RESUMEN
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.
Asunto(s)
Antocianinas/biosíntesis , Ingeniería Metabólica/métodos , Nicotiana/metabolismo , Proantocianidinas/biosíntesis , Antocianinas/genética , Antirrhinum/genética , Vías Biosintéticas/genética , Radicales Libres/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proantocianidinas/genética , Nicotiana/genética , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
Fruit set is an essential process to ensure successful sexual plant reproduction. The development of the flower into a fruit is actively repressed in the absence of pollination. However, some cultivars from a few species are able to develop seedless fruits overcoming the standard restriction of unpollinated ovaries to growth. We report here the identification of the tomato hydra mutant that produces seedless (parthenocarpic) fruits. Seedless fruit production in hydra plants is linked to the absence of both male and female sporocyte development. The HYDRA gene is therefore essential for the initiation of sporogenesis in tomato. Using positional cloning, virus-induced gene silencing and expression analysis experiments, we identified the HYDRA gene and demonstrated that it encodes the tomato orthologue of SPOROCYTELESS/NOZZLE (SPL/NZZ) of Arabidopsis. We found that the precocious growth of the ovary is associated with changes in the expression of genes involved in gibberellin (GA) metabolism. Our results support the conservation of the function of SPL-like genes in the control of sporogenesis in plants. Moreover, this study uncovers a new function for the tomato SlSPL/HYDRA gene in the control of fruit initiation.
