Enhancing Photosynthesis, Vitamin E Production and Tolerance to Herbicides By Targeting Homogentisate Catabolism Using CRISPR/Cas9 Genome Editing System in Soybean

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. In a previous study, we identified and characterized the function of homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism, through a forward genetic screen screen of a soybean fast neutron (FN) mutant population. The soybean fast neutron mutant line, MO12, produced brown seeds with higher concentrations of homogentisate and Vitamin E in seeds. In this study, we used CRISPR/Cas9 genome editing system to further study the effect of HGO genes on the homogentisate metabolism pathway in soybean. The dual gRNA CRISPR/Cas9 system was utilized to generate single and double hgo1 hgo2 mutants of soybean. The homozygous hgo1 mutant plants are phenocopies of the MO12 mutant line, whereas the hgo2 mutant plants showed a similar phenotype to the wildtype, Cv. Throne. The homogentisate content in the seed of the double hgo1 hgo2 mutant line was comparable to that of the hgo1 mutant, indicating that GmHGO2 has no significant contribution to homogentisate catabolism in soybean seeds. However, we observed improved growth performance of the double mutant in the field. The double mutant line showed increased tolerance to HPPD-inhibiting herbicide, which interferes with homogentisate biosynthesis, and an increase in photosynthetic rate compared to wild type and single mutants. Overall, our results showed that GmHGO1 is the major isoform responsible for homogentisate catabolism in seeds, but both GmHGO1 and GmHGO2 contribute to homogentisate turn-over in vegetative tissues. Moreover, our results show that targeting homogentisate catabolism is an effective approach to improving soybean agronomic traits and that CRISPR/Cas9 system is a powerful tool for plant metabolic engineering in soybean.