Metabolic engineering of proanthocyanidins through co‐expression of anthocyanidin reductase and the PAP1 MYB transcription factor

Abstract

SummaryProanthocyanidins (PAs) and their monomeric building blocks, the (epi)-flavan-3-ols, are plant antioxidants that confer multiple human health benefits. The presence of PAs in forage crops is an important agronomic trait, preventing pasture bloat in ruminant animals. However, many consumed plant materials lack PAs, and there has been little success to date in introducing monomeric or polymeric flavan-3-ols de novo into plant tissues for disease prevention by dietary means or development of 'bloat-safe' forages. We report the introduction of PAs into plants by combined expression of a MYB family transcription factor and anthocyanidin reductase for conversion of anthocyanidin into (epi)-flavan-3-ol. Tobacco leaves expressing both transgenes accumulated epicatechin and gallocatechin monomers, and a series of dimers and oligomers consisting primarily of epicatechin units. The levels of PAs reached values that would confer bloat reduction in forage species. Expression of anthocyanidin reductase in anthocyanin-containing leaves of the forage legume Medicago truncatula resulted in production of a specific subset of PA oligomers.

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

“…However, except for putative flavan-3-ol-like compounds in the hairy root cultures, no definitively identified PAs were detected, as was also the case following the overexpression of MtANR and VvANR; although the levels of anthocyanins in petals were reduced at the same time as an increase of DMACA-reactive PA-like compounds, no solid evidence supported the appearance of a measurable pool of flavan-3-ol monomer (Xie et al, 2003(Xie et al, , 2004Bogs et al, 2005). This was again the case when MtANR and CsANRs were overexpressed in PAP1 tobacco lines that accumulated massive levels of anthocyanins and clearly detectable levels of DMACAreactive PA-like material (Xie et al, 2006). This apparent paradox may be explained if the monomers produced are used immediately (through coupled or channeled reactions) for the production of PA-like compounds using endogenous modification and polymerization enzymes or if ANR possesses additional functions for producing novel DMACA-reactive compounds in vivo.…”

“…To determine whether tea ANR or LAR genes have better potential for metabolic engineering of PAs in a system that can potentially supply excess upstream precursors, they were next transformed into purple tobacco plants that accumulate massive levels of anthocyanins as a result of expression of the Arabidopsis Production of Anthocyanin Pigment1 (PAP1) gene (Borevitz et al, 2000;Xie et al, 2006). The PAP1-expressing tobacco can theoretically provide leucoanthocyanidin and anthocyanidin as endogenous substrates for LAR and ANR, respectively.…”

“…Thirty phosphinothricinresistant lines expressing each of the three target genes in PAP1 tobacco were detected by PCR and RT-PCR screening (Supplemental Fig. S5B), and, for each tea gene, the three lines with highest transcript level were selected for further analysis, together with PAP1-expressing and wild-type tobacco lines and a previously generated transgenic line coexpressing PAP1 and M. truncatula ANR (Xie et al, 2006) as a positive control. Anthocyanin levels decreased significantly in all CsLAR-and CsANR-expressing lines compared with PAP1 tobacco, and the same was observed for the PAP1 3 MtANR line (Fig.…”

Fruit skin color and the role of anthocyanin

“…A linear set of events can be described; fruit skin color variation is strongly associated with genetic variation and a combination of bioinformatics aspects (Borevitz et al 2000;Xie et al 2006). Anthocyanin biosynthesis is one of the most active research areas due not only to visual appeal, but also to the nutritional value of colored fruits.…”

Cloning, expression and molecular modeling of the anthocyanidin reductase (FaANR) gene during strawberry fruit development

“…ent developmental stages of the berry (Downey et al, 2003;Kennedy et al, 2000). Proanthocyanidins have been reported to be synthesized during the first phase of grape berry development, and there is a decline in extractable PAs after the onset of ripening and throughout ripening, which is thought to be the result of complexation of the PA polymers with other cellular components (Kennedy et al, 2000(Kennedy et al, , 2001Xie et al, 2006). The initial higher concentration of PAs (Davood and Osamu, 2006;Whale and Singh, 2007) may thus be explained on the basis of expression of FaANR limited to earlier stages of fruit development in apple.…”