In silico, in vitro and in vivo characterisation of thiamin binding proteins from plant seeds.

Thiamin, an essential micronutrient, is a cofactor for enzymes involved in the central carbon metabolism and amino acids pathways. Despite efforts to enhance thiamin content in rice by incorporating thiamin biosynthetic genes, increasing thiamin content in endosperm remains challenging, possibly due to a lack of thiamin stability and/or a local sink. The introduction of storage proteins has been successful in biofortification strategies and similar efforts targeting thiamin led to a 3-4-fold increase in white rice. However, only one thiamin-binding protein (TBP) sequence has been described in plants, more specifically from sesame seeds. Therefore, we aimed to identify and characterize TBPs, as well as to evaluate the effect of their expression on thiamin concentration, using an approach integrating in silico, in vitro and in vivo methods. We identified putative TBPs from Oryza sativa (rice), Fagopyrum esculentum (buckwheat) and Zea mays (maize) and pinpointed the thiamin-binding pockets through molecular docking. FeTBP and OsTBP contained one pocket with binding affinities similar to E. coli TBP, a well-characterized thiamin-binding protein, supporting their function. In vivo expression studies of TBPs in tobacco leaves and rice callus resulted in increased thiamin levels, with FeTBP and OsTBP showing the most pronounced effects. Additionally, thermal shift assays confirmed the thiamin binding capabilities of FeTBP and OsTBP, as observed by the significant increases in melting temperatures upon thiamin binding, indicating protein stabilization. These findings offer new insights into diversity and function of plant TBPs and highlight the potential of FeTBP and OsTBP to modulate thiamin levels in crop plants.