 (tds-4) have been identified and characterised in Arabidopsis and alfalfa, both at the biochemical and molecular level (Misyura et al, 2012;Bowerman et al, 2012;Abrahams et al, 2002;Buer and Djordjevic, 2009). All these mutants were characterised by altered flavonoid profiles and deposition patterns in the grain.…”

Variation in morphology, plant habit, proanthocyanidins, and flavonoids within a Lottus germplasm collection

“…In fact, more than 200 Lotus species represent an untapped source of additional novel Lotus genes for comparative genomics with those from L. c. var. japonicus, Arabidopsis and Medicago (Abrahams et al 2002;Schnittger and Hulskamp 2002;reviewed in Marles et al 2003;Baudry et al 2004;Aziz et al 2005;Cannon et al 2005;Dixon and Sharma 2005).…”