Structure-Based Directed Evolution of Rice 4-Hydroxyphenylpyruvate Dioxygenase Confers Enhanced Herbicide Tolerance

Precise gene modification is pivotal in molecular breeding. 4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a crucial role in the development of herbicide-resistant crops. But how to modify HPPD and thereby confer herbicide resistance in plants remains unclear. This research delineates the structure of <i>Oryza sativa</i> HPPD (<i>Os</i>HPPD) complexed with Methyl-Benquitrione (MBQ) and identifies potential residues that confer herbicide resistance. Enzymatic tests of the C-terminal α9 helix variants showed that K418D, E423Q, E423M, E423S, E423T, E423L, E423W, E432I, E432M, E432W, and E432F demonstrated preserved catalytic efficiency. The herbicide resistance test displayed that K418D, E423Q, E423M, E432I, and E432M acquired 1.5- to 3-fold enhancements against mesotrione, topramezone, MBQ, and A1 in vitro. Especially, the forced expression of E432M in <i>Arabidopsis thaliana</i> significantly elevated herbicide resistance compared to the wild type. These findings highlight the potential of specific <i>Os</i>HPPD modifications in developing crops with enhanced herbicide resistance, providing a foundation for future genetic engineering strategies.