Functional characterization of BbCHI3 in regulating flavonoid biosynthesis and abiotic stress tolerance in Blumea balsamifera.

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Learn more: PMC Disclaimer | PMC Copyright Notice BMC Plant Biol . 2026 Apr 20;26:942. doi: 10.1186/s12870-026-08721-5 Functional characterization of BbCHI3 in regulating flavonoid biosynthesis and abiotic stress tolerance in Blumea balsamifera Shan Sha 1, # , Ju Li 1, # , Kailang Mu 1 , Changmao Guo 1 , Shi Yao 1 , Qiumei Luo 1 , Pingxuan Xie 1 , Changliu Shao 1 , Zhengwei Zhang 1 , Wei Sun 3 , Yuan Yuan 2, ✉ , Zhigang Ju 1, ✉ , Yuxin Pang 1, ✉ Author information Article notes Copyright and License information 1 Pharmacy School, Guizhou University of Traditional Chinese Medicine, Guiyang, 550025 China 2 School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006 China 3 College of Life Sciences, Guizhou Normal University, Guiyang, 550025 China ✉ Corresponding author. # Contributed equally. Received 2026 Feb 7; Accepted 2026 Apr 6; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13224582  PMID: 42010492 Abstract Blumea balsamifera is a renowned medicinal plant in the Miao ethnic tradition, with flavonoids constituting its primary pharmacologically active components. However, the functional mechanisms of the key enzymes involved in their biosynthesis remain poorly understood. In this study, we identified the chalcone isomerase (CHI) gene family from the B. balsamifera transcriptome and selected a pivotal member, BbCHI3 , for in-depth functional investigation. Phylogenetic and sequence analyses indicated that BbCHI3 belongs to the type I CHI subfamily and possesses conserved catalytic active sites. Expression pattern analysis revealed that BbCHI3 is predominantly expressed in flowers and leaf bud of B. balsamifera , and its transcription is strongly induced by various stress treatments, including high salinity (HS), polyethylene glycol (PEG), abscisic acid (ABA), methyl jasmonate (MeJA), and herbivory by Spodoptera litura (SL), suggesting a crucial role in plant stress and defense responses and secondary metabolite accumulation. Subcellular localization results demonstrated that the BbCHI3 protein is localized in the cytoplasm. In vitro enzyme activity assays confirmed that recombinant BbCHI3 protein participates in the biosynthesis of various flavonoid compounds in B. balsamifera , including anthocyanidins, flavones, and flavonols. Furthermore, heterologous complementation experiments revealed that overexpression of BbCHI3 successfully restored anthocyanin accumulation in the cotyledons and seed coats of the Arabidopsis thaliana chi loss-of-function mutant ( tt5 ). Additionally, heterologous expression and functional assays in Escherichia coli BL21(DE3) demonstrated that BbCHI3 confers enhanced tolerance to extremely high salt stress. This study systematically elucidates the core function of BbCHI3 in the flavonoid biosynthesis pathway of B. balsamifera , providing a key genetic resource for deciphering the molecular basis of its Daodi (geo-authentic) quality and for the targeted breeding of superior varieties through metabolic engineering. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-026-08721-5. Keywords: Blumea balsamifera L, Chalcone isomerase, Flavonoid biosynthesis, Abiotic stress, Functional complementation, Blumeatin Introduction Blumea balsamifera (L.) DC., a perennial woody herb of the Asteraceae family, is widely distributed in tropical and subtropical regions. It is a characteristic medicinal plant used in the traditional medicine of the Miao, Li, and Zhuang ethnic groups, renowned for its significant therapeutic effects in dispelling wind and dampness, warming the middle jiao to relieve diarrhea, and promoting blood circulation to detoxify the body [ 1 – 3 ]. B. balsamifera is not only the sole botanical source for extracting natural borneol (L-borneol), aina powder (a traditional preparation), and B. balsamifera oil, but its unique flavonoid constituents, represented by blumeatin, have also garnered considerable attention due to their excellent anti-inflammatory, antioxidant, and hepatoprotective activities [ 4 – 7 ]. Currently, numerous clinical formulations have been developed using B. balsamifera as a raw material, such as Yanlishang Dropping Pills, Xinweitong Capsules, and Jin Houjian Spray. Additionally, it holds substantial economic and social value in the fragrance, essence, and cosmetics industries [ 8 , 9 ]. In recent years, due to industrial exploitation, habitat degradation, and over-harvesting, the wild resources of B. balsamifera have become increasingly depleted [ 10 ]. Therefore, elucidating the biosynthetic mechanisms of its core bioactive compounds and employing molecular biology approaches to directionally enhance their content constitute critical scientific objectives for addressing resource shortages and improving the quality of this medicinal herb. Although flavonoids constitute the primary pharmacologically active basis of B. balsamifera and are involved in physiological processes such as plant stress responses [ 11 , 12 ], the elucidation of their biosynthetic pathways currently relies predominantly on predictions from transcriptomic data. Consequently, the specific key enzyme genes and their regulatory mechanisms remain poorly characterized. Chalcone isomerase is the second rate-limiting enzyme in the flavonoid biosynthesis pathway and the first key enzyme identified. Its primary function is to catalyze the stereospecific isomerization of chalcone into a flavanone (e.g., naringenin) [ 13 , 14 ]. During plant evolution, the CHI gene family has undergone significant functional divergence, primarily into four classes (Type I-IV) [ 15 ]. Type I CHIs are ubiquitous in vascular plants and are responsible for constructing the flavonoid skeleton, whereas Type II CHIs are unique to leguminous plants and participate in isoflavone biosynthesis [ 16 , 17 ]. In contrast, Type III and Type IV CHIs are non-catalytic members with diverse functions [ 18 , 19 ]. The expression and function of CHI genes are regulated by various factors, including plant hormones [ 20 ]. For example, drought stress activates the flavonoid synthesis pathway, leading to a significant the upregulation of CHI in Glycyrrhiza uralensis [ 21 ], and ABA treatment induces upregulation of CHI-1 genes in banana [ 22 ]. Although the CHI gene family has been systematically identified in various plants such as Arabidopsis thaliana [ 23 ], Oryza sativa [ 24 ], Zea mays [ 25 ], Gossypium hirsutum [ 26 ], and Astragalus membranaceus [ 27 ], its identification and characterization in the Asteraceae species B. balsamifera remain unreported. Nevertheless, CHI is ubiquitous in B. balsamifera and is highly conserved, consistent with reports in other higher plants based on the KEGG database [ 28 ]. In B. balsamifera , flavonoid biosynthesis is primarily associated with the ko00941 pathway (Fig. 1 ). Within this pathway, CHI catalyzes the intramolecular cyclization of various chalcones (e.g., pinocembrin chalcone, isoliquiritigenin, butein, naringenin chalcone, and homoeriodictyol chalcone) to form flavanones, thereby providing essential substrates for the biosynthesis of diverse flavonoids, including anthocyanins. However, it remains unknown which specific CHI isoform plays the dominant role in the biosynthesis of flavonoids—particularly the precursors of blumeatin—and how its expression and activity are regulated by environmental factors to influence the quality of this medicinal herb. Fig. 1. Open in a new tab Flavonoid metabolic pathway in B. balsamifera Therefore, this study first performed a systematic identification and evolutionary analysis of the CHI gene family in B. balsamifera