Naringin has been studied for decades as a pharmaceutical treasure. It belongs to the flavanone glycosides, a family of plant secondary metabolites linked in the scientific literature to anti-inflammatory, antioxidant, and metabolic benefits, and it is the dominant bitter compound in the rind and flesh of Huajuhong fruit, a traditional Chinese medicine material. The enzymes that build naringin step by step were already well characterized: phenylalanine ammonia-lyase, chalcone synthase, flavone synthase, and two glycosyltransferases that decorate the flavanone backbone with sugars. What remained murky was the regulatory layer above those enzymes, the machinery that decides when the whole pathway should be switched on in response to environmental and hormonal cues.

The research team, led by Genlin Mao, Diyang Qiu, and Ruiyi Fan from the Guangdong Academy of Agricultural Sciences and Jingchu University of Technology, focused on jasmonate, a plant hormone best known for coordinating defense responses against herbivores and pathogens. When they treated pomelo seedlings with methyl jasmonate, a cell-permeable jasmonate analog widely used as an elicitor, naringin levels surged 3.45-fold. Crucially, the gene encoding CgMYC2, a member of the basic helix-loop-helix transcription factor family, responded even faster, rising 6.6-fold before the peak expression of the five core biosynthetic genes. That timing mattered: the putative regulator switched on before its presumed targets, exactly the sequence expected of a genuine upstream controller rather than a downstream passenger.

To place CgMYC2 within the canonical jasmonate signaling cascade, the researchers turned to protein interaction assays. In the standard model of jasmonate perception, the hormone is sensed by a receptor complex that then degrades members of the JAZ family of repressor proteins. Those JAZ proteins, when present, physically clamp down on MYC transcription factors and prevent them from activating genes. Using pull-down and co-immunoprecipitation experiments, the team confirmed that CgMYC2 directly interacts with CgJAZ3, a JAZ repressor from pomelo. This interaction anchors CgMYC2 firmly in the jasmonate pathway: when jasmonate levels rise, JAZ proteins are destroyed, CgMYC2 is released, and the biosynthetic genes can be transcribed.

The next question was whether released CgMYC2 actually touches the DNA of the naringin pathway genes. The answer came from a battery of complementary techniques. Yeast one-hybrid assays confirmed binding to the promoter of Cg1,2RhaT, the rhamnosyltransferase that performs the final sugar-transfer step in naringin synthesis. Electrophoretic mobility shift assays went further, demonstrating that CgMYC2 binds directly, and in a G-box-dependent manner, to the promoters of all five core biosynthetic genes: CgPAL5, CgCHS, CgFNS, Cg7GlcT, and Cg1,2RhaT. The G-box is a short DNA motif, the canonical binding site for bHLH transcription factors, and mutating or masking it abolished the interaction, establishing the molecular grammar of the connection.

Binding alone does not prove activation, so the team ran dual-luciferase transactivation assays, a technique in which a promoter is wired to a light-producing reporter enzyme. CgMYC2 transactivated all five promoters, with the strongest effect on CgCHS, the chalcone synthase gene that catalyzes the first committed step of flavonoid biosynthesis. As chromatin-level support, a single-sample CUT&Tag profiling experiment revealed that CgMYC2-associated genomic regions were enriched for G-box motifs across jasmonate-responsive and secondary-metabolic loci, painting a genome-wide picture of the regulator in action. Together, these assays build a causal chain from hormone perception to promoter occupancy to transcriptional output.

Genetic loss-of-function evidence sealed the argument. Using virus-induced gene silencing, a technique that co-opts a plant viral mechanism to knock down endogenous gene expression, the researchers suppressed CgMYC2 in pomelo seedlings. Naringin content dropped by roughly 21 percent, and the expression of the biosynthetic genes fell in parallel. The partial rather than complete reduction suggests redundancy among MYC-family members or contributions from other regulatory pathways, but the direction and coherence of the effect confirm that CgMYC2 is a positive, load-bearing component of the naringin regulatory network.

Perhaps the most striking result came from moving the gene into a completely different species. When the team overexpressed CgMYC2 heterologously in tomato, the transgenic plants activated their own flavonoid pathway and accumulated elevated levels of sixteen different flavonoid compounds. This cross-species functionality indicates that the MYC2-to-G-box regulatory logic is evolutionarily conserved, consistent with earlier work in tomato where an SlMYC2-SlMYB12 module orchestrates fruit flavonoid metabolism, and with jasmonate-driven MYC2 regulation of anthocyanin accumulation in Arabidopsis. A regulatory principle discovered in a medicinal pomelo thus appears to be a general design feature of plant secondary metabolism.

