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. 2026 Apr 27;26:1009. doi: 10.1186/s12870-026-08827-w

Mechanistic insights into adventitious root formation in Cinnamomum kanehirae stem cuttings based on phenotypic, physiological, and transcriptomic analyses

Yuntong Zhang 1,2,#, Xinliang Liu 1,#, Yueting Zhang 1, Xiaoying Dai 1, Jun Wang 2, Yongjie Zheng 1,✉, Ting Zhang 1,✉
PMCID: PMC13255347  PMID: 42045823

Abstract

Cinnamomum kanehirae is a species of high economic worth, as it produces abundant aromatic oils for industrial use and its heartwood acts as an optimal substrate for cultivating the medicinal fungus Antrodia cinnamomea. The limited understanding of the underlying molecular and physiological mechanisms restricts the successful propagation of C. kanehirae. This study systematically investigated adventitious roots (ARs) formation in C. kanehirae through anatomical observation, physiological measurement, and transcriptomic analysis. Anatomical results demonstrated that ARs originate from callus tissue and develop through three distinct stages: callus formation, root primordium initiation, and root elongation. Nutrient metabolites, enzyme activities, and endogenous hormones play crucial roles in these processes. A notable decline in soluble sugar content during early AR formation indicated intensive energy utilization for cell division. During root primordium initiation and AR elongation, fluctuations in malondialdehyde levels and the activities of superoxide dismutase and peroxidase reflected the complex regulatory role in oxidative stress in root induction. Transcriptomic analysis identified differentially expressed genes that were significantly enriched in hormone signal transduction, energy metabolism, and circadian rhythm pathways. Auxin and abscisic acid emerged as central regulators within the hormone signaling network. Weighted gene co-expression network analysis further identified hub genes, including CkPYL4, CkOPR2, CkOFP7, CkMYB306, and CkRbohA, involved in hormonal signaling and redox regulation. These findings elucidate the coordinated anatomical, physiological, and molecular mechanisms governing AR formation in C. kanehirae, establishing a theoretical foundation for improving cutting propagation in woody plants.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08827-w.

Keywords: Morphogenesis, Adventitious root, Endogenous hormones, RNA-seq, WGCNA

Introduction

Cinnamomum kanehirae, an evergreen broad-leaved tree of the Lauraceae family, is predominantly distributed in southeastern China, and it flourishes in well-lit, warm, and humid environments [1]. This highly valuable ornamental species provides timber for furniture and woodcarving, while its roots, stems, and leaves contain rich aromatic oils, with safrole serving as a significant industrial raw material [2]. The heartwood of C. kanehirae provides an ideal substrate for cultivating Antrodia cinnamomea, a precious medicinal fungus known for its diverse pharmacological properties, including anticancer, immunomodulatory, and anti-inflammatory effects [3, 4]. Recent excessive exploitation has led to a sharp decline in natural populations of C. kanehirae [5]. Given its slow natural regeneration rate, the species faces potential extinction, necessitating the development of effective artificial propagation methods. Cultivating C. kanehirae through artificial propagation not only contributes to the conservation of this valuable species but also helps meet the market demand for A. cinnamomea. Sexual reproduction in C. kanehirae presents significant challenges, as its seeds demonstrate poor quality, high hollowness rates, low germination rates, and susceptibility to pest and disease infestations, substantially limiting this propagation approach [6].

Considering these reproductive constraints and diminishing natural resources, establishing efficient asexual propagation systems is crucial for conserving and sustainably utilizing C. kanehirae. Methods including stem cutting, grafting, and tissue culture have emerged as primary propagation strategies. However, issues such as graft incompatibility and tissue browning limit these approaches effectiveness, making stem cutting the most viable method for C. kanehirae. In this propagation, Adventitious root (AR) formation serves as a crucial determinant of successful cutting propagation, ensuring clonal stability and plant uniformity, therefore, understanding the mechanisms underlying AR formation is of particular importance. In addition, previous studies have shown that AR formation in Cinnamomum species follows an induced developmental pattern, in which root primordia are initiated after cutting rather than being preformed [7, 8]. Therefore, elucidating the mechanisms of AR formation in C. kanehirae is important not only for improving its asexual propagation and supporting the conservation and utilization of this valuable species, but also for advancing the understanding of adventitious rooting mechanisms in woody plants.

