A research team led by Professor Guangtao Zhu from the College of Energy and Environmental Science, Yunnan Normal University, published a study titled “The transcription factor WRKY41-FLAVONOID 3′-HYDROXYLASE module fine-tunes flavonoid metabolism and cold tolerance in potato” in Plant Physiology.
1. Cold Induces ScF3′H Expression in Wild Potato Species
RT-qPCR analysis revealed that under cold stress, the F3′H gene in the wild species Solanum commersonii (CM) was continuously upregulated, while no significant changes were observed in the cultivated species S. tuberosum (DM) (Figure 1A). Cloning analysis showed that ScF3′H shared 99% amino acid sequence similarity with StF3′H from the cultivated species.
Figure 1:Genome editing of scf3′h in S. commersonii using the CRISPR/Cas9 system.
2. ScF3′H Enhances Cold Tolerance in Potato
CRISPR/Cas9-generated ScF3′H knockout mutants (f3′h-1, f3′h-2, f3′h-3) exhibited significantly reduced anthocyanin content and increased electrolyte leakage under cold stress, indicating compromised cold tolerance (Figure 1B–F). Conversely, overexpression of ScF3′H in the cultivated potato cultivar “Desiree” led to enhanced anthocyanin accumulation, reduced reactive oxygen species (ROS) levels, and improved cold tolerance (Figure 2A–G).
Figure 2:Overexpression of ScF3′H enhances cold tolerance in cultivated potato.
3. ScF3′H Regulates Flavonoid Metabolic Flux
Metabolomic analysis showed that overexpression of ScF3′H resulted in the upregulation of 56 flavonoid metabolites and downregulation of 79. Notably, kaempferol derivatives were reduced, while quercetin derivatives and anthocyanins accumulated significantly (Figure 3A–F). Due to the multiple hydroxyl groups on its B-ring, quercetin exhibits stronger ROS-scavenging activity, making it a key metabolite in cold tolerance enhancement.
Figure 3:ScF3′H overexpression promotes the synthesis of quercetin-O-glycosides and downstream anthocyanins.
4. ScWRKY41 Binds to the W-box Region in the ScF3′H Promoter
Promoter analysis identified a 284 bp region in the ScF3′H promoter of the wild species containing two W-box elements essential for cold responsiveness (Figure 4A–B). RNA-seq analysis identified 17 WRKY genes responsive to cold, among which ScWRKY41 was specifically upregulated under cold stress. Yeast one-hybrid (Y1H) assays confirmed its binding to the W-box elements (Figure 4C–G).
Figure 4:Cold-induced ScWRKY41 binds to the ScF3′H promoter.
5. ScWRKY41 Recruits ScHAC1 to Activate ScF3′H Expression
ScWRKY41 localizes to the nucleus and functions as a transcriptional activator (Figure 5A–B). Transient expression assays demonstrated that ScWRKY41 significantly enhanced ScF3′H promoter activity (Figure 5C–D). Chromatin immunoprecipitation (ChIP-qPCR) confirmed that ScWRKY41 directly binds to the promoter region (Figure 5F).
Figure 5:ScWRKY41 activates ScF3′H expression.
Further analysis revealed that ScWRKY41 interacts with the histone acetyltransferase ScHAC1, facilitating H3K27 acetylation and promoting ScF3′H transcription (Figure 6A–F).
Figure 6:ScWRKY41 recruits ScHAC1 to enhance H3K27ac levels in the ScF3′H promoter.
Key Findings
This study uncovers the molecular mechanism by which the ScWRKY41-ScF3′H module enhances cold tolerance through flavonoid hydroxylation. Cold stress induces the expression of ScWRKY41, which binds to W-box elements and recruits ScHAC1 to promote histone acetylation and activate ScF3′H transcription. This leads to increased accumulation of quercetin derivatives (Figure 7). This regulatory mechanism is highly conserved in wild potato species but is limited in cultivated varieties due to promoter deletion. The findings provide a theoretical basis for utilizing wild gene resources to improve cold resistance in cultivated potatoes.
Figure 7:Proposed model: The ScWRKY41-HAC1 complex regulates flavonoid metabolism in response to cold stress.