The Role of Quercetin in Response to Biotic and Abiotic Stress in Tartary Buckwheat

Under the increasingly severe challenges of global climate change, there is an
urgent need to expand the reservoir of elite genetic resources for crop improvement.
Buckwheat, a dual-purpose crop in the Polygonaceae family and Fagopyrum genus,
includes two main cultivated species: Tartary buckwheat (Fagopyrum tataricum)
and common buckwheat (Fagopyrum esculentum). Tartary buckwheat contains
notably higher levels of flavonoids (approximately 100 folds more than that in
common buckwheat), affording it significant nutritional and medicinal value.
Quercetin, one of the major flavonoids in Tartary buckwheat, exhibits potent
antioxidant activity that can alleviate human inflammation and enhance plant stress
resistance. However, the role of quercetin in Tartary buckwheat and its underlying
molecular regulatory mechanisms remain unclear. In this study, we performed a
genome-wide association study (GWAS) on quercetin content across 186 Tartary
buckwheat accessions and identified two significant association peaks, which were
subsequently subjected to in-depth investigation.
On chromosome 3, we identified a Polyethylene Glycol (PEG) induced NAC
domain-containing protein that phylogenetically clustered with the NAC2 subfamily,
and we named it FtNAC2. Haplotype analysis revealed two Single Nucleotide
Polymorphism (SNPs) in the FtNAC2 promoter that divided accessions into three
major haplotypes, with Hap2 exhibiting higher promoter activity and relative
expression. Subcellular localization and transactivation assays confirmed FtNAC2 as
a nuclear-localized transcriptional activator. Functional characterization demonstrated that FtNAC2 overexpression promoted quercetin accumulation in Tartary buckwheat hairy roots and enhanced drought tolerance by reinforcing reactive oxygen species (ROS) scavenging capacity—a finding further validated in Arabidopsis. DNA affinity purification sequencing (DAP-seq) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses identifiedFtF3'H and FtF3'5'H as potential downstream targets, showing that FtNAC2 activates their promoters to regulate quercetin biosynthesis.
On chromosome 6, we uncovered a CYP450 gene (named “FtCYP81”) strongly upregulated under both jasmonic acid (JA) treatment and Rhizoctonia solani infection, as confirmed by qRT-PCR. This endoplasmic reticulum-localized oxygenase gene enhanced quercetin accumulation in hairy roots and improved pathogen tolerance in Arabidopsis transgenic lines, primarily through reduced cell damage. Haplotype analysis indicated that a 277-bp insertion-deletion (INDEL) in the HapL promoter caused reduced promoter activity and the loss of a key MYBHv1 binding site (MBS) cis-element. DNA pull-down combined with JA-responsive transcriptome data identified two MYB proteins, and subsequent luciferase assays suggested FtMYB2 as a putative regulator, confirmed by yeast one-hybrid assays.
FtMYB2, a JA- and R. solani -inducible transcriptional activator, promoted quercetin accumulation and FtCYP81 expression in hairy roots, and enhanced R. solani tolerance in Arabidopsis. These results indicate that FtMYB2 activates the FtCYP81 promoter via binding to the MBS element, whereas the indel in HapL impairs this activation.
Previous studies have identified numerous virulence proteins in R. solani that may
perturb plant immunity. Through interaction screening, we detected a strong
interaction between FtMYB2 and a chimeric spermidine synthase/saccharopine
dehydrogenase (named “nSpe-Sdh”), validated by Bimolecular Fluorescence
Complementation (BiFC), Yeast Two-Hybrid (Y2H), and pull-down assays. Interestingly, Electrophoretic Mobility Shift Assay (EMSA) revealed that FtMYB2 alone could not bind the MbS element in vitro, but the addition of the nSpe-Sdh protein facilitated this binding. This suggests that during infection, R. solani not only perturbs JA-mediated signaling but also, via nSpe-Sdh, recruits FtMYB2 to the MBS element of the FtCYP81 promoter, activating gene expression to promote quercetin accumulation and enhance pathogen tolerance—representing a coordinated plant defense response to pathogen invasion.
In summary, our findings elucidate that FtNAC2 and FtCYP81 participate in both
biotic and abiotic stress responses by modulating quercetin accumulation in Tartary
buckwheat through distinct regulatory networks. These results provide molecular
insights into the differential quercetin accumulation among cultivated populations
and offer valuable gene resources for breeding improved germplasm with enhanced
agronomic traits in future.