During cutting propagation, cuttings detect specific stress signals, triggering AR formation from differentiated cell layers in non-root tissues [9]. Tissue injury, endogenous hormone changes, and pathogenic microorganisms significantly influence AR formation [10]. At the cutting site, cell wall and plasma membrane damage leads to excessive reactive oxygen species (ROS) accumulation, causing oxidative stress [11]. Plants activate antioxidant defense systems to counter this stress, where superoxide dismutase (SOD) initially converts superoxide anions into hydrogen peroxide, and peroxidase (POD) maintains ROS homeostasis by scavenging hydrogen peroxide [12].

Plant hormones act as key signaling molecules during AR formation, regulating AR initiation, development, and final root establishment through complex regulatory networks [13]. Among them, auxin is the primary plant growth regulator, governing AR formation [14]. Local auxin biosynthesis and polar transport at the cutting base result in its basal accumulation, initiating cell-fate reprogramming [15]. The indole-3-pyruvate monooxygenase YUCCA (YUC) gene family facilitates auxin biosynthesis, while PIN-FORMED (PIN) proteins regulate its polar transport through multiple regulatory factors [16, 17]. Several transcription factors, including auxin/Indole-3-acetic acid (AUX/IAA), auxin response factor (ARF), WUSCHEL-related homeobox (WOX), and Lateral Organ Boundaries Domain (LBD), respond to auxin induction, regulating downstream target gene expression and influencing AR formation [18–20]. Auxin also interactions with other phytohormones through intricate cross-regulatory networks. Its antagonistic relationship with cytokinin plays a decisive role in AR formation [21]. Elevated auxin levels activate ARFs, which suppress ISOPENTENYL TRANSFERASE 3 (IPT3) and ISOPENTENYL TRANSFERASE 5 (IPT5) expression, thereby inhibiting cytokinin biosynthesis and promoting AR initiation [22].

Abscisic acid (ABA) functions as an internal signal activating specific response genes under environmental stress [23]. Wounding during cuttings triggers rapid changes in ABA concentration, activating injury response pathways [24]. ABA is synthesized via oxidative cleavage of C40 carotenoids, producing xanthoxin, which is converted to ABA aldehyde and subsequently oxidized to ABA [25]. Increasing intracellular ABA levels enable pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory component of ABA receptor (RCAR) receptors to inhibit protein phosphatase 2 C (PP2C) activity, thereby activating sucrose non-fermenting 1-related protein kinase 2 (SnRK2) kinases, which phosphorylate ABA-responsive element binding factor (ABF)/ABA-responsive element-binding protein (AREB) transcription factors [26]. Beyond this canonical ABA signaling cascade, myeloblastosis (MYB) transcription factors have emerged as critical downstream regulators and modulators of ABA responses [27]. Jasmonic acid (JA), a lipid-derived phytohormone, acts in concert with ABA to regulate wound and stress responses. Through their synergistic interaction, JA and ABA jointly help maintain cellular homeostasis and modulate auxin-related signaling pathways, thereby influencing callus formation and adventitious root initiation. Moreover, increasing evidence suggests that ABA can enhance JA biosynthesis; for instance, in rice, ABA upregulates allene oxide cyclase (AOC) expression and consequently elevates endogenous JA levels [28].

As the primary propagation method for C. kanehirae, stem cutting necessitates a comprehensive understanding of AR formation. This study integrates anatomical observations, physiological measurements, and transcriptomic analysis to examine AR development across different stages. Temporal gene expression dynamics were explored through trend analysis, while weighted gene co-expression network analysis (WGCNA) was employed to correlate physiological traits with gene modules and identify key regulatory genes involved in AR formation. The findings provide new insights into the molecular and physiological mechanisms governing AR development in C. kanehirae.

Materials and methods

Plant materials

Healthy, dormant branches were collected from a three-year-old C. kanehirae mother tree maintained at the Jiangxi Provincial Academy of Forestry (27°53′N, 116°47′E). Stem cuttings, each bearing one leaf and measuring ~ 18 cm in length, were submerged in water for 30 min prior to use. For AR formation analysis, basal stem segments excised from stem cuttings (~ 0.5 cm in length) were collected at 0, 5, 10, 15, 20, 25, and 30 days of post-initiation. A portion of these samples was fixed in Formalin–Acetic Acid–Alcohol fixative (FAA) solution (70% ethanol: acetic acid: formaldehyde = 90:5:5) for anatomical analysis. The remaining samples were flash-frozen in liquid nitrogen, homogenized, and stored at −80 °C for subsequent physiological measurements and transcriptome sequencing. For each time point, three independent biological replicates were collected from separate stem cuttings derived from the same mother tree and grown under identical conditions to ensure reproducibility.