based on transcriptomic data and integrated bioinformatics. Subsequently, a pivotal member, BbCHI3 , was selected for in-depth functional characterization. Its spatiotemporal expression patterns and responses to various stimuli—including abiotic stresses (salt, drought, ABA), the defense-related hormone MeJA, and insect herbivory—were profiled using RT-qPCR. The biological function of BbCHI3 was further validated through a series of experiments: heterologous expression in a prokaryotic system followed by in vitro enzyme activity assays, determination of its subcellular localization, and heterologous complementation in the (A) thaliana tt5 mutant. Moreover, heterologous expression in E. coli demonstrated that BbCHI3 confers tolerance to extreme salt stress. Collectively, our findings not only refine the flavonoid biosynthetic pathway in (B) balsamifera but also provide a key genetic resource for deciphering how environmental factors shape the quality of Daodi medicinal herbs and for facilitating molecular breeding. Materials and methods Plant materials B. balsamifera experimental materials used in this study were originally collected from the banks of the Hongshui River in Luodian County, Guizhou Province, China (25°14′35″N, 106°31′23″E). The species was identified by Associate Professor Ju Zhigang of Guizhou University of Traditional Chinese Medicine as Blumea balsamifera (L.) DC., family Asteraceae, following the taxonomic criteria of Flora Reipublicae Popularis Sinicae. The identified plants were subsequently transplanted to and maintained in a glass greenhouse at the Medicinal Plant Cultivation Experimental Base, Huaxi Campus, Guizhou University of Traditional Chinese Medicine. For expression analysis in different tissues and organs, healthy, pest- and disease-free roots, stems, leaves, and flowers of B. balsamifera (L.) DC. (family Asteraceae) were collected from mature plants older than two years. For expression analysis across different leaf developmental stages, leaf samples at the leaf bud (I), young leaf (II), expanding leaf (III), mature leaf (IV), and senescent leaf (V) stages were obtained from the same cohort of mature plants (over two years old). For abscisic acid (ABA) and polyethylene glycol (PEG) treatments, detached leaves from four-month-old in vitro plantlets were incubated in ABA and PEG solutions for 6, 12, 24, and 48 h, respectively. For salt stress and methyl jasmonate (MeJA) treatments, young leaves of B. balsamifera at the eight- to twelve-leaf stage were used, and Spodoptera litura larvae were reared to the third instar for herbivory assays. All materials were immediately flash-frozen in liquid nitrogen upon collection or after stress treatment and stored at -80 °C for use within one week. A. thaliana ecotype Columbia-0 was used as a heterologous system. Seeds were germinated, and plants were grown in a controlled environment chamber at 22 °C with a 16-hour light/8-hour dark photoperiod. Leaves from 3- to 4-week-old plants were harvested for protoplast isolation and subsequent transient transfection assays to determine protein subcellular localization. Gene identification and cloning Candidate CHI genes were identified from a de novo assembled transcriptome of B. balsamifera . A local BLAST search was performed using (A) thaliana CHI protein sequences as queries (E-value < 1e − ⁵). Concurrently, a hidden Markov model (HMM) search was conducted using the Pfam profile for chalcone isomerase (PF02431). The resulting candidate sequences were subjected to conserved domain analysis using NCBI-CDD to confirm the presence of characteristic CHI domains. Sequences lacking these domains were excluded, yielding a final set of BbCHI genes. Gene-specific primers were designed to amplify the full-length coding sequences. These sequences were amplified from (B) balsamifera leaf cDNA by PCR and subsequently cloned into the target vector using a seamless assembly method (e.g., Gibson Assembly). Bioinformatics analysis of the BbCHI gene family The physicochemical properties of the identified BbCHI proteins, including amino acid number, molecular weight, and isoelectric point, were analyzed using TBtools. Their subcellular localization was predicted using WoLF PSORT II, ChloroP, and Plant-mPLoc. Post-translational modification sites (phosphorylation and N-glycosylation) were predicted with NetPhos 3.1 Server and NetNGlyc, respectively. Secondary structures were predicted using SOPMA, and the stereochemical quality of the protein models was assessed using the SAVES server. A maximum likelihood phylogenetic tree was constructed from aligned protein sequences using the TBtools “One Step Build a ML Tree” function with 5,000 bootstrap replicates and was subsequently annotated with Adobe Illustrator. Multiple sequence alignment of BbCHI proteins was performed with Clustal W2 ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). Conserved motifs were identified using the MEME suite ( http://meme-suite.org/tools/meme ), (parameters: number of motifs = 4). Domain architecture and motifs were visualized with TBtools. For molecular docking, homology models of the BbCHI proteins were built using SWISS-MODEL. The 3D conformers of four chalcone substrates structures (pinocembrin chalcone, naringenin chalcone, homoeriodictyol chalcone, and eriodictyol chalcone) were retrieved from PubChem. Protein structures were prepared for docking (e.g., removal of water, addition of charges) using PyMOL. Docking simulations were conducted with AutoDock Vina, and the resulting binding poses were visualized in PyMOL. Codon usage bias analysis of the BbCHI gene family Codon usage patterns of the BbCHI gene family were analyzed using CodonW software and the online CUSP program. The analysis key parameters were calculated, including: overall nucleotide compositions, the effective number of codons (ENC), GC content at the three codon positions (GC1, GC2, and GC3s), base frequencies at the synonymous third position (A3, T3, G3, C3), and relative synonymous codon usage (RSCU). Visualization included ENC-plot, parity rule 2 (PR2) bias plot, and neutrality plot, generated using Origin 2024. Statistical correlations among these indices were assessed with SPSSv. 21.0. Codon usage data for the BbCHI genes were extracted via the EMBOSS Explorer ( http://www.bioinformatics.nl/emboss-explorer/ ). For comparative purposes, genome-wide codon usage tables for five model organisms ( http://www.kazusa.or.jp/codon/ ): E. coli , Saccharomyces cerevisiae , A. thaliana , Nicotiana tabacum , and O. sativa : were obtained from the Codon Usage Database. These datasets enabled a direct comparison of codon usage bias between the BbCHI genes and the reference genomes. Gene expression analysis To identify flavonoid biosynthesis genes and analyze their expression, transcriptome sequencing was performed on B. balsamifera leaves at different developmental stages. Expression levels of BbCHI genes across these stages are presented as log₂(FPKM + 1) values. Their expression patterns were further examined by RT-qPCR in various tissues, at different leaf stages, and under multiple stress conditions. For stress treatments, healthy, uniform leaves were used. Salt stress was applied by treating leaves with 150 or 200 mmol·L⁻¹ NaCl solution for 12 days; untreated leaves served as controls. For biotic stress assays, leaves sprayed with deionized water for 72 h served as the control group. The Treatment included: (1) spraying with 1.00 mmol·L⁻¹ MeJA for 72 h; (2) spraying with deionized water for 66 h followed by a 6 h feeding period with second-instar SL larvae; and (3) spraying with 1.00 mmol·L⁻¹ MeJA for 66 h prior to 6 h of larval feeding. For hormone and drought stress, leaves were treated with 0.30 mmol·L⁻¹ ABA or 