Unlocking purple: Two metabolic genes found essential for petunia flower color formation

Anthocyanins are major pigments responsible for the vivid colors of ornamental flowers. However, the metabolic origin of the precursor phosphoenolpyruvate (PEP), which fuels anthocyanin biosynthesis in petals, has remained unclear. This study identifies PhENO1 and PhPPT as the key gene pair that jointly supplies PEP to the shikimate pathway in petunia. When these two genes were simultaneously silenced, flowers shifted from vibrant purple to a lighter shade, accompanied by substantial reductions in PEP, shikimate, flavonoids, aromatic amino acids, and total anthocyanins. By contrast, silencing either gene alone did not affect flower color. These findings clarify how primary metabolism feeds secondary metabolite synthesis, offering a new mechanistic understanding of floral pigmentation.

Flower color is influenced by anthocyanins, which originate from the phenylpropanoid pathway. The shikimate pathway supplies phenylalanine, the fundamental precursor for anthocyanin formation. This process requires precursor phosphoenolpyruvate (PEP), which may be produced in plastids by enolase, transported from the cytoplasm via the PEP/phosphate translocator, or generated by pyruvate orthophosphate dikinase. However, which PEP source dominates in petals of ornamental flowers has remained uncertain. Previous genetic studies suggested that different plant tissues and species rely on distinct PEP-generation routes. These knowledge gaps make it difficult to fully understand or precisely modulate floral pigmentation. Due to these unresolved questions, in-depth research on the metabolic origin of PEP during flower coloration is needed.

Researchers from South China Agricultural University conducted a study published (DOI: 10.1093/hr/uhaf040) on 1 May 2025 in Horticulture Research, focusing on the metabolic basis of petunia flower coloration. The team examined the functional roles of PhENO1, PhPPT, and PhPPDK in supplying PEP for anthocyanin biosynthesis in Petunia hybrida. They applied virus-induced gene silencing to analyze how single and combined gene suppression affects floral pigmentation. The findings reveal that only the co-silencing of PhENO1 and PhPPT leads to visibly lighter flower color and significantly reduced pigment accumulation.

The study first identified PhENO1, PhPPT, and PhPPDK genes in petunia and examined their protein localization. PhENO1 and PhPPDK localized to plastids, while PhPPT1 was associated with PEP transport between cytoplasm and plastids. Expression profiling confirmed that all three genes are active during flower development, particularly when pigment synthesis peaks.

Virus-induced gene silencing experiments showed that suppressing any single gene did not affect flower color. However, simultaneously silencing PhENO1 and PhPPT resulted in a clear and reproducible change in floral appearance—from deep purple to noticeably lighter purple. Biochemical measurements demonstrated substantial decreases in PEP (−30%), shikimate (−25%), flavonoids (−33%), phenylalanine, tryptophan, and tyrosine (up to −97%). Total anthocyanin levels declined by more than 40%.

Interestingly, expression levels of key structural genes in the shikimate and anthocyanin biosynthesis pathways remained unchanged, indicating that the reduced pigment phenotypes were driven not by transcriptional regulation, but by metabolic limitation of precursor supply.

The findings support a model in which PEP for anthocyanin synthesis is primarily jointly provided by PhENO1-mediated glycolysis and PhPPT-mediated plastid transport, rather than by PhPPDK.

“The study reveals a direct metabolic bottleneck in petunia anthocyanin synthesis,” the authors noted. “By demonstrating that PhENO1 and PhPPT collaboratively ensure sufficient PEP supply, we clarify a long-standing question about how primary carbon metabolism supports floral pigment accumulation. This knowledge provides a foundation for manipulating flower color through targeted metabolic engineering rather than altering downstream structural genes. The work highlights that pigment intensity is not only about biosynthetic enzymes, but also about precursor availability.”

Understanding the metabolic sources that enable anthocyanin biosynthesis opens new avenues for breeding ornamental plants with customizable flower colors. Targeted modulation of PhENO1 and PhPPT expression could allow controlled adjustment of pigment levels without altering genetic pathways responsible for flower development or structural traits. The findings also have broader relevance across crop and horticultural species, as the shikimate pathway underlies production of phenolics, fragrances, antioxidants, and defense compounds. Future applications may include enhancing nutritional flavonoids in edible flowers, improving plant stress tolerance, and designing ornamentals with new color gradients or intensities.

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References

DOI

10.1093/hr/uhaf040

Original Source URL

https://doi.org/10.1093/hr/uhaf040

Funding information

This study was supported by the National Natural Science Foundation of China (32271939, 32202527 and 31870692).

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.