title: Metabolic engineering of anthocyanins in dark tobacco varieties.
abstract: In this study, we investigate the metabolic engineering of anthocyanins in two dark tobacco crops (Narrow Leaf Madole and KY171) and evaluate the effects on physiological features of plant photosynthesis. Arabidopsis PAP1 (production of anthocyanin pigment 1) gene (AtPAP1) encodes a R2R3-type MYB transcript factor that is a master component of regulatory complexes controlling anthocyanin biosynthesis. AtPAP1 was introduced to Narrow Leaf Madole and KY171 plants. Multiple transgenic plants developed red/purple pigmentation in different tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the expression levels of six pathway genes were increased two- to eight-fold in AtPAP1 transgenic plants compared with vector control plants. Dihydroflavonol reductase and anthocyanidin synthase genes that were not expressed in wild-type plants were activated. Spectrophotometric measurement showed that the amount of anthocyanins in AtPAP1 transgenic plants were 400-800 µg g-1fresh weight (FW). High-performance liquid chromatography (HPLC) analysis showed that one main anthocyanin molecule accounted for approximately 98% of the total anthocyanins. Tandem MS/MS analysis using HPLC coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry identified the main anthocyanin as cyanidin 3-O-rutinoside, an important medicinal anthocyanin. Analysis of photosynthesis rate, chlorophylls and carotenoids contents showed no differences between red/purple transgenic and control plants, indicating that this metabolic engineering did not alter photosynthetic physiological traits. This study shows that AtPAP1 is of significance for metabolic engineering of anthocyanins in crop plants for value-added traits.
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: Xianzhi
authors.initials: X
authors.lastname: He
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Research and Development, R. J. Reynolds Tobacco Company, Winston-Salem, NC, 27102, USA.
authors.forename: Darlene
authors.initials: D
authors.lastname: Lawson
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: De-Yu
authors.initials: DY
authors.lastname: Xie
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D000872:Anthocyanins / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002338:Carotenoids / Q000378:metabolism; D002734:Chlorophyll / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D005838:Genotype; D060847:Metabolic Engineering; D010105:Oxygenases / Q000235:genetics; D000073718:Pancreatitis-Associated Proteins; D010641:Phenotype; D010788:Photosynthesis; D010858:Pigmentation; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000429:Alcohol Oxidoreductases; D000872:Anthocyanins; D029681:Arabidopsis Proteins; D002338:Carotenoids; D002734:Chlorophyll; D010105:Oxygenases; D000073718:Pancreatitis-Associated Proteins; D010940:Plant Proteins; D014157:Transcription Factors; C502612:AT1G56650 protein, Arabidopsis; C000612141:REG3A protein, human; C056570:dihydroflavanol 4-reductase; C118159:anthocyanidin synthase; C428983:cyanidin 3-rutinoside