30% (w/v) PEG-6000 solution for 6, 12, 24, and 48 h, with untreated leaves harvested at the corresponding time points as controls. Three independent biological replicates were performed. Relative gene expression was calculated using the 2⁻ ΔΔCt method. All data are presented as mean ± standard error (SE). Responses to methyl jasmonate (MeJA) and Spodoptera litura herbivory were analyzed using independent samples t-tests conducted with SPSS version 21.0 software ( P < 0.05). All other data were assessed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, with statistical significance set at P < 0.05, and data were visualized as bar graphs with Origin 2024 or GraphPad Prism. Cis-regulatory elements in the 2,000 bp promoter regions upstream of the BbCHI3 gene were predicted using PlantCARE, and their distribution was visualized as a heatmap using TBtools. Recombinant BbCHI3 protein: in vitro enzyme assay and salt stress response in E. coli The BbCHI3 coding sequence was cloned into the pET32a vector using BamHI/EcoRI sites and transformed into E. coli . Optimal conditions (temperature, time, IPTG concentration) for expressing the recombinant His-tagged BbCHI3 were determined. Large-scale cultures were induced under these conditions. Cells were harvested by centrifugation, resuspended in PBS, and lysed by sonication. The protein was purified from the lysate by immobilized metal affinity chromatography (IMAC) using a stepwise imidazole gradient. Fractions containing BbCHI3 were assayed by SDS-PAGE, pooled, and concentrated. Enzymatic activity of the purified BbCHI3 was determined by incubation the enzyme with chalcone substrates (pinocembrin chalcone, naringenin chalcone, homoeriodictyol chalcone, and eriodictyol chalcone). Reaction products were separated and quantified by high-performance liquid chromatography (HPLC) [ 29 ]. Single colonies of the control (BL21-pET32a) and BbCHI3 -expressing (BL21-pET32a- BbCHI3 ) strains were grown in LB-ampicillin to mid-log phase (OD600 ~ 0.5), induced with optimal IPTG, and grown to an OD 600 of ~ 1.2. Cultures were serially diluted (10⁰ to 10 − ⁵), and 7 µL aliquots were plated on LB-ampicillin agar without NaCl (control) and with 150 or 200 mmol·L − ¹ NaCl. Plates were incubated overnight at 37 °C. Control and BbCHI3 -expressing strains were inoculated into LB-ampicillin liquid medium containing 200 mmol·L − ¹ NaCl. Growth (OD 600 ) was monitored hourly for 12 h, with three biological replicates per strain. In planta functional analysis of BbCHI3 via heterologous complementation For functional complementation, the BbCHI3 coding sequence was inserted into the XbaI/and BamHI sites of the pBI121 binary vector. The recombinant plasmid was transformed into Agrobacterium tumefaciens strain GV3101. Tran sformation of the A. thaliana chi mutant ( tt5 ) was performed using the floral dip method. Transgenic T1 plants were selected on appropriate antibiotics selection, and genomic DNA was extracted from putative transgenic plants using the CTAB method for PCR confirmation. Phenotypic analysis, including pigment restoration in seedlings and seeds) was conducted using a stereomicroscope. For metabolic profiling, seedlings of wild-type (WT), chi mutant, and BbCHI3 T2 transgenic lines were ground to a fine powder in liquid nitrogen. Flavonoids were extracted with acidified methanol (methanol: concentrated HCI, 99:1, v/v) at 4 °C for 16 h. Extracts were clarified by centrifugation, and the supernatants were analyzed by high-performance liquid chromatography (HPLC) [ 29 ]. Quantification of flavonoids was performed using an external standard curve generated using pinocembrin standards. Subcellular localization of BbCHI3 To generate a C-terminal GFP fusion protein, the BbCHI3 coding sequence (without the stop codon) was amplified and inserted into the NdeI and ClaI sites of a pUC19-based GFP expression vector (pUC19-HA-GFP). The construct was transformed into Escherichia coli DH5α, and positive clones were verified by Sanger sequencing. Transfection-grade plasmid DNA was prepared using the EndoFree Maxi Plasmid Kit. The verified pUC19- BbCHI3 -GFP plasmid was co-transfected with a chloroplast-localized RFP marker into isolated Arabidopsis thaliana mesophyll protoplasts using a PEG-mediated transfection. Protoplasts were incubated in the dark at 22 °C for 18–22 h before imaging. Imaging was performed on a laser scanning confocal microscope [ 30 ]. GFP was excited at 488 nm, and RFP at 546 nm. Co-localization of the GFP and RFP signals was indicated as chloroplast targeting, while distinct signals suggested cytoplasmic localization. Results Analysis of physicochemical properties and structural characteristics of gene family proteins Based on the transcriptomic data of B. balsamifera, four members of the CHI gene family were identified via BLAST alignment and conserved PFAM domain screening. These genes were designated BbCHI1 through BbCHI4 according to their transcriptome IDs. Analysis of the predicted BbCHI proteins (Table 1 ) revealed the following physicochemical characteristics: they consist of 211 to 405 amino acids; their predicted molecular masses range from 23.63 to 45.67 kDa; the average isoelectric point (pI) is 6.95; the number of positively charged residues (Arg + Lys) ranges from 21 to 51, while the number of negatively charged residues (Asp + Glu) ranges from 26 to 44; all have negative grand average of hydropathy (GRAVY) scores, classifying them as hydrophilic proteins. Subcellular localization predictions suggested a primary localization to the chloroplast, cytoskeleton, and nucleus. Analyses using SignalP 4.1 and TMHMM Server version 2.0 indicated that all BbCHI proteins lack a signal peptide (Supplementary Fig. S1A) and transmembrane domains (Supplementary Fig. S1B). Phosphorylation sites in the BbCHI proteins (BbCHI1-4) were predicted using the NetPhos 3.1 Server (Supplementary Fig. S1C). The predicted number of phosphorylation sites ranged from 23 to 42, with serine residues being the most frequent target. Analysis via the NetNGlyc website predicted potential N-glycosylation sites in BbCHI2 and BbCHI4 (Supplementary Fig. S1D), while BbCHI1 and BbCHI3 lacked predicted N-glycosylation sites. Secondary structure prediction using SOPMA (Supplementary Table S1) indicated that the BbCHI proteins are primarily composed of α-helices, extended strands, random coils, and β-sheets. Among these, α-helices were the predominant element, contributing to the stability of the protein backbone; catalytic active sites are frequently situated within such helical regions. Furthermore, conformational analysis performed with the SAVES server (Supplementary Fig. S1E) revealed that the φ and ψ dihedral angles of amino acid residues predominantly occupy the allowed regions of the Ramachandran plot, supporting the structural validity of the models for subsequent experimental investigations. Table 1. Physicochemical properties of the CHI family proteins from B. balsamifera Protein Naa (aa) MW (kDa) PI Arg + Lys Asp + Glu Overall average hydrophilicity GRAVY Subcellular localization BbCHI1 211 23.63 4.7 21 34 -0.12 88.25 Cytoskeleton BbCHI2 287 31.04 9.33 34 26 -0.075 93.8 Chloroplast BbCHI3 226 24.51 4.97 22 27 -0.029 85.93 Cytoskeleton BbCHI4 405 45.67 8.79 51 44 -0.317 51.82 Nucleus Open in a new tab Multidimensional phylogenetic and structural variation analysis of the CHI Gene Family in B. balsamifera To elucidate the evolutionary relationships of the CHI gene family, a phylogenetic tree was constructed using 25 CHI protein sequences from B. balsamifera and other representative species. The analysis classified the CHIs into four distinct subfamilies (Fig. 2 A), with Type II enzymes forming the largest clade. This phylogenetic framework provides