FAD and NADPH binding sites of YUCCA6 are essential for chaperone activity and oxidative stress tolerance in Arabidopsis thaliana

Phytohormone auxin plays a pivotal role in governing plant growth, development, and responses to abiotic stresses. YUCCA6 (YUC6), an auxin biosynthetic enzyme belonging to the flavin monooxygenase (FMO) subfamily, converts indole-3-pyruvic acid to indole-3-acetic acid. Our prior investigation uncovered that YUC6 also functions as a thiol-reductase and chaperone in a Cys85-dependent manner, resulting in conferred tolerance to nickel heavy metal stress and drought and delayed leaf senescence. Notably, the conserved co-factor binding sites (FAD and NADPH) in YUC6, shared with FMOs and thioredoxin reductase, prompted our exploration into their significance for holdase chaperone activity and oxidative stress tolerance in Arabidopsis. We demonstrate that YUC6 transcripts are upregulated in response to methyl viologen (MV)-induced oxidative stress, implicating YUC6 in oxidative stress response. Mutations in co-factor binding sites markedly diminish the chaperone activity of YUC6, and reduce the YUC6-mediated oxidative stress tolerance in Arabidopsis. Furthermore, YUC6 proteins exist as oligomeric states under native conditions, formed by disulfide-bond bridges. Oligomeric YUC6 displays enhanced chaperone activity compared to its monomeric YUC6. We found here that co-factor binding sites of YUC6 are necessary for its chaperone properties.