Abstract
Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce “bloat-safe” plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed.
Highlights
The colorless flavonoid polymers called proanthocyanidins (PAs) or condensed tannins (CTs), and their monomeric building blocks, the-flavan-3-ols (+) catechin and (-)-epicatechin, are important bioactive compounds both for human health and agriculture, and their production in crop plants using metabolic engineering would permit the design of à-la-carte nutraceutical foods
Our results indicated that the increased expression of both AmDelila and AmRosea1 transcription factors (TFs), is able to upregulate the expression of genes involved in the anthocyanin biosynthetic pathway chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), DFR1 and anthocyanidin synthase (ANS), activating the F3H, DFR1 and ANS genes coding for three central enzymes of the flavonoid biosynthetic pathway [58]
Our results showed that the expression of genes encoding the enzymes CHS, CHI, F3 ́H, DFR1 and ANS, were induced in the transgenic plants indicating a correlation exists between the expression levels of the TFs AmRosea1 and AmDelila, the purple phenotype and the expression of genes involved in the anthocyanin biosynthetic pathway
Summary
The colorless flavonoid polymers called proanthocyanidins (PAs) or condensed tannins (CTs), and their monomeric building blocks, the (epi)-flavan-3-ols (+) catechin and (-)-epicatechin, are important bioactive compounds both for human health and agriculture, and their production in crop plants using metabolic engineering would permit the design of à-la-carte nutraceutical foods. Several types of transcription factors (TFs), including basic helix-loop-helix (bHLH), MYB and WD40, control the expression of genes coding for enzymes of this pathway [4]. These TFs act together to regulate the expression of genes encoding enzymes such as chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT), [5,6,7,8,9,10,11,12]. The conversion of cyanidin to the flavan-3-ol (-)-epicatechin, a building block of PAs, has been accomplished by such a strategy [1, 14]
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