The smoke tree is a striking shrub best known for the wispy, smoke-like plumes that give it its common name, but its heartwood has long attracted attention from natural products chemists. Heartwood extracts of C. coggygria are rich in flavonoids, a sprawling family of plant secondary metabolites that includes many compounds with documented anticancer activity in laboratory settings. Earlier work had established that butin, butein, and sulfuretin exhibit cytotoxic effects against leukemia cells, yet the molecular mechanisms underlying these effects in K562 cells—a widely used model of chronic myeloid leukemia—remained poorly defined. The new study set out to close that gap by treating the cells with the purified compounds as well as with crude heartwood extracts prepared with ethanol, water, and a methylene chloride/methanol mixture.

The experimental design combined classical pharmacology with modern genomics. Flow cytometry was used to determine how the treatments altered cell cycle distribution and whether they triggered the activation of apoptosis, the programmed cell death pathway that many anticancer therapies seek to engage. To pin down the mode of cell death, the researchers employed caspase inhibition, a technique that reveals whether cell death depends on the caspase enzymes that execute apoptosis. The centerpiece of the study, however, was the transcriptomics: three independent mRNA sequencing experiments were performed on treated K562 cells, and the resulting data were subjected to differential gene expression analysis followed by pathway-level analyses using the Reactome and KEGG databases through gene set enrichment analysis, or GSEA. Network and pathway-cluster analyses then tied the individual enriched pathways together into coherent biological stories.

The headline finding is that all three flavonoids push leukemia cells in a broadly similar direction—toward growth suppression, a metabolic downshift, and the activation of adaptive stress-response networks—but each compound leaves its own distinctive fingerprint on the transcriptome. Butein emerged as the most transcriptionally aggressive of the three, inducing 809 differentially expressed genes. Butin followed with 439 differentially expressed genes, and sulfuretin produced 299. Despite these differences in breadth, the team identified 134 differentially expressed genes shared across all three treatments, suggesting a common core response to this family of flavonoids that may underlie their shared antileukemic activity.

Butin’s transcriptomic signature tells a story of systematic shutdown. Treatment with this compound produced a transcriptional shift marked by the suppression of RNA processing, mRNA splicing, translation initiation, and mitochondrial bioenergetics. In practical terms, the compound appears to choke off the leukemia cell’s ability to read out its genetic information, mature its messenger RNAs, and manufacture new proteins, while simultaneously starving the cell’s power plants. The enrichment analysis pointed specifically to downregulation of oxidative phosphorylation, the OXPHOS system that generates most of a cell’s ATP, and of the tricarboxylic acid cycle, or TCA cycle, which feeds that system. For rapidly dividing cancer cells, which depend heavily on coordinated protein synthesis and mitochondrial metabolism, this dual hit represents a serious metabolic and biosynthetic crisis.

Butein, the most potent transcriptional perturber, induced a strikingly coordinated downregulation of many of the same programs. Mitochondrial respiration, RNA maturation, mRNA maturation, and mRNA transport were all suppressed after butein treatment, indicating that the compound disrupts both the energy supply and the information-processing pipeline of the leukemic cell. The overlap between butin and butein is notable from a chemical biology standpoint: both compounds share a core flavonoid scaffold, and their convergent effects on mitochondrial and RNA-related pathways suggest that these processes may be particularly vulnerable targets for this class of molecules. At the same time, the fact that butein altered nearly twice as many genes as butin hints at additional, compound-specific activities that remain to be fully characterized.

Sulfuretin stood apart. Instead of the broad biosynthetic and metabolic shutdown seen with its two chemical cousins, sulfuretin displayed a distinct pattern characterized by positive enrichment of stress, inflammatory, immune, and proteasome-related pathways. In other words, treated cells mounted what looks like an adaptive stress response, ramping up programs associated with inflammation and protein quality control. Alongside this activation, the analysis revealed negative enrichment of RB1/E2F-associated G1/S transition programs—a reference to the molecular circuitry that governs the transition from the G1 phase of the cell cycle into DNA synthesis. Suppressing this program suggests that sulfuretin may hold leukemia cells at the G1 checkpoint, preventing them from committing to another round of cell division.

These findings matter because they move the field beyond the simple observation that a plant compound kills cancer cells in a dish. By resolving the transcriptomic architecture of the response, the study offers mechanistic hypotheses that can be tested directly: whether the compounds induce reactive oxygen species through mitochondrial dysfunction, whether the proteasome activation seen with sulfuretin reflects a genuine unfolded protein burden, and whether the shared 134-gene core response contains biomarkers that predict sensitivity to flavonoid treatment. The authors also emphasize that the framework they have built is not limited to leukemia. Because the transcriptomic signatures point to fundamental processes—protein synthesis, mitochondrial respiration, cell cycle control—their approach can be translated to other types of malignancy, where the same compounds might exploit different vulnerabilities depending on the tissue of origin.

