A newly identified ethylene–melatonin cascade boosts polyphenol biosynthesis in grape seeds

Ethylene is known to influence fruit physiology, but its specific role in regulating phenolic metabolism in grape seeds has remained unclear. This study identifies a signaling cascade in which ethylene enhances melatonin biosynthesis by inducing VvASMT through the transcription factor VvERF5. Increased melatonin then activates VvERF104, which binds a melatonin-responsive promoter element to stimulate VvMYB14 expression. Once activated, VvMYB14 directly regulates key structural genes—including VvPAL, VvC4H, and VvCHS—resulting in broad remodeling of the phenylpropanoid pathway and changes in phenolic metabolite profiles. These findings provide new mechanistic insight into hormone crosstalk governing grape seed secondary metabolism.

Phenolic compounds stored in grape seeds are central to fruit quality, antioxidant activity, and wine characteristics. Ethylene and melatonin both influence berry development and secondary metabolism, yet the molecular steps connecting these signals to phenylpropanoid biosynthesis have not been fully delineated. Previous studies implicated VvMYB14 in melatonin-mediated proanthocyanidin synthesis, but the upstream regulators controlling its activation remained unknown. The lack of clarity surrounding how ethylene interacts with melatonin, how these signals converge on transcriptional regulators, and how they jointly modulate phenolic pathways has limited a deeper understanding of hormone crosstalk in grape berries. These unresolved questions motivate the need for a detailed mechanistic investigation.

A research team from Shandong Agricultural University reported (DOI: 10.1093/hr/uhaf061) in Horticulture Research on June 1, 2025, a comprehensive signaling model that explains how ethylene regulates phenolic biosynthesis in grape seeds. Through transcriptomics, metabolomics, promoter dissection, and genome-wide DNA-binding assays, the study identifies an ethylene-triggered ERF5–melatonin–ERF104 pathway that activates the transcription factor VvMYB14. VvMYB14 subsequently modulates major phenylpropanoid genes, fundamentally shaping polyphenol accumulation during seed development.

The study first characterized temporal patterns of ACC (the ethylene precursor) and melatonin during seed development. ACC peaked earlier than melatonin, and ethylene treatment elevated melatonin levels while selectively upregulating VvASMT. Screens for upstream regulators revealed that VvERF5 binds the ethylene-responsive element in the VvASMT promoter and activates its transcription. Overexpression and suppression in grape seeds, calli, and Arabidopsis confirmed that VvERF5 enhances melatonin biosynthesis.

To determine how melatonin induces VvMYB14, the authors dissected a 2.2-kb promoter region and identified a specific melatonin-responsive element (MTRE). Yeast one-hybrid, EMSA, and luciferase assays verified that melatonin-induced VvERF104 binds this MTRE and activates VvMYB14. Suppression of VvERF104 reduced melatonin- and ethylene-induced activation of VvMYB14, highlighting its essential role.

RNA-seq of VvMYB14-overexpressing seeds revealed thousands of differentially expressed genes, with strong enrichment in phenylpropanoid and flavonoid pathways. Targeted metabolomics identified altered levels of phenolic acids, stilbenes, and other metabolites. DAP-seq mapped over 10,000 VvMYB14 binding sites and identified a highly enriched MEME-1 motif. Functional assays confirmed that VvMYB14 directly regulates VvPAL, VvC4H, and VvCHS, shifting metabolic flux toward cinnamic acid derivatives and stilbenes.

Together, these findings define a cohesive ERF5–melatonin–ERF104–MYB14 regulatory circuit.

“Our work clarifies a long-standing gap in understanding how ethylene influences phenolic metabolism,” said the study's corresponding author. “By revealing that VvERF5 initiates melatonin production and VvERF104 transmits melatonin signals to activate VvMYB14, we now have a unified model connecting hormone signaling to phenylpropanoid regulation. This cascade not only explains the coordinated activation of pathway genes such as VvPAL, VvC4H, and VvCHS but also provides molecular targets for improving grape quality and polyphenol composition.”

The discovery of this ethylene-mediated signaling cascade offers new strategies for enhancing grape phenolic profiles. By manipulating key regulators—including VvERF5, VvASMT, VvERF104, or VvMYB14—breeding programs may fine-tune phenolic content to improve wine flavor, seed antioxidant capacity, or stress resilience. The identification of functional promoter elements and transcriptional targets also provides actionable tools for engineering secondary metabolism. Beyond grapes, this hormone-interaction model serves as a framework for studying phenylpropanoid regulation in other horticultural crops, enabling broader applications in fruit quality enhancement and metabolic optimization.

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References

DOI

10.1093/hr/uhaf061

Original Source URL

https://doi.org/10.1093/hr/uhaf061

Funding information

This work was financially supported by the National Key Research and Development Program of China (2022YFD2100100), Fruit Industry Technology System of Shandong Province (SDAIT-06-03), Key Research and Development Program of Shandong Province (2023TZXD015, 2022TZXD0011) and the National Natural Science Foundation of China (32072537).

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.