Genome-Wide Identification of the CHS Gene Family in Platycodon grandiflorus and Expression Analysis Under Multiple Phytohormone Treatments

Plants are chemical factories, constantly manufacturing a vast array of compounds to survive, grow, and defend themselves. Among these chemical tools are flavonoids, a large group of molecules that give flowers their vibrant colors, protect leaves from harsh sunlight, and help plants fight off diseases. For humans, these same compounds are often the source of the healing properties found in medicinal herbs. To make these flavonoids, plants rely on a specific assembly line of enzymes, with one particular enzyme acting as the gatekeeper. This gatekeeper, known as chalcone synthase, performs the first critical step, combining smaller molecular building blocks to create the foundation for all flavonoid production. Without this enzyme, the entire factory line stops, and the plant cannot produce these vital protective chemicals.

Understanding how a plant controls this gatekeeper is key to understanding how it produces its medicinal value. In the case of the balloon flower, a perennial herb used for centuries in traditional East Asian medicine to treat coughs and inflammation, scientists have long known it contains valuable flavonoids. However, the specific genetic instructions that tell the plant when and how much of this gatekeeper enzyme to make remained a mystery. Researchers at the Sichuan College of Traditional Chinese Medicine set out to map the entire family of genes responsible for this enzyme in the balloon flower, looking at how these genes are arranged, how they have changed over time, and how they react to different chemical signals within the plant.

The team began by scanning the complete genetic code of the balloon flower to find every instance of the gene that codes for chalcone synthase. They identified twenty distinct genes, which they named from one to twenty. These genes are not scattered randomly; they are unevenly spread across nine of the plant's chromosomes, with a heavy concentration on chromosomes one, two, and three. On some of these chromosomes, the genes are clustered together in tight groups, suggesting they were created when sections of the genome were copied and pasted during the plant's evolutionary history. This pattern of duplication appears to be the main reason the balloon flower has so many copies of this gene, rather than the entire genome doubling over, which happens in some other plant species.

When the researchers looked closely at the proteins these genes create, they found that most are water-loving molecules designed to work inside the fluid part of the cell, though some are predicted to function near the cell's outer boundary or inside the energy-producing chloroplasts. By comparing the balloon flower genes to those of other plants, including the model plant Arabidopsis and the medicinal ginseng, the team discovered that the balloon flower's genes fall into two main evolutionary groups. One group looks very similar to the standard gatekeeper enzymes found in many other plants, possessing a compact and efficient structure. The second group, however, has a slightly different shape, with extra extensions at the ends that make the protein more elongated. This structural difference suggests that while all twenty genes can start the process of making flavonoids, they may have specialized roles or slightly different efficiencies.

The study also examined the control switches located just before each gene, known as promoters. These switches determine when a gene turns on. The researchers found that these switches are packed with instructions that respond to stress and hormones. Specifically, they identified numerous signals that react to methyl jasmonate, a natural plant hormone that often signals the need for defense or the production of medicinal compounds. They also found signals for other hormones like gibberellin, auxin, and salicylic acid, as well as elements that respond to drought, cold, and light. This complex arrangement of switches implies that the plant has a sophisticated system for deciding exactly which version of the enzyme to produce based on its current environment and needs.

To see how these genes actually behave, the researchers treated young balloon flower plants with different hormones and measured the activity of eight representative genes. The results showed a clear pattern of specialization. When the plants were sprayed with methyl jasmonate, most of the tested genes became highly active, confirming that this hormone is a powerful trigger for the flavonoid production line. However, the response to other hormones was much more varied. Some genes lit up strongly when exposed to gibberellin or auxin, while others remained quiet or responded only to salicylic acid. One gene, in particular, showed no significant change in activity regardless of the treatment, suggesting it might have a constant, baseline role rather than a reactive one.

This work provides the first complete map of the chalcone synthase family in the balloon flower, revealing a system that is both robust and flexible. The presence of twenty genes, created largely through segmental duplication, gives the plant a rich toolkit to manage its production of flavonoids. The fact that different genes respond to different hormonal signals means the plant can fine-tune its chemical output depending on whether it is under attack, growing rapidly, or simply maintaining its daily functions. By understanding this genetic landscape, scientists now have a solid foundation for future studies aimed at boosting the medicinal compounds in this valuable plant, potentially leading to better ways to cultivate and utilize its healing properties.