Directed Evolution Identifies Herbicide-Resistant Variants of 4-Hydroxyphenylpyruvate Dioxygenase

Herbicide application is an integral component of contemporary agriculture, offering a critical means to manage weed populations and safeguard crop productivity. However, the pervasive use of these chemicals has raised environmental concerns and accelerated the evolution of herbicide-resistant weeds, which poses a significant challenge to crops, the environment, and human health. In particular, the development of herbicide resistance among weeds poses a significant challenge to sustainable agricultural production worldwide. Soybean (Glycine max L.) as a major global crop plays a pivotal role in food security, feed production, and industrial applications. The increasing demand for soybeans underscores the importance of enhancing its productivity through innovative approaches. One such approach involves modifying the HPPD (4-hydroxyphenylpyruvate dioxygenase, EC 1.13.11.27) gene in soybeans to confer resistance against herbicides like mesotrione, a member of the HPPD inhibitor class. HPPD is a key enzyme in the biosynthesis of carotenoids and tocopherols in plants as well as a target of a class of triketone herbicides. The evolution of protein structure simulation techniques, coupled with advanced MD simulations of protein-ligand complexes, presents a formidable platform for deciphering and optimizing biomolecular interactions. By simulating the binding of mesotrione to various HPPD variants, the key residues involved in the binding process can be further understood, which paves the way for the engineering of crops with enhanced herbicide resistance. This study aims to explore the modification of HPPD in soybean to enhance resistance to mesotrione by using gene editing technologies. To identify mutations in the soybean HPPD gene that confer herbicide resistance while maintaining its native activity, we established a highthroughput mutant screening system in E. coli. By employing this approach, nine single nucleotide polymorphisms resulting in amino acid substitutions were found as key contributors to mesotrione resistance. Furthermore, various combinations of these mutations in HPPD exhibited synergistic effects in enhancing mesotrione resistance. To validate the functionality of these HPPD variants, we carried out genetic complementation of the Arabidopsis athppd mutant, and our results demonstrated that the modified HPPD enzymes retain sufficient activity to support normal plant growth and development. When these HPPD variants were overexpressed in Arabidopsis, the transgenic plants exhibited an elevated level of herbicide resistance compared to those expressing the wild-type soybean HPPD. By performing molecular dynamics simulations on the wild-type HPPD and five of its mutant variants, we conducted an in-depth residue energy decomposition analysis, van der Waals (VdW) interaction changes, and binding free energies. This comprehensive computational approach enabled us to derive results that are consistent with those obtained from our experimental studies. These findings provide valuable insights into the identified amino acid residues in HPPD as potential targets for gene editing to develop herbicide-resistant soybean cultivars and perhaps other crops in the future.