a basis for inferring functional divergence. Among the BbCHIs, BbCHI1, BbCHI2, and BbCHI4 belong to Type II, whereas BbCHI3 clustered with Type I chalcone isomerases, including that from A. thaliana . To assess conserved functional and structural features, a multiple sequence alignment was performed comparing the amino acid sequences of BbCHIs with orthologs from A. thaliana , O. sativa , Z. mays , and Medicago truncatula (Fig. 2 B). Notably, BbCHI3 contains conserved active-site residues at seven positions: R42, G43, L44, F53, T54, I56, L107, Y112, K115, V116, and G119 (or other residue at position 119; please verify), which are involved in (2S)-naringenin formation. However, a substitution was identified within the conserved Type I active-site motif; specifically, the canonical isoleucine (I) residue is replaced by methionine (M). In contrast, BbCHI1 shares only three of these conserved residues (F53, T54, I56, and Y112), and its overall active-site architecture shows greater similarity to that of AtCHIL. By comparison, BbCHI2 and BbCHI4 lack conservation at all these key residue positions. Analysis of conserved domains indicated that all BbCHI proteins belong to the chalcone isomerase superfamily (Fig. 2 C). Furthermore, MEME analysis identified four distinct conserved motifs (Motifs 1–4) within these proteins (Fig. 2 D). All BbCHI proteins contain Motif 1; however, BbCHI4 harbors all four motifs, whereas BbCHI1 contains only Motif 1. Fig. 2. Open in a new tab A Phylogenetic analysis of CHI proteins. The tree includes BbCHI proteins from B. balsamifera and CHI proteins from other plant species, constructed using TBtools with bootstrap support values from 5,000 replicates indicated as percentages at the nodes. BbCHIs identified in this study are marked by black pentagrams. Different CHI subfamilies (Type I-IV) are color-coded. Species abbreviations:Bb, B. balsamifera ; At, Arabidopsis thaliana ; Os, Oryza sativa ; Nt, Nicotiana tabacum ; Zm, Zea mays ; Pt, Populus trichocarpa ; Gm, Glycine max ; Ch, Gossypium hirsutum ; Vv, Vitis vinifera ; Pv, Phaseolus vulgaris ; Ps, Paeonia suffruticosa ; Le, Solanum lycopersicum ; Li, Litchi chinensis ; Lj, Lotus japonicus . B Multiple sequence alignment of CHI proteins highlighting conserved residues. Colors denote specific functional features: purple, residues involved in (2S)-naringenin binding; green, active-site residues involved in hydrogen bonding; orange, residues associated with substrate preference for naringenin chalcone and isoliquiritigenin. Sequence conservation is indicated below the alignment: asterisk “*”, identical residues; colon “:”, conserved substitutions; period “.”, semi-conserved substitutions. C Schematic representation of conserved protein domains. D Schematic representation of conserved protein motifs, with Motifs 1–4 shown in distinct colors Codon usage bias in the BbCHI gene family of B. balsamifera and implications for heterologous expression ENC-plot analysis (Fig. 3 A) showed that one BbCHI gene lay near the expected curve, while the others were distributed below it. The overall distribution trend paralleled the standard curve, suggesting that the codon usage of most BbCHI genes is primarily shaped by mutational bias, with limited influence from natural selection. PR2 plot analysis (Fig. 3 B) revealed that nucleotide compositions clustered in the lower quadrants, indicating a slight bias towards pyrimidines (T, C) over purines (A, G) at the synonymous third codon position, which further support the dominant role of mutational bias. Neutrality plot analysis (Fig. 3 C) demonstrated that most points lay above the regression line. The slope of the regression between GC3 and GC12 was 0.902 ( R ² = 0.797), indicating a weak relationship, suggesting that nucleotide composition at the three codon positions is largely independent. Collectively, these results suggest that codon usage in the BbCHI gene family is primarily governed by mutational bias with minimal to minimal selective constraint. Fig. 3. Open in a new tab A ENC-plot analysis. NC, effective number of codons; GC3s, GC content at the third synonymous codon position. B PR2-plot analysis. A3s, T3s, G3s, and C3s, the contents of adenine, thymine, guanine, and cytosine, respectively, at the third synonymous codon position. The lines x = 0.5 and y = 0.5 serve as reference for assessing codon usage bias. C Neutrality plot analysis. GC3, GC content at the third synonymous codon position; GC12, mean GC content at the first and second codon positions. D Correlation analyses among codon usage bias indices. E Analysis of relative synonymous codon usage (RSCU). F Comparative analysis of codon usage bias between B. balsamifera and other species. Ec, Escherichia coli ; Nt, Nicotiana tabacum ; At, Arabidopsis thaliana ; Sc, Saccharomyces cerevisiae ; Os, Oryza sativa Correlation analyses among codon usage bias indices for the B. balsamifera CHI gene family (Fig. 3 D) revealed several key relationships. T3s showed a significant negative correlation with both GC3s and G3s, indicating that strong GC-biased mutation dominates third-position nucleotide composition, favoring T-to-C/G substitutions. C3s exhibited significant positive correlations with GC3s, overall GC content, and the frequency of optimal codons (Fop), indicating that GC-biased mutation elevates C content and that C-ending codons are favored (considered “optimal”). GC content showed significant positive correlations with both the codon bias index (CBI) and Fop, suggesting a potential link to translational efficiency. Furthermore, CBI and Fop showed a highly significant positive correlation, underscoring the link between codon bias and optimal codon usage. Analysis of relative synonymous codon usage (RSCU) values (Fig. 3 E) showed that BbCHI1, BbCHI2, BbCHI3, and BbCHI4 contained 25, 29, 22, and 27 codons with RSCU > 1, respectively, indicating high-frequency usage and a preference for A/U endings. In contrast, the numbers of codons with RSCU = 1.0 were 4, 6, 10, and 4, respectively, suggesting unbiased (neutral) usage for these codons. Due to the lack of an efficient genetic transformation system in B. balsamifera , functional studies of its genes typically depend on heterologous expression. To guide the selection of an appropriate expression host, we analyzed the relative codon adaptation between the BbCHI genes and five model organisms. Codons exhibiting significant adaptation differences (adaptation ratio of ≤ 0.5 or ≥ 2.0) were identified and counted (Fig. 3 F). The analysis revealed 14 and 8 codons with substantial divergence between BbCHI and the genomes of E. coli and Saccharomyces cerevisiae , respectively. This indicates that E. coli is a suitable prokaryotic expression host. If S. cerevisiae is to be employed as a eukaryotic expression host, codon optimization of the CHI gene tailored to the codon usage bias of yeast would be advisable. For plant hosts, 8, 6, and 21 codons showed significant divergence from A. thaliana , Nicotiana tabacum , and O. sativa , respectively. These results suggest that O. sativa is a preferred heterologous host for the functional expression of BbCHI genes. Analysis of tissue-specific expression patterns and responses to abiotic and biotic stresses RNA-seq was employed to profile the expression patterns of the BbCHI gene family across five distinct developmental stages of B. balsamifera leaves (morphological characteristics illustrated in Fig. 4 A). Transcriptomic analysis revealed that BbCHI1 , BbCHI2 , and BbCHI3 exhibited elevated transcript abundance at stage I, with moderate expression detected at stages III and IV (Fig. 4 B). In contrast, BbCHI4 displayed maximal expression at stage V and intermediate levels at stage II. Notably, BbCHI3 maintained