The study also illustrates the power of pathway-level analysis over simple gene lists. Differential expression analysis alone can identify thousands of changed transcripts, but GSEA against curated databases such as Reactome and KEGG, combined with network and pathway-cluster analyses, revealed that the changed genes were not scattered at random. Instead, they converged on coherent biological modules—RNA processing, translation initiation, OXPHOS, TCA cycle, G1/S transition, proteasome function—that together explain how a flavonoid treatment can translate into cytotoxicity. The team’s careful handling of technical variation, documented in supplementary analyses showing sample clustering before and after batch correction, adds confidence that the observed signatures reflect biology rather than experimental noise.

For now, the work remains at the level of cell culture, and the usual caveats apply: concentrations that are cytotoxic in a dish do not automatically translate into safe and effective drugs, and the pharmacokinetics of flavonoids in the human body are notoriously complex. But the study delivers something that earlier cytotoxicity screens could not—a transcriptomic framework for further investigation of the antileukemic properties of butin, butein, and sulfuretin. As interest in natural products as sources of anticancer lead compounds continues to grow, the smoke tree’s heartwood chemistry has just become considerably more interesting, and the road from Balkan shrub to molecular medicine has gained a well-marked first milestone.

Subject of Research: Transcriptomic effects of Cotinus coggygria flavonoids on K562 myeloid leukemia cells

Article Title: Transcriptome analysis of K562 myeloid leukemia cells treated with butin, butein, and sulfuretin from the heartwood of Cotinus coggygria Scop.

Article References: Transcriptome analysis of K562 myeloid leukemia cells treated with butin, butein, and sulfuretin from the heartwood of Cotinus coggygria Scop.. (n.d.). https://doi.org/10.1186/s12906-026-05570-5

Image Credits: AI Generated

DOI: 10.1186/s12906-026-05570-5

Keywords: Cotinus coggygria, flavonoids, butin, butein, sulfuretin, K562 leukemia cells, transcriptome analysis, RNA sequencing, gene set enrichment analysis, apoptosis, mitochondrial respiration, natural products

A research team led by Ruifeng Guo and Yonglin Wang of Beijing Forestry University, together with colleagues at the Beijing Academy of Agriculture and Forestry Sciences and the United States Department of Agriculture, set out to map the microbial communities living inside smoke tree branches. The researchers sampled twelve trees, six healthy and six diseased, separating each branch into two distinct compartments: the outer epidermis, or bark, and the inner xylem, the vascular tissue that transports water. From each tree they collected branches in three health states: branches from completely healthy trees, symptomless branches from diseased trees, and the visibly diseased branches themselves. Using amplicon sequencing of bacterial 16S rRNA and fungal ITS regions, they generated a detailed census of the endophytic microbiota inhabiting each tissue type.

The sequencing data revealed that Verticillium wilt profoundly reshapes the internal microbial world of the tree. Bacterial communities in diseased xylem actually showed higher Shannon diversity than in healthy xylem, suggesting that infected trees may recruit additional microorganisms as a defensive response. Fungal communities told the opposite story: diversity dropped significantly in the epidermis and xylem of diseased branches. Principal coordinate analysis and permutational multivariate analysis of variance confirmed that disease status significantly altered community composition in nearly every compartment. Critically, when the researchers re-ran their analyses after excluding V. dahliae sequences, the pattern held, meaning the pathogen’s influence on the fungal community extended well beyond its own abundance. Quantitatively, disease explained roughly 40 percent of the variation in fungal communities but only about 11 percent in bacterial communities, indicating that fungi are far more sensitive to the wilt epidemic than bacteria.

To probe the stability of these communities, the team constructed co-occurrence networks linking microbial taxa based on correlated abundances. The fungal networks in diseased branches had fewer connections, fewer negative correlations, and markedly lower robustness when nodes were removed in simulations of species extinction. Both diseased and symptomless branches from infected trees showed elevated vulnerability, meaning the loss of a single taxon could disproportionately collapse network efficiency. Fungal networks proved consistently more fragile than bacterial ones, echoing earlier findings in pepper stems and olive roots where disease also destabilized fungal assemblages. This loss of connectivity, a form of microbial dysbiosis, suggested that wilt disease does not merely add a pathogen to an otherwise intact community; it actively dismantles the ecological architecture that keeps endophytes in check.

