Structural, evolutionary and functional insights into the NAC gene family in Stevia rebaudiana and identification of important genes regulating steviol glycosides biosynthesis

NAC (NAM, ATAF1/2 and CUC2) proteins represent one of the largest transcription factor families, specific to plants and are critically involved in regulating plant growth, development and secondary metabolite production. Stevia (Stevia rebaudiana Bertoni) is a highly valuable crop due to the presence of natural sweeteners- steviol glycosides (SGs)- in its leaves. Identifying key NAC genes that synergistically influence the SG production is crucial for the genomic-assisted breeding of stevia. In this analysis, a total of 113 SrNACs were found and grouped into 14 subgroups, with classification corroborated by their phylogenetic relationships, gene structure and conserved motif patterns. Low Ka/Ks ratios indicated strong purifying selection, highlighting high protein conservation and functional constraint on these genes. Gene synteny studies demonstrated the highest synteny with corresponding genes in Helianthus annuus. To explore functional associations, RNA-seq analysis combined with WGCNA identified nine pathway genes (DXS, DXR, HDR, GGDPS, ent-KS, UGT85C2, UGT91D2, UGT74G1 and UGT76G1) co-expressed with twenty-five SrNACs and revealed a broad co-regulatory network, potentially involved in the SG biosynthesis. Further, qPCR-based expression profiling of three candidate SrNACs (SrNAC21, SrNAC22 and SrNAC52) showed a strong correlation with five SG biosynthesis pathway genes (DXS, HDR, ent-KS, UGT85C2 and UGT76G1). These trends were further validated by HPTLC-based quantification of SGs across different samples. SrNAC21, SrNAC22 and SrNAC52 may serve as key regulators of SG biosynthesis as they possess strong correlation with five SG biosynthesis pathway genes, namely DXS, HDR, ent-KS, UGT85C2 and UGT76G1, suggesting a potential regulatory significance. This first comprehensive study of SrNACs offers key insights into their role in SG biosynthesis, laying a foundation for functional characterization and future efforts to enhance metabolic engineering in S. rebaudiana.