relatively high expression throughout all examined leaf developmental stages, suggesting its involvement in the entire leaf ontogeny of B. balsamifera and implicating a predominant role in the biosynthesis of flavonoid metabolites.RT-qPCR was performed to assess BbCHI3 expression (Fig. 4 C), the results confirmed high expression at stage I, consistent with RNA-seq data; moderate expression was detected at stages II and V, whereas negligible expression was observed at stages III and IV, diverging from the transcriptional patterns revealed by RNA-seq analysis. Furthermore, RT-qPCR analysis across major organs (Fig. 4 D) revealed that BbCHI3 was detected in all tissues examined but exhibited differential expression, with transcript abundance was significantly highest in FL, lowest in Rt and intermediate in St and leaves, suggesting a role in flavonoid-mediated pigmentation. The transcriptional response of BbCHI3 to various stresses was also investigated. Under heat stress (HS) treatment, BbCHI3 expression was significantly upregulated (~ 4.3-fold) compared to the control (Fig. 4 E). In contrast, under extreme high-salt (EHS) treatment, its expression was downregulated and did not differ significantly from the control. MeJA treatment significantly induced BbCHI3 expression (1.8-fold, P < 0.001), whereas herbivory by SL resulted in a gradual suppression (Fig. 4 F). Notably, the MeJA + SL co-treatment triggered a dramatic upregulation (21.2-fold, P < 0.0001). Furthermore, treatment with 0.30 mmol·L⁻¹ ABA led to a significant downregulation of BbCHI3 expression (Fig. 4 G), suggesting that ABA negatively regulates its expression. Under PEG-simulated drought stress (Fig. 4 H), BbCHI3 expression showed a dynamic, biphasic response: an initial significant increase was followed by a decline and a subsequent rise to a second peak. To further investigate the potential biological functions of the BbCHI3 gene, cis‑regulatory elements within the 2000 bp upstream sequence were systematically identified using the PlantCARE database. A total of 29 cis‑regulatory elements were predicted (Fig. 4 I). These elements were primarily classified into three major categories: plant hormone response elements (e.g., ABA and MeJA‑responsive elements), plant growth and development (light‑responsive elements and core promoter elements), and stress responses (including elements related to abiotic stresses (e.g., drought) and biotic stress/defense responses). These findings suggest that the BbCHI3 gene may play regulatory roles in diverse biological processes and throughout plant development. Fig. 4. Open in a new tab A Morphological characteristics of B. balsamifera leaves at five distinct developmental stages. I-V represent the leaf bud, young leaf, tender leaf, mature leaf, and senescent leaf stages, respectively. B Transcriptomic profiles of the B. balsamifera CHI gene family across different leaf developmental stages. C Expression pattern of the BbCHI3 gene across different leaf developmental stages, analyzed by one-way ANOVA. D Tissue-specific expression pattern of the BbCHI3 gene, analyzed by one-way ANOVA. E Response of the BbCHI3 gene to salt stress, analyzed by one-way ANOVA. F Response of the BbCHI3 gene to MeJA and SL herbivory stress, analyzed using independent samples t-test. G Response of the BbCHI3 gene to ABA and drought stress, analyzed by one-way ANOVA. H Response of the BbCHI3 gene to drought stress simulated by 30 % PEG, analyzed by one-way ANOVA. Note: Different letters indicate significant differences ( P < 0.05) in gene expression compared to the control group; indicates significant ( P < 0.05) and indicates highly significant ( P < 0.01). RNA-seq data were normalized using log2 transformation, and RT-qPCR data were analyzed using SPSS software. I Analysis of cis-acting elements in the BbCHI3 promoters In vitro enzymatic characterization of BbCHI3 To characterize the catalytic function of the B. balsamifera chalcone isomerase, in vitro enzymatic assays were performed with the recombinant protein. First, molecular docking was used to predict the binding modes of BbCHI proteins to four key substrates and to identify potential catalytic residues (Fig. 5 ). The results indicated that all BbCHI proteins could form hydrogen bonds with these substrates. The calculated binding energies were negative (ranging from − 4.84 to -1.63 kcal/mol), indicating favorable binding interactions. Detailed binding site information is provided in Supplementary Table S2. The docking analysis suggested that the BbCHI proteins have higher affinity for pinocembrin chalcone and homoeriodictyol chalcone than for naringenin chalcone and eriodictyol chalcone. Based on these results and its high expression level, BbCHI3 was selected for further experimental analysis. The recombinant plasmid pET32a- BbCHI3 was expressed in E. coli . Optimal conditions for recombinant protein induction—temperature, induction time, and IPTG concentration—were determined. Maximum expression was achieved at 16 °C for 18 h with 0.8 mmol·L⁻¹ IPTG (Supplementary Fig. S2). Under these conditions, soluble recombinant BbCHI3 was successfully purified (Fig. 6 A). Eluates from 50 mmol·L⁻¹ and 80 mmol·L⁻¹ imidazole steps were pooled, dialyzed, and concentrated to yield a high-purity protein preparation (Fig. 6 B). Enzymatic reactions were then performed using the purified BbCHI3 protein and, as a control, the protein from the empty pET32a vector, with the four substrates. Reaction products were analyzed by HPLC. The in vitro activity assay (Fig. 6 C) demonstrated that BbCHI3, but not the control, catalyzed the complete conversion of naringenin chalcone to (2S)-naringenin, confirming its Type I chalcone isomerase activity. Furthermore, BbCHI3 also catalyzed the conversion of pinocembrin chalcone to (2S)-pinocembrin, as well as the conversion of homoeriodictyol chalcone and eriodictyol chalcone to homoeriodictyol and eriodictyol, respectively. These results indicate that BbCHI3 is a multifunctional enzyme. Fig. 5. Open in a new tab Molecular docking of BbCHI proteins to four chalcone substrates: pinocembrin chalcone, naringenin chalcone, homoeriodictyol chalcone, and eriodictyol chalcone. The left panel shows the overall docking pose, and the right panel provides a detailed view of the ligand-binding interactions. Supplementary Table S2 lists the corresponding binding energies and analyzes the key amino acid residues in the active site Fig. 6. Open in a new tab A Induction and purification of the BbCHI3 protein. M: Protein marker; Lanes 1-5: Eluate with 10 mM imidazole; Lanes 6-10: Eluate with 20 mM imidazole; Lanes 11-15: Eluate with 50 mM imidazole; Lanes 16-20: Eluate with 80 mM imidazole; Lanes 21-25: Eluate with 100 mM imidazole; Lanes 26-30: Eluate with 150 mM imidazole. B SDS-PAGE analysis of samples from the BbCHI3 purification and concentration process. M, protein ladder; lane 1, pET32a-BL21 (induced, +IPTG); lane 2, BbCHI3-pET32a-BL21 (uninduced); lane 3, total lysate of induced BbCHI3-pET32a-BL21; lane 4, concentrated recombinant BbCHI3 protein; lane 5, insoluble fraction (precipitate) from induced BbCHI3-pET32a-BL21. C HPLC chromatograms from in vitro enzyme assays. Reactions contained BbCHI3 or the pET32a empty vector control protein and one of four chalcone substrates: pinocembrin chalcone, naringenin chalcone, homoeriodictyol chalcone, or eriodictyol chalcone. Assays were performed in triplicate; representative chromatograms are shown. The x-axis indicates retention time (min), and the y-axis indicates relative abundance Functional validation of B. balsamifera chalcone isomerase by heterologous complementation in Arabidopsis For functional characterization in planta , A. thaliana was used as a heterologous host. The