Within this destabilized landscape, one genus stood out. Differential abundance analysis and random forest classification both identified Botryosphaeria as a top biomarker of diseased branches, enriched alongside Verticillium itself. Linear correlation analysis showed that the relative abundance of Verticillium was positively and significantly associated with Botryosphaeria, with an R-squared of 0.47, while it was negatively correlated with Alternaria and Cladosporium. The team isolated a strain of Botryosphaeria dothidea from infected branches and found that it showed no antagonistic activity against V. dahliae in culture, ruling out simple competition. Spatially, the two fungi occupied different niches: B. dothidea concentrated in the bark while V. dahliae dominated the xylem. Field collections from four Beijing districts reinforced the association, with both fungi recovered together from 37 percent of 76 diseased branch samples and V. dahliae alone from 63 percent.

The decisive test came in the greenhouse. When smoke tree seedlings were inoculated with B. dothidea alone, nothing happened; the trees stayed healthy. When they were inoculated with V. dahliae alone, characteristic wilt symptoms appeared. But when seedlings received V. dahliae first and B. dothidea second, wilting was dramatically more severe than with either pathogen alone. Quantitative PCR revealed the mechanism behind this synergy: V. dahliae biomass was unchanged by the presence of its partner, but B. dothidea biomass surged in co-inoculated plants. In other words, the vascular pathogen was not simply teaming up with an equal partner; it was unlocking the door for a latent endophyte to multiply and inflict damage it could never achieve on its own.

Physiological and histochemical assays traced how the trees lost their ability to resist the second invader. Catalase and peroxidase, the antioxidant enzymes that scavenge reactive oxygen species during immune responses, were most active in plants challenged with V. dahliae alone and lowest in co-inoculated plants. Malondialdehyde content and relative conductivity, both markers of cell membrane damage, peaked in co-inoculated seedlings. DAB staining showed that V. dahliae infection drove substantial hydrogen peroxide accumulation, while trypan blue staining revealed extensive cell death, and microscopy documented solid blockage of xylem vessels. Together these results indicated that the first infection had already crippled the tree’s defense system before the second fungus even arrived.

Transcriptome sequencing provided the molecular explanation. In V. dahliae-infected branches, 2,800 predicted genes were significantly downregulated and 1,871 were upregulated compared with controls. Among the downregulated set, 394 genes in a single expression cluster were tied to plant defense, including disease resistance genes, protein kinases, jasmonic acid-responsive factors, chitinase-binding proteins, terpenoid synthases, cellulose synthases, protease inhibitors, redox regulators, and transcription factors. KEGG enrichment showed that hormone signal transduction, phenylpropanoid biosynthesis, MAPK signaling, brassinosteroid and monoterpenoid biosynthesis, and benzoxazinoid production were all significantly suppressed. Gene ontology analysis likewise flagged reduced transmembrane receptor kinase activity and compromised cell wall and cytoskeleton components. B. dothidea-infected branches showed a strikingly similar transcriptional signature, with the same defense and hormone pathways downregulated, confirming that both fungi exploit overlapping vulnerabilities in the host.

The study reframes B. dothidea, long recognized as a worldwide latent pathogen of woody plants that waits for drought, wounds, or stress before striking, as an opportunistic accomplice whose pathogenicity is triggered by another microbe rather than by abiotic stress alone. It also adds smoke tree wilt to a growing list of plant diseases, from grafted grapevine decline to soybean root rot, in which co-infection produces symptoms far worse than the sum of the parts. For forest managers, the practical implications are significant: because the two fungi occupy different tissues, control strategies may need to pair fungicides with measures that boost overall host vigor, keeping the latent bark dweller in its harmless endophytic state. The researchers are already testing plant immune activators in smoke tree plots to see whether shoring up the tree’s own defenses can break the deadly synergy, offering hope that the red hills of Beijing can be protected not by attacking one pathogen, but by managing the entire microbial community within the tree.

Subject of Research: Synergistic co-infection of smoke trees by Verticillium dahliae and the latent bark fungus Botryosphaeria dothidea

Article Title: The bark latent fungus Botryosphaeria dothidea exacerbates branch dieback following the infection with Verticillium dahliae

Article References: Guo, R., Li, Y., Tang, C., Zhao, Y., Wang, M., Qiao, G., Klosterman, S. J., & Wang, Y. (2026). The bark latent fungus Botryosphaeria dothidea exacerbates branch dieback following the infection with Verticillium dahliae. Stress Biology, 6(1), Article 13. https://doi.org/10.1007/s44154-026-00288-3

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00288-3

Keywords: Verticillium dahliae, Botryosphaeria dothidea, smoke tree, Cotinus coggygria, Verticillium wilt, plant microbiome, endophytes, co-infection, branch dieback, xylem, RNA-seq, forest pathology