Morphological and anatomical observation

Paraffin sections were prepared to observe anatomical changes during AR formation. Tissue segments of ~ 5 mm in length were cut from the basal region of explants and fixed in FAA for 48 h at 4 ℃ to maintain cellular integrity. The samples were dehydrated through a graded ethanol series, embedded in paraffin, stained with safranin and fast green, mounted in neutral resin, and examined under a DFC500 optical microscope (Leica Microsystems, Wetzlar, Germany). Representative anatomical structures were photographed and documented for analysis.

Physiological and biochemical analysis

Soluble sugar and protein levels were quantified using the anthrone and Coomassie brilliant blue colorimetric methods, respectively. Endogenous hormone levels, including IAA, indole-3-butyric acid (IBA), gibberellin (GA), ABA, and zeatin riboside (ZR), in the phloem of the basal stem segments were measured by high-performance liquid chromatography-mass spectrometry. Malondialdehyde (MDA) content was determined by the thiobarbituric acid method. The enzymatic activities of SOD, polyphenol oxidase (PPO), and POD were measured using standard colorimetric assays. Each analysis included six biological replicates.

RNA-seq and data analysis

Total RNA was extracted using the Omega Plant RNA Kit (Omega Bio-Tek, Norcross, GA, USA), and its integrity was confirmed by 1% agarose gel electrophoresis. RNA purity and concentration were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). High-quality RNA samples were utilized for cDNA library construction, performed by Wuhan Benagen Technology Co., Ltd. Following quality assessment, libraries were sequenced on the DNBSEQ-T7 high-throughput platform, with three biological replicates per group. Raw reads were filtered to remove sequences containing more than 5% ambiguous bases (N), those with ≥ 50% of bases having a Phred quality score ≤ 5, reads contaminated with adaptor sequences, and PCR duplicate reads, and the resulting high-quality clean reads were retained in FASTQ format for subsequent analyses [29]. Clean reads were mapped to the Cinnamomum kanehirae reference genome using STAR [30]. The reference genome was previously assembled and annotated [31]. The quality of clean reads was further assessed based on Q20 and Q30 scores, GC content, and mapping rate to ensure high sequencing accuracy and reliable alignment. Sequencing quality was evaluated through randomness and saturation analyses. Transcript quantification was performed using RSEM, generating both read count and FPKM values. Paired-end reads derived from the same fragment were treated as a single sequencing unit for quantification (Table S1). In addition, Pearson correlation analysis among biological replicates demonstrated high consistency, confirming the reproducibility of the transcriptomic data.

Differential gene expression and functional enrichment analysis

Differentially expressed genes (DEGs) across rooting stages were identified using DESeq2 with thresholds of |log2FC| > 1 and adjusted p < 0.05. Clustering patterns of DEGs were analyzed using STEM software, followed by functional enrichment analysis [32]. Enrichment of DEGs was analyzed against the KEGG database, and metabolic overview maps were generated using Mapman 3.7.0 [33]. The Pearson correlation coefficient was used to evaluate the association between expression change of DEGs in the auxin, cytokinin and ABA signaling pathway and the corresponding endogenous hormone levels.

Weighted Gene Co-Expression Network Analysis (WGCNA) analysis

WGCNA was conducted as previously reported [34]. To identify potential hub genes within the co-expression network, genes in the top 20% ranked by intramodular edge weight were first selected as highly co-expressed candidates. The resulting network was then analyzed in Cytoscape (version 3.10.3), where nodes were ranked according to degree centrality, and the top 60 genes were retained as core hub genes. Functional annotation and supporting literature were subsequently used to identify genes potentially involved in AR formation.

RNA-seq data validation

Some AR-associated DEGs were also analyzed in triplicate with three independent biological replicates using quantitative real-time PCR (qRT-PCR) with gene-specific primers designed using Primer Premier 5 and synthesized by Ruibiotech (Beijing, China) (Table S2). Primer specificity was confirmed by melting curve analysis. Amplification efficiency was evaluated using a standard curve generated from serial cDNA dilutions. Gene expression relative to internal control actin was calculated using the 2−ΔΔCt [35]. The expression trends obtained by qRT-PCR were consistent with the RNA-seq results.

