Abstract
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.
Highlights
Flavonoids are one of the most important secondary metabolite groups in plant, among them, anthocyanins and proanthocyanidins are two important branch products in flavonoid pathway
Similar results were demonstrated in Medicago truncatula, more proanthocyanidins are accumulated in the leaves of PAP1 and MtANR over-expression plants compared with the wild type [11]. These results suggested that ectopic accumulation of proanthocyanidins in plant, in particular in leaves, may be feasible by co-expression of PAP1 and anthocyanidin reductase (ANR) genes
Anthocyanidins could be catalyzed by ANR to form proanthocyanidin monomers; presumably, in the presence of ANR, more proanthocyanidins will be produced when there is massive anthocyanidins accumulated, but the competition between the biosynthesis of proanthocyanidins and anthocyanins could be a block for the engineering of proanthocyanidins in plant as they share the same early biosynthetic pathway (Fig 1) [17,24]
Summary
Flavonoids are one of the most important secondary metabolite groups in plant, among them, anthocyanins and proanthocyanidins are two important branch products in flavonoid pathway. Anthocyanins contribute to plant coloration and survival [1], and proanthocyanidins provide defense against plant pathogens and other diseases [2,3]. The existence of F3’5’H in many other plants, such as tea plant, grape and Medicago truncatula could promotes further hydroxylation at the 5’ position in the B-ring of dihydroquercetin and quercetin, to form dihydromyricetin and myricetin [8] (Fig 1). No F3’5’H gene exists in Arabidopsis, which could not synthesize any flavonoid derivatives with trihydroxyl groups on the B-ring as tea plant or grapes that are rich in proanthocyanidins in leave tissues
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