The practical implications extend in several directions. For breeders of Huajuhong and other citrus, CgMYC2 offers a molecular marker and a potential editing target for tuning naringin content, whether the goal is maximizing medicinal quality or reducing bitterness in juice cultivars. The study also connects to a broader agricultural context: recent work has shown that stabilizing MYC2 in citrus can confer resistance to Huanglongbing, the devastating citrus greening disease, suggesting that a single regulatory node influences both defense chemistry and quality traits. More broadly, jasmonate elicitation is already used to boost secondary metabolite production in medicinal plants, and knowing the specific transcription factor that gates a target pathway makes such elicitation strategies far more predictable and engineerable.

Scientifically, the study exemplifies how modern plant biology dissects a regulatory question by triangulating across scales: hormone physiology, gene expression kinetics, protein-protein interaction, protein-DNA binding, chromatin profiling, and transgenic functional tests all converge on the same answer. CgMYC2 emerges as a bridge between jasmonate perception and naringin biosynthesis, a single point of control where an environmental signal becomes a chemical phenotype. For a compound with documented pharmacological relevance and a central role in the identity of one of China’s most valued medicinal fruits, that bridge is likely to attract intense attention from both basic researchers and the citrus industry in the years ahead.

Subject of Research: Jasmonate-regulated transcriptional control of naringin flavonoid biosynthesis in Citrus grandis 'Tomentosa'

Article Title: CgMYC2 directly activates jasmonate-induced naringin biosynthesis in Citrus grandis 'Tomentosa'

Article References: CgMYC2 directly activates jasmonate-induced naringin biosynthesis in Citrus grandis 'Tomentosa'. (n.d.). https://doi.org/10.1007/s00299-026-03960-0

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03960-0

Keywords: CgMYC2, naringin, jasmonate, Citrus grandis, flavonoid biosynthesis, bHLH transcription factor, JAZ repressor, G-box, methyl jasmonate, plant hormone signaling, secondary metabolism, virus-induced gene silencing

The experiment itself was elegantly simple. The researchers rolled a fabric pattern wheel along both sides of the midvein of young rice leaves, creating two parallel rows of tiny punctures that mimic the physical damage insects and pathogens inflict. They then harvested leaf samples at 0.25, 0.5, 1, 3, 8, and 24 hours after wounding, flash-freezing each batch in liquid nitrogen. Crucially, they also collected leaves from completely untouched plants at every one of those same time points. That decision turned out to be one of the most consequential design choices in the study, because the untreated controls exposed something the researchers had not set out to find: even healthy, undamaged rice plants undergo dramatic swings in gene expression depending on the time of day.

Principal component analysis of the RNA sequencing data cleanly separated wounded samples from controls, confirming that injury triggers a massive transcriptional reprogramming. But the control samples also split apart from one another, with the dusk samples, collected eight hours into the light cycle, standing apart most sharply. Circadian clock genes such as LUX ARRYTHMO and CIRCADIAN CLOCK ASSOCIATED 1 rose and fell across the day in the untouched plants, yet showed no significant difference between wounded and time-matched control leaves. The practical implication is stark: studies that compare wounded plants only against a single zero-hour baseline risk mislabeling ordinary circadian fluctuations as wound responses. By using time-matched controls at every point, this team could filter out that noise and isolate the true wound signal.

What emerged from that filtered signal was a response that begins almost instantaneously. Within fifteen minutes of wounding, 607 genes were already upregulated, and the number of differentially expressed genes climbed steadily, peaking somewhere between three and eight hours after injury. At the heart of the early response sat the jasmonate pathway, the best-known wound-signaling system in plants. The researchers identified 18 jasmonate biosynthetic genes and 13 catabolic genes among the upregulated set. The metabolite measurements told a matching story: the jasmonate precursor 12-oxophytodienoic acid peaked as early as fifteen minutes after wounding, while jasmonic acid itself and its bioactive conjugate jasmonoyl-L-isoleucine began accumulating within the first quarter hour and peaked at one hour.

Then came the shutdown. Jasmonate catabolites, specifically 12-hydroxy-JA and 12-hydroxy-JA-Ile, accumulated later, peaking at eight and three hours respectively, as the plant began breaking down the very hormone it had just mobilized. This is not waste; it is regulation. Jasmonate signaling is powerful but costly, and unchecked accumulation stunts growth, a phenomenon known as the growth-defense tradeoff. Enzymes of the CYP94 cytochrome P450 subfamily and jasmonate oxidases convert the active hormone into hydroxylated and carboxylated forms that no longer activate defense genes. In Arabidopsis, mutants lacking three jasmonate oxidases grow more slowly than wild-type plants, underscoring why rice deploys this metabolic brake. The new data show the brake engaging on schedule, roughly three hours after the alarm first sounds.

