Natural Variation in the FtNAC2 Promoter Regulates Quercetin Accumulation and Drought Tolerance in Tartary Buckwheat

Tartary buckwheat, valued for its nutritious and medicinal quercetin. Following two independent domestication events, distinct quercetin accumulation patterns have emerged between the southwestern (SL) and northern (NL) landrace populations. However, the genetic mechanisms underlying these metabolic divergences remain elusive. Here, we identified the transcription factor FtNAC2 through genome-wide association study (GWAS) of quercetin content in 480 accessions of Tartary buckwheat. Haplotype analysis identified two single nucleotide polymorphisms (SNPs) in the FtNAC2 promoter that defined three major haplotypes, with higher promoter activity and gene expression observed in Hap2. Functional characterization revealed that FtNAC2 promotes quercetin accumulation in Tartary buckwheat hairy roots and potentially serves as a multifunctional regulator influencing both drought tolerance in buckwheat and seed size in Arabidopsis. Transcriptome co-clustering and pull-down mass spectrometry (MS) indicated FtNAC52 as a potential regulatory partner of FtNAC2. DNA affinity purification sequencing (DAP-seq) and quantitative reverse transcription PCR (qRT-PCR) analyses demonstrated that FtNAC2 promoted quercetin biosynthesis by upregulating FtF3'H and FtF3'5'H genes. Collectively, our results elucidated how FtNAC2 influences quercetin content variation in Tartary buckwheat, providing molecular insights into the differential quercetin accumulation between cultivated populations.