Statistical analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Least Significant Difference (LSD) test in SPSS software (version 27.0). Normality and homogeneity of variance were assessed prior to ANOVA. Differences were considered statistically significant at P < 0.05. Figures were generated using GraphPad Prism (version 9.0).

Results

Anatomical and morphological dynamics during AR formation

Morphological observations characterized the sequential process of AR formation in C. kanehirae cuttings. Initially, swelling appeared at the basal cutting site (Fig. 1B), followed by the emergence of small protuberances on the surface (Fig. 1C) and subsequent penetration of ARs through the epidermis (Fig. 1D). Anatomical analysis identified three distinct stages of AR development. During the callus induction stage, cortical cells underwent active division and dedifferentiation, forming tightly arranged parenchyma cells with large nuclei that developed into callus tissue (Fig. 1F). In the root primordium initiation stage, the callus tissue differentiated to form root primordia (Fig. 1G). The AR elongation stage was characterized by continuous cell division and proliferation within the root primordia, culminating in mature ARs that penetrated the epidermis (Fig. 1H).

Fig. 1.

Fig. 1

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Anatomical and morphological dynamics in cutting base during AR formation. A-D Morphological observations. E-H Anatomical observations. A, E CK0: No root primordia or morphological changes at the cutting base; (B, F) CK1: Callus formation originating from cortical cells; (C, G) CK2: Root primordia formation within the callus; (D, H) CK3: Adventitious roots emerging through the epidermis

Dynamics of nutrient content and enzyme activity during AR formation

Soluble sugar content decreased significantly at the callus induction stage but increased and stabilized during root primordium initiation and AR elongation. Conversely, soluble protein content rose sharply in the early AR formation stage, then declined significantly. MDA, an indicator of membrane damage and ROS accumulation, showed no significant changes during callus induction but increased markedly during root primordium initiation and AR elongation. Antioxidant enzyme activities demonstrated distinct temporal patterns. POD activity decreased significantly during callus induction, then increased and stabilized. SOD activity remained constant in the early AR formation stage, declined significantly during root primordium initiation, and increased again during AR elongation. PPO activity displayed a pattern similar to POD, declining initially, rising markedly during root primordium initiation, and stabilizing thereafter (Fig. 2).

Fig. 2.

Fig. 2

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Variations in nutrient content and enzyme activity at different stages. Data are expressed as mean ± SD. Statistical significance among stages was determined by one-way ANOVA followed by LSD test, after verifying normality and homogeneity of variance. Different lowercase letters indicate statistically significant differences among stages at P < 0.05

Variations in endogenous hormones during AR formation

Across distinct AR formation stages, endogenous hormone contents demonstrated characteristic dynamic changes. IAA and its precursor IBA exhibited contrasting patterns: IAA levels decreased significantly during callus induction, increased markedly during root primordium initiation, then declined substantially during AR elongation. Conversely, IBA content showed a consistent decrease throughout AR formation, indicating potential rapid conversion to IAA or utilization during root induction. ZR content demonstrated an inverse pattern compared to IAA, while the IAA/ZR ratio closely paralleled IAA fluctuations, suggesting the auxin-to-cytokinin balance critically regulates stage-specific AR development. GAs followed a pattern similar to IAA, whereas the IAA/GA ratio increased dramatically during callus induction, decreased during root primordium initiation, and maintained relative stability during AR elongation, indicating coordinated auxin-GA interaction in root development regulation. ABA levels increased continuously throughout AR formation, emphasizing its role in stress adaptation and developmental control. The significant reduction in the IAA/ABA ratio suggests that elevated ABA signaling may serve as an adaptive stress response, facilitating hormonal balance coordination and enhancing AR formation efficiency (Fig. 3).

Fig. 3.

Fig. 3

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Endogenous hormone levels during AR formation. Data are expressed as mean ± SD. Statistical significance among stages was determined by one-way ANOVA followed by LSD test, after verifying normality and homogeneity of variance. Values with different lowercase letters are significantly different (P < 0.05)

Transcriptomic profiling across four AR formation stages

Samples from four representative stages (CK0–CK3) were selected for RNA-seq, based on morphological, anatomical, and hormonal data, with three biological replicates per stage. Replicates showed strong consistency (Fig. 4A–B). Each of the twelve libraries produced over 40 million clean reads. Sequencing quality was high (Q20 > 98.37%, Q30 > 94.15%), with GC content ranging from 45.82% to 46.12% (Table S1). Most reads (91.25–92.66%) mapped uniquely to the C. kanehirae reference genome, while 4.98–6.15% mapped to multiple locations.

