Betalain,an economically valuable water-soluble natural plant pigment,is prized for its strong antioxidant activity,making it popular as a dietary supplement and a visual marker for plant transformation.However,market...Betalain,an economically valuable water-soluble natural plant pigment,is prized for its strong antioxidant activity,making it popular as a dietary supplement and a visual marker for plant transformation.However,market demand significantly outstrips current production capacity.This study reports the development of an efficient push-and-pull multigene strategy based on polycistronic expression and metabolic flux regulation to enhance betalain biosynthesis in transgenic maize(Zea mays L.)endosperm.We engineered a novel enhanced RUBY(eRUBY)system derived from the original polycistronic RUBY construct(CYP76AD1P2ADODA1P2ADOPA5GT unit,abbreviated CDG)by introducing arogenate dehydrogenase(ADHα)to increase the L-tyrosine substrate supply.All the genes were driven by the endosperm-specific promoter.Fusion of ADHαinto a single polycistronic eRUBY construct(CDGA)produced significantly higher betanin(6.88 mg g−1 dry weight)and isobetanin(1.81 mg g−1 dry weight)levels than in CDG+A,which stacked the ADHαcassette independently with CDG.The high betalain accumulation in CDGA lines(which also exhibited higher transgene copy number)resulted in a 2.85–7.58-fold improvement in endosperm antioxidant capacity compared to WT(versus 2.48–2.80-fold in CDG+A).Importantly,transgenic plants maintained a normal phenotype.Transcriptome and metabolome analyses further indicated that metabolism of phenylalanine,alanine,aspartate,and glutamate contributes to betalain production.Hybridization with sweet corn successfully created a high-sugar eRUBY maize variety.Collectively,these results demonstrate the successful development of a novel maize germplasm with significantly enhanced nutritional value through high betalain accumulation.展开更多
基金supported by grants from the Biological Breeding-National Science and Technology Major Project(2024ZD04077)the Invigorate the Seed Industry of Guangdong Province(2024-NPY-00-044)+3 种基金the National Natural Science Foundation of China(32272120)the Guangxi Science and Technology Major Project(GKAA24206023)the National Key Research and Development Program of China(2024YFF1000800)the Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops Major Project(FCBRCE-202502,FCBRCE-202504).
文摘Betalain,an economically valuable water-soluble natural plant pigment,is prized for its strong antioxidant activity,making it popular as a dietary supplement and a visual marker for plant transformation.However,market demand significantly outstrips current production capacity.This study reports the development of an efficient push-and-pull multigene strategy based on polycistronic expression and metabolic flux regulation to enhance betalain biosynthesis in transgenic maize(Zea mays L.)endosperm.We engineered a novel enhanced RUBY(eRUBY)system derived from the original polycistronic RUBY construct(CYP76AD1P2ADODA1P2ADOPA5GT unit,abbreviated CDG)by introducing arogenate dehydrogenase(ADHα)to increase the L-tyrosine substrate supply.All the genes were driven by the endosperm-specific promoter.Fusion of ADHαinto a single polycistronic eRUBY construct(CDGA)produced significantly higher betanin(6.88 mg g−1 dry weight)and isobetanin(1.81 mg g−1 dry weight)levels than in CDG+A,which stacked the ADHαcassette independently with CDG.The high betalain accumulation in CDGA lines(which also exhibited higher transgene copy number)resulted in a 2.85–7.58-fold improvement in endosperm antioxidant capacity compared to WT(versus 2.48–2.80-fold in CDG+A).Importantly,transgenic plants maintained a normal phenotype.Transcriptome and metabolome analyses further indicated that metabolism of phenylalanine,alanine,aspartate,and glutamate contributes to betalain production.Hybridization with sweet corn successfully created a high-sugar eRUBY maize variety.Collectively,these results demonstrate the successful development of a novel maize germplasm with significantly enhanced nutritional value through high betalain accumulation.
