Anthocyanins are widely distributed flavonoid pigments that produce red, purple, and blue coloration in flowers and fruits. Their biosynthesis is typically activated by the MBW complex, composed of MYB, bHLH, and WD40 proteins. While numerous studies have identified anthocyanin activators, less is known about how plants prevent abnormal or excessive pigment deposition. Red-skinned pear cultivars offer an excellent model, as spontaneous bud mutations often produce striking differences in peel color. However, the underlying molecular basis for these natural color variations remains insufficiently explained. Due to these challenges, there is a need for in-depth investigation into the regulatory mechanisms that suppress pigment formation in pears.
Researchers from Nanjing Agricultural University and collaborating institutions reported (DOI: 10.1093/hr/uhaf071) on March 3, 2025, in Horticulture Research a detailed functional characterization of the pear transcription factor PuKAN4. By integrating transcriptomics, gene overexpression assays, dual-luciferase reporter tests, and protein-interaction analyses, the study reveals that PuKAN4 acts as a potent repressor of anthocyanin biosynthesis. The work shows how PuKAN4 interacts with major anthocyanin regulators—including PuMYB10, PuMYB114, and PubHLH3—to shape the development of red pigmentation in pear fruit.
The team began by comparing the green-skinned cultivar ‘Nanguo’ with its natural red bud mutant ‘Hongnanguo’. Anthocyanin measurements confirmed markedly higher pigment accumulation in the red mutant. Transcriptome profiling identified PuKAN4 as a significantly upregulated gene in red fruit peels. Functional assays in tobacco, pear calli, and pear fruit demonstrated that overexpression of PuKAN4 sharply reduced anthocyanin content and downregulated key structural genes (PuDFR, PuANS, PuUFGT).
Dual-luciferase assays showed that PuKAN4 alone does not activate pigment-related promoters but strongly represses the activation triggered by PuMYB10 and PuMYB114. Protein–protein interaction analyses—including yeast two-hybrid, GST pull-down, and luciferase complementation—confirmed that PuKAN4 physically binds PuMYB10 and PuMYB114. Importantly, this interaction does not prevent MYB–PubHLH3 complex formation but diminishes its transcriptional output.
Promoter dissection revealed a MYB-recognition CAACCA element in the PuKAN4 promoter. Both PuMYB10 and PuMYB114 strongly activated promoter fragments containing this motif, demonstrating that these MYB activators directly induce PuKAN4 expression. This establishes a regulatory feedback loop: MYB activators stimulate anthocyanin biosynthesis while simultaneously inducing PuKAN4, which then restrains excessive pathway activation. The presence of a conserved EAR repression motif supports PuKAN4’s function as an active suppressor.
“Fruit coloration is a key trait for consumer preference, but pigment production must be precisely regulated to maintain metabolic balance,” said the corresponding author. “Our discovery of the PuKAN4 feedback loop explains how pears prevent excessive anthocyanin accumulation, which could otherwise disrupt carbon allocation and metabolic stability. This mechanism highlights the sophisticated coordination between activators and repressors in perennial fruit species and advances our understanding of how plants fine-tune secondary metabolic pathways.”
This research provides a valuable genetic framework for breeding pear cultivars with enhanced and stable red coloration. Understanding how PuKAN4 modulates anthocyanin biosynthesis enables more targeted manipulation of pigment pathways using conventional breeding, molecular markers, or gene-editing approaches. The identification of an activator–repressor feedback loop offers insights applicable to other fruit crops where color quality and metabolic homeostasis are important concerns. Ultimately, unraveling the function of PuKAN4 helps pave the way for precision horticulture strategies that improve fruit appearance, stress resilience, and marketability.
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References
DOI
10.1093/hr/uhaf071
Original Source URL
https://doi.org/10.1093/hr/uhaf071
Funding information
This work was funded by the National Science Foundation of China (31820103012), the Earmarked Fund for China Agriculture Research System (grant no. CARS-28), the Earmarked Fund for Jiangsu Agricultural Industry Technology System (JATS [2023]412), Jiangsu Specially-Appointed Professor Project (2018), and Jiangsu Agricultural Science and Technology Innovation Fund [Grant No. CX(23)3016].
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.