Fig. 4.

Fig. 4

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Sample sequencing results. A Heatmap of sample correlation. B Boxplot of gene expression levels (FPKM). C Venn diagram illustrating DEGs across the three AR formation stages. D Scatterplot of DEGs across multiple comparisons, where the y-axis represents log2(FC), and the x-axis displays the comparison groups

DEGs were identified using the criteria of p < 0.05 and |log₂ (fold change) | > 1(Fig. 4C). In the CK0 vs. CK1 comparison, 2,537 genes were significantly upregulated and 3,415 were downregulated. In the CK1 vs. CK2 comparison, fewer DEGs were detected, with 1,229 upregulated and 522 downregulated. The largest number of DEGs was found between CK2 and CK3, including 2,249 upregulated and 1,340 downregulated genes (Fig. 4D).

Temporal dynamics of DEGs

The STEM algorithm identified 20 expression profiles across four AR formation stages (Fig. 5). Seven profiles, P0, P2, P5, P7, P16, P17, and P19, demonstrated statistically significant enrichment (p < 0.05), revealing distinct gene expression trends associated with specific developmental stages. Genes in P0, P2, P5, and P7 exhibited low expression during primordia induction, while those in P16, P17, and P19 were highly expressed. During primordium formation, P0, P17, and P19 maintained consistent expression trends, in contrast to the opposing patterns in P2 and P7. P5 and P16 maintained relatively stable expression across all stages.

Fig. 5.

Fig. 5

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Temporal expression patternanalysis and KEGG enrichment of significantly clustered profiles identified by STEM. Expression profiles were clustered across four time points (CK0-CK3). Each profile represents a distinct temporal expression pattern of DEGs. Profile numbers and corresponding p-value are shown above and within each panel, respectively. Significantly enriched profiles are highlighted, and KEGG pathway enrichment analysis was performed for genes within the significantly enriched profiles. Bubble color indicates -log10(p-value), with deeper green indicating higher significance. Bubble size represents number of enriched DEGs per pathway

KEGG enrichment analysis revealed that DEGs in P0 were mainly involved in circadian rhythm and photosynthesis pathways. P7 DEGs were primarily distributed across secondary metabolic pathways, including phenylpropanoid, flavonoid, sesquiterpenoid, and triterpenoid biosynthesis, and primary metabolic pathways, including tryptophan and α-linolenic acid metabolism. P16 DEGs were associated with diverse functions, notably signal transduction (plant hormone signaling and MAPK cascade), redox-regulation (glutathione metabolism), and secondary metabolism (zeatin biosynthesis).

MapMan and KEGG enrichment analysis of DEGs

Combined KEGG enrichment and MapMan visualization revealed key pathways and functional modules underlying AR formation (Fig. 6A-C). In CK0 vs. CK1, DEGs were enriched in Metabolism, OS, and Environmental Information Processing (EIP). Photosynthesis-related and circadian rhythm pathways were predominant in Metabolism and OS, respectively, while plant hormone signal transduction and MAPK signaling pathways dominated EIP. Phenylpropanoid biosynthesis showed the highest enrichment in CK1 vs. CK2 and CK2 vs. CK3 comparisons, with plant hormone signal transduction particularly prominent in CK2 vs. CK3.

Fig. 6.

Fig. 6

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MapMan and KEGG enrichment analyses of DEGs across three stages. A CK0 vs. CK1, (B) CK1 vs. CK2, and (C) CK2 vs. CK3. The MapMan regulation overview maps illustrate differences in DEGs expression levels among the three stages, with blue indicating higher expression and red indicating lower expression. In the KEGG bubble plots, the x-axis represents the GeneRatio (the proportion of DEGs in a given pathway relative to all DEGs), and the y-axis lists the KEGG pathways. Bubble color denotes the adjusted p-value, while bubble size indicates the number of DEGs in the corresponding pathway

MapMan regulation overview revealed dynamic changes across several functional modules. Redox-related processes, including heme, thioredoxin, ascorbate/glutathione, glutaredoxin, and dismutase/catalase, demonstrated marked effects. During CK0 vs. CK1, hormone signaling exhibited distinct expression patterns: ABA-related genes showed predominantly upregulation, while GA-related genes displayed downregulation. Additionally, transcription factor activity, protein modification, and protein degradation modules showed notable responses. Conversely, CK2 vs. CK3 analysis revealed largely downregulated ABA-related genes, upregulated BA-related genes, and bidirectional regulation of GA-related genes.

