N-glucosyltransferase GbNGT1 from Ginkgo complement auxin metabolic pathway

Abstract

As a group of the most important phytohormone, auxin homeostasis is regulated in a complex manner. Generally, auxin conjugations especially IAA glucosides are dominant on high auxin level conditions. Former terminal glucosylation researches mainly focus on O-position, while IAA-N-glucoside or IAA-Asp-N-glucoside has been neglected since their found in 2001. In our study, IAA-Asp-N-glucoside was firstly found specifically abundant (as high as 4.13 mg/g) in ginkgo seeds of 58 cultivars from Ginkgo Resource Nursery built in 1990. Furthermore, a novel N-glucosyltransferase GbNGT1, which could catalyze IAA-Asp and IAA to form their corresponding N-glucoside, was identified through differential transcriptome analysis and in vitro enzymatic test. The enzyme was demonstrated to possess specific catalyze capacity toward the N-position of IAA-amino acid or IAA among 52 substrates, and was typical of acid tolerance, metal ion independence and high temperature sensitivity. Docking and site-directed mutagenesis of this enzyme confirmed that E15G mutant could almost abolish enzyme catalytic activity towards IAA-Asp and IAA in vitro and in vivo. The IAA modification of GbNGT1 and GbGH3.5 was verified by transient expression assay in Nicotiana benthamiana. In conclusion, our results complement the terminal metabolic pathway of auxin, and the specific catalytic function of GbNGT1 towards IAA-amino acid provide a new way to biosynthesis indole-amide compounds.HighlightThe N-glucosylation of IAA or IAA-amino acids in auxin metabolism had been neglected over decades, our work for GbNGT1 redeems the missing chain of auxin metabolic pathway.