RESUMO
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.
Assuntos
Antocianinas/biossíntese , Engenharia Metabólica/métodos , Nicotiana/metabolismo , Proantocianidinas/biossíntese , Antocianinas/genética , Antirrhinum/genética , Vias Biossintéticas/genética , Radicais Livres/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proantocianidinas/genética , Nicotiana/genética , Fatores de Transcrição/genética , Regulação para CimaRESUMO
To generate a forage crop with increased biomass density that retains forage quality, we have genetically transformed lines of alfalfa (Medicago sativa L.) expressing antisense constructs targeting two different lignin pathway biosynthetic genes with a construct for down-regulation of a WRKY family transcription factor that acts as a repressor of secondary cell wall formation in pith tissues. Plants with low-level expression of the WRKY dominant repressor construct produced lignified cell walls in pith tissues and exhibited enhanced biomass and biomass density, with an increase in total sugars in the cell wall fraction; however, lines with high expression of the WRKY dominant repressor construct exhibited a very different phenotype, with loss of interfascicular fibres associated with repression of the NST1 transcription factor. This latter phenotype was not observed in transgenic lines in which the WRKY transcription factor was down-regulated by RNA interference. Enhanced and/or ectopic deposition of secondary cell walls was also seen in corn and switchgrass expressing WRKY dominant repressor constructs, with enhanced biomass in corn but reduced biomass in switchgrass. Neutral detergent fibre digestibility was not impacted by WRKY expression in corn. Cell walls from WRKY-DR-expressing alfalfa plants with enhanced secondary cell wall formation exhibited increased sugar release efficiency, and WRKY dominant repressor expression further increased sugar release in alfalfa down-regulated in the COMT, but not the HCT, genes of lignin biosynthesis. These results suggest that significant enhancements in forage biomass and quality can be achieved through engineering WRKY transcription factors in both monocots and dicots.
Assuntos
Biomassa , Lignina/metabolismo , Medicago sativa/fisiologia , Parede Celular/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Medicago sativa/genética , Panicum/genética , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/citologia , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Zea mays/genéticaRESUMO
El rol que asume la persona que coordina, es único y diferencial en cada uno de los grupos a los que pertenece; de igual manera, cada grupo operativo tiene aspectos y elementos al interior de su dinámica, que lo hacen innovador y diferente a los otros en los cuales se haya estado inmerso con anterioridad.
The role assumed by the person who coordinates is unique and differential in each of the groups to which it belongs; Similarly, each operating group has aspects and elements within its dynamics, which make it innovative and different from the others in which it has previously been immersed.