BbCHI3 gene was introduced into the Arabidopsis chi mutant ( tt5 ) via floral dip transformation. Positive transgenic lines were identified and characterized by stereomicroscopy. Compared to wild-type (WT) and chi mutant plants, BbCHI3 transgenic lines exhibited higher transcript levels of the transgene. Notably, these seedlings exhibited restoration of purple pigmentation in cotyledons and hypocotyls, contrasting with the green mutant phenotype (Fig. 7 A). Their seed coat color was also restored from yellow to a brown shade similar to that of WT (Fig. 7 B). PCR genotyping confirmed successful transformation. A control amplification with β-actin primers was successful in all lines, whereas BbCHI3 -specific primers yielded a product only in transgenic plants, not in WT or chi mutants (Fig. 7 C). For flavonoid quantification, a standard curve was established using pinocembrin as a representative standard (Supplementary Fig. S3). Concentrations were calculated based on the HPLC peak areas of detected flavonoids to this curve. Flavonoids, including eriodictyol (3’,5,5’,7-tetrahydroxyflavanone), naringenin, homoeriodictyol, and eriodictyol, were extracted from seedlings of WT, chi mutant, and T2 generation BbCHI3 transgenic Arabidopsis . HPLC analysis detected all four compounds across genotypes, but their concentrations were significantly higher in BbCHI3 transgenic plants compared to both WT and mutant plants (Fig. 7 D). These results demonstrate that BbCHI3 is functional in planta and support its role in the biosynthesis of anthocyanidins, blumeatin, anthocyanins, and flavonols in B. balsamifera. Fig. 7. Open in a new tab A Seedling phenotypes of wild-type (WT), chi mutant ( tt5 ), and BbCHI3 T2 transgenic Arabidopsis lines, showing complementation of purple pigmentation in cotyledons and hypocotyls. B Seed coat phenotypes of the corresponding lines, showing color restoration from yellow (mutant) to brown (WT-like). C DNA verification. WT: wild-type Arabidopsis thaliana ; tt5: CHI mutant Arabidopsis thaliana ; OX1, OX2, OX3, three independent homozygous BbCHI3 overexpression lines, respectively. D HPLC-based metabolic profiling of flavonoids in BbCHI3 transgenic seedlings. Quantified concentrations are provided in Supplementary Table S3 (experiments performed in triplicate). Representative chromatograms (line plots) are shown; blue lines indicate the signal intensity of detected compounds. The x-axis is retention time (min); the y-axis is relative abundance Subcellular localization of BbCHI3 The subcellular localization of BbCHI3 was first predicted using online tools. ChloroP predicted a putative chloroplast transit peptide, suggesting possible chloroplast targeting. However, other tools yielded conflicting predictions: WoLF PSORT suggested cytoskeletal localization, whereas Plant-mPLoc indicated plastid or plasma membrane localization. To resolve these discrepancies experimentally, a BbCHI3-GFP fusion construct was generated. The BbCHI3 coding sequence (without its stop codon) was fused in-frame to the 5’ end of the GFP coding sequence in the pUC19 vector under the control of the 35S promoter. This 35S::BbCHI3-GFP construct and a 35 S::GFP control vector were co-transfected with a free RFP marker into Arabidopsis protoplasts for transient expression. Subcellular Localization was analyzed by confocal laser scanning microscopy (Fig. 8 ). The GFP fluorescence from BbCHI3-GFP showed no overlap with the RFP signal, demonstrating cytoplasmic localization of BbCHI3. This cytoplasmic localization is consistent with reports for CHI proteins in other plants. For example, Arabidopsis CHI is a soluble cytoplasmic protein [ 31 ], and and can form dynamic metabolon complexes with other flavonoid enzymes in the cytoplasm to enhance catalytic efficiency [ 32 ]. Thus, the cytoplasmic localization of BbCHI3 provides direct cytological evidence supporting its role in flavonoid biosynthesis in B. balsamifera . Fig. 8. Open in a new tab Subcellular localization of BbCHI3. Bright field, bright-field image; RFP, red fluorescence (excitation at 546 nm), used as a cytoplasmic marker; BbCHI3-GFP, green fluorescence (excitation at 488 nm), showing localization of the BbCHI3-GFP fusion protein; Merge, pseudo-color overlay of the three channels. Scale bar, 25 μm Analysis of salt stress tolerance conferred by heterologous BbCHI3 expression in E. coli To investigate whether heterologous BbCHI3 expression confers salt stress tolerance, we compared the growth of E. coli strains harboring the empty pET32a vector (control) or the recombinant pET32a- BbCHI3 vector under increasing NaCl concentrations. The results showed that under 150 mmol·L⁻¹ NaCl treatment, the control strain BL21-pET32a exhibited marginally better growth than the recombinant strain BL21-pET32a- BbCHI3 (Fig. 9 A). Conversely, under the more severe stress condition of 200 mmol·L⁻¹ NaCl, the BL21-pET32a- BbCHI3 strain displayed significantly enhanced growth compared to the control, demonstrating a protective role of BbCHI3 under high-salinity conditions. We then performed a detailed growth curve analysis under continuous exposure to 200 mmol·L⁻¹ NaCl stress (Fig. 9 B). During the first 1–2 h (lag phase), the OD₆₀₀ values of both strains were similar. From 3-7 h (exponential phase), the BbCHI3 -expressing strain showed significantly higher growth, confirming the protective role of BbCHI3. From 8-12 h, both strains reached a similar OD₆₀₀ plateau, consistent with the entry into stationary phase due to nutrient limitation. Fig. 9. Open in a new tab A Spot assay for salt tolerance. Bacterial cultures of the control (BL21-pET32a) and BbCHI3 -expressing (BL21-pET32a- BbCHI3 ) strains were serially diluted (10-fold steps). Spots correspond to the undiluted culture (10⁰, OD600 = 1.2) and dilutions of 10-¹, 10-², 10-3, 10-⁴, and 10-⁵ on LB agar plates containing the indicated NaCl concentration. B Growth kinetics under salt stress. Growth (OD600) of the control and BbCHI3 -expressing strains was monitored over time in liquid LB medium under 200 mmol•L⁻¹ NaCl stress. The line graph depicts the growth curves, and the bar chart (inset) shows the final optical densities Discussion Molecular evolutionary and functional divergence of the chalcone isomerase gene in B. balsamifera The chalcone isomerase gene family has undergone significant functional divergence during plant evolution. In this study, BbCHI3 shares five key active-site residues (Arg, Thr, Tyr, Asn, and Ser) with AtCHI. Phylogenetically, BbCHI3 clusters within a clade containing Type I CHIs from monocots, such as maize, suggesting that it is a canonical Type I chalcone isomerase. This conclusion was further supported by subsequent in vitro enzyme activity assays, which demonstrated that the BbCHI3 protein efficiently catalyzes the conversion of naringenin chalcone to (2S)-naringenin. Moreover, HPLC analysis confirmed the accumulation of (2S)-naringenin in the transgenic complementation system, providing additional support for its functional identity as an active enzyme. Notably, the clustering of BbCHI3 with monocot CHIs suggests that its functional divergence may have occurred prior to or during the early stages of, the divergence between monocots and dicots, indicating a high degree of conservation. In contrast, BbCHI1, BbCHI2, and BbCHI4 are phylogenetically placed within a clade containing Type II and Type IV CHIs. Given that Type II CHIs are predominantly legume-specific and dedicated to isoflavone biosynthesis [ 33 , 34 ], these BbCHIs are more likely to be Type IV enzymes or to represent non-catalytic ancestral forms. Conserved domain analysis confirms their membership in the Chalcone_3 superfamily. Notably, BbCHI1 harbors a specific domain ( PLN02804 ) reported in cotton