Developing carotenoids-enhanced tomato fruit with multi-transgene stacking strategies.

As natural dominant pigments, carotenoids and their derivatives not only contribute to fruit color and flavor quality but are regarded as phytochemicals beneficial to human health because of various bioactivities. Tomato is one of the most important vegetables as well as a main dietary source of carotenoids. So, it's of great importance to generate carotenoid-biofortified tomatoes. The carotenoid biosynthesis pathway is a network co-regulated by multiple enzymes and regulatory genes. Here, we assembled four binary constructs containing different combinations of four endogenous carotenoids metabolic-related genes, including SlOR, SlDXS, SlPSY, and SlBHY by using a high efficiency multi-transgene stacking system and a series of fruit-specific promotors. Transgenic lines overexpression SlOR alone, three genes (SlOR/SlDXS/SlPSY), two genes (SlOR/SlBHY), and all these four genes (SlOR/SlDXS/SlPSY/SlBHY) were enriched with carotenoids to varying degrees. Notably, overexpressing SlOR alone showed comparable effects with simultaneous overexpression of the key regulatory enzyme coding genes SlDXS, SlPSY, and SlOR in promoting carotenoid accumulation. Downstream carotenoid derivatives zeaxanthin and violaxanthin were detected only in lines containing SlBHY. In addition, the sugar content and total antioxidant capacity of these carotenoids-enhanced tomatoes was also increased. These data provided useful information for the future developing of biofortified tomatoes with different carotenoid profiles, and confirmed a promising system for generation of nutrients biofortified tomatoes by multiple engineering genes stacking strategy.