Genome-Wide Characterization of the Soybean GmCXE Gene Subfamily Reveals GmCXE54 as a Candidate Gene for Root Isoflavone Accumulation

Abstract

Carboxylesterases (CXEs) participate in diverse plant metabolic processes, including isoflavone biosynthesis. However, the soybean GmCXE subfamily remains poorly characterized, especially in relation to root isoflavone accumulation and the response to Fusarium oxysporum. Here, fifty-six putative GmCXE genes were identified in the soybean genome and classified into three major phylogenetic clades. Analyses of gene structure, conserved motifs, protein domains, and promoter cis-elements revealed conserved features as well as potential functional divergence among subfamily members. Collinearity and duplication analyses indicated that segmental duplication was the main driver of GmCXE subfamily expansion. Tissue-specific expression profiling and RT-qPCR validation selected five root-expressed genes as candidates associated with isoflavone accumulation. SNP variation analysis and allelic group analysis of 209 soybean accessions further prioritized GmCXE54 as a candidate gene for root isoflavone accumulation. Allelic groups defined by a putative promoter SNP, Chr.20-rs39215413, showed significant differences in root daidzein and total isoflavone contents, with accessions carrying the C allele exhibiting higher levels of both traits than those carrying the T allele. Functional analysis in soybean hairy roots showed that GmCXE54 overexpression increased daidzein and total isoflavone accumulation. At 3 h after F. oxysporum inoculation, GmCXE2, GmCXE39, and GmCXE54 were induced, with GmCXE54 showing the strongest response in the resistant accession ZD27. These findings clarify GmCXE subfamily evolution and identify GmCXE54 as a candidate gene associated with root isoflavone accumulation and early F. oxysporum response, offering new perspectives for improving soybean isoflavone-related traits and investigating root response mechanisms.