Ocimum kilimandscharicum 4CL11 negatively regulates root formation by inhibiting polar auxin transport via flavonoids accumulation

Abstract

4-Coumarate-CoA Ligase (4CL) is an important enzyme in the phenylpropanoid biosynthesis pathway. Multiple 4CLs are identified in Ocimum species; however, their function remains enigmatic. In this study, we independently overexpressed three Ok4CL isoforms from Ocimum kilimandscharicum (Ok4CL7, -11 and -15) in the plant species Nicotiana benthamiana, Solanum tuberosum and Arabidopsis thaliana for functional characterization. Interestingly, Ok4CL11 overexpression causes a rootless or reduced root growth phenotype, whereas overexpression of other two isoforms (Ok4CL7 and -15) resulted in normal adventitious root growth. Overexpression of Ok4CL11 in N. benthamiana leads to abundance of flavonoid-glycosides (i.e. kaempferol-3,7-O-bis-alpha-L-rhamnoside [K3,7R], quercetin-3-O-rutinoside [Q3R]), reduces auxin levels, and disrupts auxin transport and signaling, which cumulatively results in a rootless phenotype. In silico studies show that auxin transporters (PINs/LAXs) have higher binding affinity to K3,7R than to auxins, suggesting a potential disruption of auxin transport by the flavonoid-glycosides accumulated in Ok4CL11-OE lines. Our study reveals a unique indirect function of Ok4CL11 as a negative regulator of root growth, likely involving inhibition of auxin transport. As Ok4CL11 strongly affects root development, it is imperative to understand tissue-specific regulation of its gene expression and could be utilized for agricultural benefits.