to mediate hormonal responses and positively regulate flavonoid accumulation [ 35 ]. Similarly, BbCHI2 contains the PLN03174 domain, which is also present in Actinidia kolomikta [ 36 ]. We propose that these non-catalytic members may serve auxiliary functions, potentially by assembling into metabolon complexes with chalcone synthase or other flavonoid biosynthetic enzymes. Such interactions could help stabilize metabolic flux or fine-tune anthocyanin accumulation. Future investigations should aim to decipher the precise coordinating roles of these non-catalytic subunits within the broader flavonoid metabolic network of B. balsamifera . Spatiotemporal expression and potential functions of BbCHI3 Plants employ spatiotemporal control of secondary metabolite accumulation as a key adaptive strategy [ 37 , 38 ]. Flavonoid composition and levels are significantly influenced by the expression and regulation of chalcone isomerase [ 39 ]. Our results demonstrate that BbCHI3 displays strong tissue specificity, with maximal transcript abundance in FL. This pattern is consistent with reports for AaCHI in Artemisia annua [ 40 ] and implies that high floral BbCHI3 expression facilitates the production of anthocyanins and flavonols for pollinator attraction and UV protection. Conversely, minimal expression in Rt likely reflects a lower requirement for pigmented flavonoids in belowground tissues, where metabolic resources may be preferentially allocated to lignin or phytoalexin biosynthesis. During leaf development, BbCHI3 expression peaked at the metabolically active bud stage (stage I) and declined upon maturation. This dynamic expression profile suggests that young tissues utilize elevated expression of key biosynthetic genes to pre-assemble flavonoid-derived defense compounds, thereby mitigating biotic and abiotic threats during vulnerable developmental windows. Environmental plasticity plays a pivotal role in defining the quality of Daodi medicinal materials [ 41 ]. Our data show that high-salinity stress significantly upregulates BbCHI3, promoting flavonoid accumulation to scavenge reactive oxygen species (ROS) and mitigate salt-induced oxidative damage [ 42 ]. Conversely, under extreme salinity, BbCHI3 expression is suppressed, indicating a physiological ceiling for this adaptive response. Such repression aligns with reports that severe salt stress disrupts cellular metabolism and broadly inhibits gene expression [ 43 ]. Intriguingly, heterologous expression of BbCHI3 conferred marked tolerance to extreme salt stress in E. coli . This suggests that the protein itself is highly stable, and its downregulation in plants likely results from transcriptional repression rather than protein inactivation, These findings provide crucial leads for subsequent in-depth functional characterization of this chalcone isomerase. However, owing to the inherent limitations of the prokaryotic heterologous expression system, the current evidence only supports the preliminary inference that BbCHI3 may be implicated in salt stress tolerance regulation, which remains insufficient to substantiate its direct role in salinity resistance. Future investigations should integrate multi-dimensional evidence, including plant physiological and biochemical parameter determination, as well as validation through gene-edited mutants of B. balsamifera , to construct a comprehensive evidentiary chain from correlation to functionality, thereby elucidating the precise mechanistic role of this gene in flavonoid-mediated oxidative stress responses. In B. balsamifera , the stress hormone ABA represses in a time-dependent manner CHI gene expression, potentially shifting metabolic resources away from specialized metabolism toward essential stress-coping mechanisms. Under drought-mimicking conditions, BbCHI3 expression displayed a multiphasic pattern. This induction is associated with stomatal closure and elevated flavonoid levels, positioning BbCHI3 as a participant in drought adaptation [ 44 ]. A key finding of this study is the synergistic interaction between MeJA and SL herbivory. Whereas herbivory alone did not induce, and even slightly suppressed, BbCHI3 expression, MeJA pre-treatment prior to insect attack triggered a dramatic ~ 21.2-fold upregulation. When the larvae of SL fed on tomato leaves treated with MeJA-treated, their growth and development as herbivorous insects were significantly inhibited [ 45 ]. This potent priming effect corresponds to the MeJA-responsive and defense-related cis -elements in the BbCHI3 promoter, indicating that exogenous MeJA primes the defense system of B. balsamifera . Consequently, upon subsequent herbivory, BbCHI3 is robustly induced, diverting metabolic resources into defensive flavonoid production to generate antifeedant or toxic effects [ 46 ]. Collectively, these findings elucidate how MeJA elicitors could be applied to simultaneously bolster herbivore resistance and elevate the levels of medicinal flavonoids in B. balsamifera . Elucidation and refinement of the flavonoid biosynthetic pathway in B. balsamifera Combining in vitro enzymology and in planta complementation, this study delineates a BbCHI3-centric flavonoid biosynthetic pathway in B. balsamifera . BbCHI3 displays canonical Type I CHI activity, efficiently converting naringenin chalcone to (2S)-naringenin—a committed step toward diverse flavonoids like anthocyanins, flavones, and flavonols. This catalytic function was confirmed by heterologous complementation: BbCHI3 overexpression restored anthocyanin accumulation and pigmentation in the Arabidopsis tt5 mutant, confirming its conserved function in planta . These findings align with reports of sweet potato CHI complementing the same mutant [ 47 ]. In contrast, CHI loss-of-function consistently reduces flavonoid content across species, altering phenotypes such as onion bulb coloration (associated with reduced quercetin) [ 48 ] and barley leaf flavonoid levels [ 49 ]. The absence of a robust transformation system for B. balsamifera currently limits functional validation to heterologous systems. Although the Arabidopsis complementation strongly supports BbCHI3’s conserved activity, it may not fully reflect its native regulatory network. Future efforts will prioritize developing efficient B. balsamifera transformation or employing VIGS/CRISPR-Cas9 for knockout or overexpression of BbCHI3 in its native background, enabling precise quantification of its role in blumeatin production and total flavonoid content production. Beyond its canonical activity, this study establishes the broad substrate specificity of BbCHI3, notably its catalysis of pinocembrin chalcone to (2S)-pinocembrin. Based on previous metabolomic data, we have proposed the biosynthetic pathway for blumeatin, a characteristic constituent of B. balsamifera . As shown in Fig. 10 , pinocembrin chalcone is first synthesized from cinnamoyl-CoA and three malonyl-CoA units by chalcone synthase CHS. This chalcone is then stereospecifically isomerized to pinocembrin by BbCHI3, followed by subsequent hydroxylation and O-methylation steps to produce blumeatin. This proposed pathway resolves a key gap in blumeatin biosynthesis. Moreover, BbCHI3 also isomerizes homoeriodictyol and eriodictyol chalcones, positioning it as a multifunctional enzyme within the flavonoid metabolic network of B. balsamifera . Fig. 10. Open in a new tab Biosynthesis of blumeatin, anthocyanins, flavonoids, and flavonols In summary, BbCHI3 is crucial not only in regulating essential physiological processes (such as anthocyanin synthesis) in B. balsamifera , but also in determining the quality of its geo-authentic medicinal material (via blumeatin biosynthesis) and its environmental stress tolerance. The findings of this study identify BbCHI3 