A novel mutation in non-constitutive lycopene beta cyclase (CstLcyB2a) from Crocus sativus modulates carotenoid/apocarotenoid content, biomass and stress tolerance in plants.

Main conclusion: Mutation at A¹²⁶ in lycopene-β-cyclase of Crocus (CstLcyB2a) sterically hinders its binding of δ-carotene without affecting lycopene binding, thereby diverting metabolic flux towards β-carotene and apocarotenoid biosynthesis. Crocus sativus, commonly known as saffron, has emerged as an important crop for research because of its ability to synthesize unique apocarotenoids such as crocin, picrocrocin and safranal. Metabolic engineering of the carotenoid pathway can prove a beneficial strategy for enhancing the quality of saffron and making it resilient to changing climatic conditions. Here, we demonstrate that introducing a novel mutation at A¹²⁶ in stigma-specific lycopene-β-cyclase of Crocus (CstLcyB2a) sterically hinders its binding of δ-carotene, but does not affect lycopene binding, thereby diverting metabolic flux towards β-carotene formation. Thus, A126L-CstLcyB2a expression in lycopene-accumulating bacterial strains resulted in enhanced production of β-carotene. Transient expression of A126L-CstLcyB2a in C. sativus stigmas enhanced biosynthesis of crocin. Its stable expression in Nicotiana tabacum enhanced β-branch carotenoids and phyto-hormones such as abscisic acid (ABA) and gibberellic acids (GA's). N. tabacum transgenic lines showed better growth performance and photosynthetic parameters including maximum quantum efficiency (Fv/Fm) and light-saturated capacity of linear electron transport. Exogenous application of hormones and their inhibitors demonstrated that a higher ratio of GA₄/ABA has positive effects on biomass of wild-type and transgenic plants. Thus, these findings provide a platform for the development of new-generation crops with improved productivity, quality and stress tolerance.