Stacking triple genes increased proanthocyanidins level in Arabidopsis thaliana

Anthocyanins and proanthocyanidins are two important plant secondary metabolites, and they contribute to plant survival and human health. In particular, proanthocyanidins could also prevent ruminants from the damage of pasture bloat. However, the improvement of proanthocyanidins content remain unsatisfied. In this study, we attempted to improve proanthocyanidins level by gene stacking in Arabidopsis thaliana as prove-of-concept. Two proanthocyanidin pathway genes from tea plant, CsF3'5'H and CsANR2, were co-expressed in the wild type and PAP1 over-expression Arabidopsis. Over-expression of CsF3'5'H slightly affected anthocyanins level in leaves and proanthocyanidins in mature seed when expressed alone in the pap1-D line. Over-expression of CsANR2 led to an obvious decrease in anthocyanins in leaves of both wild type and pap1-D lines, but increase in proanthocyanidin level in mature seeds. Over-expression of CsANR2 in pap1-D lines lead to production of DMACA-reactive soluble proanthocyanidins in leaves, but not in wild type or pap1-D lines. Anthocyanins level was decreased in the leaves of CsF3'5'H, CsANR2 and pap1-D co-expression lines, but proanthocyanidins were increased remarkably in both leaves and mature seeds in the co-expression line. It is concluded that co-expression of CsANR2 and PAP1 in Arabidopsis produce soluble proanthocyanidins in leaves, and co-expression of CsF3'5'H, CsANR2 and PAP1 lead to a significant increase in proanthocyanidins in mature seeds. The transcript levels of endogenous CHS, DFR, ANS and ANR genes in Arabidopsis were up-regulated in the triple genes co-expression line. Based on these studies, it is possible to develop new plant germplasm with improved proanthocyanidins by co-expressing of multiple genes.