To organize the thousands of responding genes into a coherent architecture, the team turned to weighted gene co-expression network analysis, feeding all 5,543 wound-induced upregulated genes into the algorithm. Nine modules emerged, and their timing divided cleanly into two waves. Three modules, colored green, yellow, and brown in the network, contained early-responsive genes peaking within the first hour. Gene ontology analysis showed these were enriched in regulatory functions: mitogen-activated protein kinase cascades, DNA-binding transcription factor activity, defense response regulation, and signaling transduction. Hub genes in these modules included kinases such as MPK3 and MKKK62 and transcription factors including WRKY24, WRKY1, WRKY71, and MYB-family members. In contrast, the turquoise and blue modules held late-responsive genes peaking after eight hours, dominated by transport processes, cell wall biosynthesis, and specialized metabolism, particularly L-phenylalanine metabolism, the gateway to the phenylpropanoid pathway.

The transcription factor census was striking in its own right: 377 upregulated and 163 downregulated TF genes spanning 50 families responded to wounding. The earliest responders, 79 of them already upregulated at fifteen minutes and peaking by half an hour, came predominantly from the ERF, WRKY, NAC, MYB, and bHLH families, and included known regulators of rice herbivore resistance such as ERF3, WRKY53, and WRKY71. Kinase genes, including members of the calcium/calmodulin-dependent protein kinase family alongside MAPK cascade components, were also induced within the first hour. Perhaps most intriguingly, the team found rapid induction of small peptide-coding genes: five rapid alkalinization factors, three plant elicitor peptide precursors, two phytosulfokines, and two PSY peptides. RALF4, RALF6, PROPEP3, and PROPEP6 transcripts jumped within fifteen minutes, consistent with the idea that damaged cells release peptide signals that are perceived by membrane receptors and amplify the wound alarm across the tissue.

The study also pulled a second hormone into the wound story. Because early wound-responsive genes were enriched in salt-stress-related terms, the researchers examined abscisic acid, the classic abiotic stress hormone. Core ABA biosynthetic genes were upregulated within an hour, with the rate-limiting enzyme gene NCED4 induced as early as fifteen minutes, and ABA itself accumulated significantly by half an hour. This fits with recent work in Arabidopsis showing that wound-induced jasmonate promotes ABA biosynthesis, which in turn sustains lignin deposition for wound healing. Meanwhile, photosynthesis-related genes, including light-harvesting complex II and magnesium-chelatase components, were significantly downregulated, reflecting the well-documented cost of defense: a wounded leaf redirects resources away from growth and toward protection and repair.

The late metabolic shift produced one of the study’s most surprising findings. In the phenylpropanoid pathway, entry-point genes, six phenylalanine ammonia-lyase genes, four cinnamate-4-hydroxylase genes, and three 4-coumarate:CoA ligase genes, were strongly upregulated, and nine phenolamides accumulated to high levels in wounded leaves. Yet flavonoid levels generally fell, even though their biosynthetic genes showed no significant change. The researchers propose a flux-shift explanation: phenolamide biosynthesis competitively consumes shared substrates such as p-coumaric acid, starving the flavonoid branch. It is a reminder that in plant metabolism, upregulating one arm of a pathway can quietly throttle another.

The final comparison carried the greatest agricultural weight. Leaf folder larvae, a major rice pest that rolls leaves and scrapes away the upper epidermis and mesophyll, cause real wounds, but the transcriptional response they provoke is strikingly incomplete. Only 41.9 percent of wound-upregulated genes also responded to leaf folder feeding. Genes co-induced by both treatments clustered around jasmonate signaling and specialized metabolism, while wound-specific genes involved transport and heat response, and leaf folder-specific genes centered on specialized metabolite biosynthesis. Most tellingly, although leaf folder feeding did trigger ABA biosynthesis, many ABA-responsive genes that mechanical wounding strongly induced remained flat after insect feeding. The likely explanation lies in the insect’s oral secretions, which are deposited into wounds during feeding and can contain effectors that suppress plant defenses, as demonstrated for the cotton bollworm proteins HARP1 and HAS1. The authors suggest similar suppressors may operate in leaf folder secretions, quietly muting a branch of the rice alarm system while the insect feeds. For breeders seeking rice varieties with durable pest resistance, the message is clear: a plant’s wound response and its herbivore response are not the same thing, and the difference may be exactly where the insects want it.

Subject of Research: Time-series transcriptomic analysis of wound-induced defense signaling and gene expression dynamics in rice leaves

Article Title: Wound-induced transcriptional dynamics in rice

Article References: Chen, Y., Jin, G., Lu, J., Lou, Y., Jiménez-Alemán, G. H., & Li, R. (2025). Wound-induced transcriptional dynamics in rice. Crop Health, 3(1), Article 15. https://doi.org/10.1007/s44297-025-00055-2

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00055-2

Keywords: rice, wounding, transcriptomics, jasmonate, plant defense, abscisic acid, leaf folder, phenylpropanoid, small peptides, gene expression, herbivory, circadian rhythm