# Genome-Wide Identification and Characterization of Isoflavone Synthase (IFS) Gene Family, and Analysis of GgARF4-GgIFS9 Regulatory Module in Glycyrrhiza glabra

## TL;DR

This study explores how a specific gene and its regulator control flavonoid production in a medicinal plant, offering new insights into plant metabolism.

## Contribution

The study identifies a regulatory module involving GgARF4 and GgIFS9 that mediates auxin-induced flavonoid synthesis in Glycyrrhiza glabra.

## Key findings

- GgARF4 directly activates the GgIFS9 promoter, which is involved in isoflavone biosynthesis.
- IAA treatment significantly induces GgIFS9 expression and flavonoid biosynthesis pathway genes.
- GUS assays confirm IAA activates the GgIFS9 promoter in tobacco plants.

## Abstract

Isoflavone synthase (IFS) is the key enzyme in isoflavonoid biosynthesis and has been functionally characterized in numerous plant species. Glycyrrhiza species, valued for their medicinal properties, accumulate flavonoids with significant physiological activities. Among these, isoflavones play crucial roles in plant growth, development and stress responses. However, the IFS gene family in Glycyrrhiza remains poorly understood. In this study, we identified 10, 9 and 9 IFS genes in G. uralensis, G. inflata and G. glabra, respectively. Phylogenetic analysis classified these genes into four distinct clades (Clade A–D). Further characterization included chromosomal localization, gene structure, conserved motifs, cis-acting elements and synteny analysis. Using yeast one-hybrid (Y1H) screening, dual-luciferase assays and an electrophoretic mobility shift assay (EMSA), these results revealed that auxin response factor 4 (GgARF4) directly binds to the isoflavone synthase 9 (GgIFS9) promoter and activates its expression. Following indole-3-acetic acid (IAA) treatment, RNA-seq revealed that in the differentially expressed genes (DEGs), the genes involved in isoflavonoid and flavonoid biosynthesis pathways were significantly enriched. The result of quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that GgIFS9 was strongly induced by IAA. β-Glucuronidase (GUS) assays confirmed that IAA activates the expression of the GgIFS9 promoter in Nicotiana tabacum. Our findings reveal that, through GgARF4 and its downstream-activated gene GgIFS9, IAA may promote flavonoid synthesis in G. glabra. This study provides novel insights into the auxin-mediated regulation of secondary metabolism in Glycyrrhiza species.