as a key target for the metabolic engineering of blumeatin content and for breeding novel B. balsamifera varieties with enhanced stress tolerance and quality. Given the increasing depletion of wild B. balsamifera resources, the BbCHI3 gene, together with the upstream BbCHS gene, can be engineered into high-yield microbial cell factories using Saccharomyces cerevisiae or Yarrowia lipolytica . This strategy would enable the industrial biomanufacturing of the high-value medicinal compound blumeatin, providing a sustainable and environmentally friendly solution to resource scarcity. Furthermore, the molecular markers developed in this study can be directly applied to screen for elite germplasm and facilitate molecular breeding in B. balsamifera , thereby promoting the modernization of its industry. Conclusions This study reports the first systematic identification of the chalcone isomerase gene family in B. balsamifera and identifies BbCHI3 as a key Type I member governing flavonoid biosynthesis. Integrated biochemical and molecular evidence establishes that the cytoplasm-localized BbCHI3 protein exhibits both canonical CHI activity (converting naringenin chalcone to (2S)-naringenin) and a broad substrate specificity, enabling the key conversion of pinocembrin chalcone to pinocembrin. This work thus delineates a critical and previously unresolved step in the biosynthesis of characteristic metabolites like blumeatin, flavones, and flavonols. BbCHI3’s activity extends to homoeriodictyol and eriodictyol chalcones, linking it to the production of related flavonoids. I n planta function was confirmed via heterologous complementation in Arabidopsis , where BbCHI3 restored anthocyanin accumulation. Its expression is spatiotemporally regulated, showing highest levels in FL and young leaves, and is induced by high salt, ABA, and MeJA. Remarkably, heterologous BbCHI3 expression conferred tolerance to extreme salt tolerance in E. coli . Collectively, BbCHI3 emerges as a dual-function hub: a metabolic hub within the flavonoid network and a stress-responsive regulator that facilitates environmental adaptation and contributes to Dao-di quality. These findings provide a robust genetic and mechanistic basis for metabolic engineering aimed at enhancing blumeatin yield and for developing stress-tolerant, high-quality B. balsamifera cultivars. Supplementary Information 12870_2026_8721_MOESM1_ESM.docx (844.9KB, docx) Supplementary Material 1: Supplementary Fig. S1. Analysis of Physicochemical Properties and Structural Characteristics of Gene Family Proteins. Supplementary Fig. S2. Optimization of induction temperature, time, and IPTG concentration for the recombinant protein BbCHI3 pET32a BL21. 12870_2026_8721_MOESM2_ESM.docx (19.3KB, docx) Supplementary Material 2: Supplementary Table. S1 Prediction of the Secondary Structures of BbCHIs. Supplementary Table. S2 The BbCHIs protein possesses four substrate-binding sites. Supplementary Table. S3 Volatile flavonoids in A. thaliana. 12870_2026_8721_MOESM3_ESM.docx (29MB, docx) Supplementary Material 3: Full-length, uncropped images gels and blots. Acknowledgements We sincerely thank the editor and reviewers for their rigorous evaluation and constructive comments. We also gratefully acknowledge Guizhou University of Traditional Chinese Medicine and Guizhou Normal University for providing laboratory facilities and administrative support for this research. Institutional review board statement Not applicable. Informed consent statement Not applicable. Abbreviations CHI Chalcone Isomerase CHS Chalcone Synthase Cinnamoyl-CoA Cinnamoyl-Coenzyme A NC Naringenin Chalcone NA Naringenin PC Pinocembrin Chalcone PA Pinocembrin HC Homoeriodictyol Chalcone HA Homoeriodictyol EC Eriodictyol Chalcone EA Eriodictyol HS High Salt EHS Extra High Salt MeJA Methyl Jasmonate SL Spodoptera litura ABA Abscisic Acid PEG Polyethylene glycol Rt Root St Stem Lf Leaf FI Flower IPTG Isopropyl-beta-D-thiogalactopyranoside TBtools Toolbox for Biologists HPLC High Performance Liquid Chromatography RT-qPCR Quantitative reverse transcription polymerase chain reaction Authors’ contributions SS and KLM jointly conceived and drafted the manuscript. SS, JL, CMG, SY and QML collaboratively performed the experimental work. PXX, CLS and ZWZ were responsible for data analysis and software implementation. WS, ZGJ and YXP reviewed and supervised the manuscript. All authors contributed to this work and approved the final version for submission. Funding This research was supported by the Guizhou Authentic medicinal Materials Germplasm Resource Bank Equipment Construction Project (Authentic medicinal Materials Advantages and Characteristics Industry Cluster (Chinese medicinal materials Industry Cluster))(3411-411000163), the State Administration of Traditional Chinese Medicine high-level key discipline construction project (zyyzdxk-2023186), and the Guizhou University of Chinese Medicine, Karst medicinal resources protection and innovative utilization of scientific and technological innovation talent team project (GZY TD Hezi [2022]001). This work was supported by the Key Project of Guizhou Provincial Basic Research Program (2026) “Study on the Structure-Activity Dependence and Molecular Regulatory Mechanisms of Same Efficacy but Different Effects’ between Qianchan Aipian and Heterologous Borneol”. Data availability Sequence data from this article can be found in this published article and its supplementary information files.Sequence data from this article can be found in the GenBank data libraries under accession numbers: BbCHI1, PZ235942 ; BbCHI2, PZ235943 ; BbCHI3, PZ235944 ; BbCHI4, PZ235945 . The accession number for the raw transcriptome data of Blumea balsamifera is SAMN40028479. Declarations Ethics approval and consent to participate Not applicable. Competing interests The authors declare no competing interests. Footnotes Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Shan Sha and Ju Li are co-first authors. Contributor Information Yuan Yuan, Email: yuanyuanhaida@126.com. Zhigang Ju, Email: juzhigangz@163.com. Yuxin Pang, Email: pyxmarx@126.com. References 1. Xie XL, Chen ZX, Pang YX, Guan LL, Guo KM. Research Progress on Resources of Blumea balsamifera . 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Supplementary Materials 12870_2026_8721_MOESM1_ESM.docx (844.9KB, docx) Supplementary Material 1: Supplementary Fig. S1. Analysis of Physicochemical Properties and Structural Characteristics of Gene Family Proteins. Supplementary Fig. S2. Optimization of induction temperature, time, and IPTG concentration for the recombinant protein BbCHI3 pET32a BL21. 12870_2026_8721_MOESM2_ESM.docx (19.3KB, docx) Supplementary Material 2: Supplementary Table. S1 Prediction of the Secondary Structures of BbCHIs. Supplementary Table. S2 The BbCHIs protein possesses four substrate-binding sites. Supplementary Table. S3 Volatile flavonoids in A. thaliana. 12870_2026_8721_MOESM3_ESM.docx (29MB, docx) Supplementary Material 3: Full-length, uncropped images gels and blots. Data Availability Statement Sequence data from this article can be found in this published article and its supplementary information files.Sequence data from this article can be found in the GenBank data libraries under accession numbers: BbCHI1, PZ235942 ; BbCHI2, PZ235943 ; BbCHI3, PZ235944 ; BbCHI4, PZ235945 . The accession number for the raw transcriptome data of Blumea balsamifera is SAMN40028479. Articles from BMC Plant Biology are provided here courtesy of BMC ACTIONS View on publisher site PDF (7.1 MB) Cite Collections Permalink RESOURCES Similar articles Cited by other articles Links to NCBI Databases Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top

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