Analysis of DEGs in key pathways

Significant enrichment of the circadian rhythm pathway was detected during early AR formation (CK0 vs. CK1). Genes associated with light perception and circadian regulation, including phytochrome A (PHYA), PHYB, and the central clock component EARLY FLOWERING 3 (ELF3), were upregulated. In contrast, LATE ELONGATED HYPOCOTYL (LHY), PSEUDO-RESPONSE REGULATOR 7 (PRR7), PSEUDO-RESPONSE REGULATOR 5 (PRR5), and negative light-signaling regulators CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), GIGANTEA (GI), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), SUPPRESSOR OF PHYA-105 1 (SPA1) were downregulated. Furthermore, the UV-B protective gene CHALCONE SYNTHASE (CHS) showed strong induction, suggesting its role in early light signal responses. These findings indicate that circadian rhythm and light signaling pathways cooperatively regulate hormonal and metabolic activities to initiate root primordium formation (Fig. 7).

Fig. 7.

Fig. 7

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DEGs associated with the circadian rhythm pathway during AR formation. Box color indicates log2 fold changes in three comparison groups: CK0 vs. CK1, CK1 vs. CK2, and CK2 vs. CK3. Orange, green, and white boxes represent significantly upregulated, significantly downregulated, and non-significantly changed genes, respectively

The analysis identified 121 DEGs in the plant hormone signal transduction pathway, with 34 DEGs specifically enriched in the auxin signaling pathway. Genes encoding the key protein YUC, essential for auxin biosynthesis, demonstrated stage-specific expression patterns. YUC genes showed significant downregulation in the CK0 vs. CK1 comparison, aligning with the decreased IAA concentration during early AR formation. In contrast, these genes exhibited marked upregulation in the CK2 vs. CK3 comparison, with an inverse relationship to the increased IAA concentration during the AR elongation stage. Additionally, DEGs encoding auxin influx carrier (AUX1/LAX), AUX/IAA, and ARF families exhibited similar expression dynamics, with most showing significant downregulation at the early stage (CK0 vs. CK1). However, genes belonging to small auxin-up RNA (SAUR) and Gretchen Hagen 3 (GH3) families demonstrated more varied expression patterns across developmental stages (Fig. 8).

Fig. 8.

Fig. 8

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Auxin-related DEGs. Box colors denote log2 fold changes for CK0 vs. CK1, CK1 vs. CK2, and CK2 vs. CK3: orange for upregulated, green for downregulated, and white for non-significant expression changes

The analysis identified 10, 10, and 15 DEGs enriched in the cytokinin, GA, and ABA signaling pathways, respectively. In the cytokinin pathway, genes encoding Cytokinin Response 1 (CRE1) and Histidine-containing Phosphotransfer (AHP) Protein were significantly upregulated in CK0 vs. CK1, consistent with the initial rise in zeatin riboside. In contrast, the negative regulator Type-A Arabidopsis Response Regulator (Type-A ARR) was downregulated. During the CK2 vs. CK3 stage, two Type-B Arabidopsis Response Regulator (Type-B ARR) genes were upregulated, corresponding to ZR accumulation during adventitious root elongation. In the GA pathway, two Phytochrome-Interacting Factor (PIF) genes were persistently downregulated throughout root development. In the ABA pathway, PP2C genes were downregulated in CK0 vs. CK1, whereas SnRK2 was upregulated at this stage but later suppressed due to PP2C inhibition. Genes encoding PYR/PYL and ABF exhibited dynamic expression patterns across different stages of AR formation (Fig. 9).

Fig. 9.

