Mutations in active surface sites of NtGGPPS1 enhance carotenoid biosynthesis and drought resistance in Nicotiana tabacum

Amino acids on the surface of enzymes play a vital role in their catalytic activity and functional properties, influencing enzymatic stability and inducing conformational changes. In this research, we demonstrate that surface mutations in geranylgeranyl diphosphate synthase1 from Nicotiana tabacum (NtGGPPS1) improve carotenoid biosynthesis and increase drought tolerance. We introduced three mutations at key surface sites: the active center V154A, the enzyme surface site V233E, and the surface complex sites V233E/I209S, creating transgenic lines OE#V154A, OE#V233E and OE#V233E/I209S, respectively. Interestingly, under standard tobacco cultivation conditions, these overexpression lines displayed improved growth and development phenotypes relative to wild-type plants, characterized by enhanced pigmentation, whereas the ntggpps1 mutant materials exhibited the opposite trends. Our studies showed that mutations in the enzyme surface sites promote plant growth, upregulate carotenoid biosynthesis-related genes, and increase drought resistance. Notably, the OE#V233E displayed significantly higher level expression of carotenoid biosynthesis genes compared to OE#NtGGPPS1 transgenic lines, effectively directing GGPP towards carotene and downstream abscisic acid production. Furthermore, it can modulate photosynthesis, promote plant growth and development, enhance antioxidant capacity, and improve drought tolerance. These findings provide new insights into GGPPS1 engineering and open avenues for developing drought-resistant crops. Our results pave the way for the structure-guided rational design and application of elite genes in higher plants.