Fig. 9

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DEGs involved in CK, GA, and ABA pathways during AR formation. Box colors denote log2 fold changes for CK0 vs. CK1, CK1 vs. CK2, and CK2 vs. CK3: orange for upregulated, green for downregulated, and white for non-significant expression changes

Correlation between endogenous hormone levels and related DEGs

Pearson’s correlation analysis was performed between IAA, ZR and ABA contents from CK0 to CK3 and the expression levels of genes in their respective pathways. IAA levels exhibited dynamic fluctuations during AR formation, and several auxin-related genes showed correlations with endogenous IAA dynamics (Fig. 10A). The auxin influx-related CkAUXs genes also showed negative correlations with IAA levels, whereas the receptor gene CkTIR1 exhibited a positive correlation. Among the canonical early auxin-responsive genes, CkSAUR71 showed a strong negative correlation, and CkGH3 was likewise negatively correlated. In contrast, expression patterns within the CkAux/IAA family remained variable, and the auxin response factor CkARF3 showed a positive correlation with IAA.

Fig. 10.

Fig. 10

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Correlation between the expression of auxin and ABA signaling pathway DEGs and endogenous hormone contents. A IAA. B ABA. Only DEGs showing an absolute correlation coefficient |R| > 0.5 are displayed. The size of each dot represents the strength of the correlation between gene expression and endogenous hormone contents; larger dots indicate stronger correlations. The color of the dots reflects the p-value, with redder colors indicating smaller p-values

ZR content showed limited correlations with genes involved in the cytokinin signaling pathway. In contrast, ABA levels exhibited a continuous increasing trend during AR formation, and the expression patterns of genes in the ABA signaling pathway were generally associated with ABA dynamics (Fig. 10B). Among them, the ABA receptor CkPYL4 showed a strong and significant correlation with ABA levels (p < 0.05). Except for CkPP2C51−2, most CkPP2C family genes, which function as negative regulators of ABA signaling, were negatively correlated with ABA dynamics. Genes belonging to the CkSnRK family, which encode core kinases in the ABA signaling pathway, and the downstream transcription factor family CkABF/ABI exhibited more variable expression patterns across developmental stages.

WGCNA of DEGs and Hormone Traits in AR Formation

Endogenous hormones demonstrated distinct dynamics across various AR formation stages. To elucidate the transcriptional regulatory patterns underlying these processes, a WGCNA was conducted using 8,510 DEGs (Fig. 11A). The analysis identified 10 co-expression modules, with three modules showing significant correlations with endogenous hormone level variations. Specifically, the concentration changes of IAA and ABA exhibited strong associations with the MEpurple module (Fig. 11B). KEGG enrichment analysis of this module revealed significant enrichment in secondary metabolic pathways, including phenylpropanoid biosynthesis, flavonoid biosynthesis, and cytochrome P450-related pathways. Additionally, tryptophan metabolism and α-linolenic acid metabolism were identified as precursor pathways for IAA and JA biosynthesis, respectively (Fig. 11C).

Fig. 11.

Fig. 11

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Module construction and hub gene identification based on WGCNA. A Modules identified using the dynamic tree cut method, with each color representing a distinct module. B Heatmap depicting correlations between modules and hormonal traits; values in each cell represent the correlation coefficient. C KEGG pathway enrichment of DEGs in the MEpurple module. D Co-expression network highlighting hub genes in the MEpurple module

To identify potential hub genes within the co-expression network, genes with the highest edge weights were selected, and the network was constructed in Cytoscape (Fig. 11D). Genes demonstrating the highest connectivity were screened, and those associated with AR formation were designated as candidate hub genes. The key gene CkPYL4, involved in ABA signaling, exhibited expression patterns consistent with ABA concentration changes, while Cinnamomum kanehirae 12-oxophytodienoate reductase 12 (CkOPR2), a key enzyme in JA biosynthesis, and Cinnamomum kanehirae respiratory burst oxidase homolog A (CkRbohA), a member of the NADPH oxidase family, displayed significant negative correlations with IAA levels. Several transcription factors emerged as critical regulatory components: the transcriptional repressor Cinnamomum kanehirae OVATE Family Protein 7 (CkOFP7) demonstrated negative correlation with IAA content, while the MYB family transcription factor CkMYB306 exhibited positive correlation with ABA levels.

Validation of gene expression

To verify RNA-Seq results, twelve DEGs involved in AR formation were analyzed by qRT-PCR. The qRT-PCR expression profiles closely mirrored those obtained from transcriptome sequencing, supporting the accuracy of the RNA-Seq data (Fig. 12).

Fig. 12.

Fig. 12

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Validation of RNA-Seq expression patterns using qRT-PCR. Orange lines represent qRT-PCR relative expression, and green bars indicate RNA-Seq FPKM values across time points

Discussion

AR formation process in C. kanahirae

AR formation represents a crucial developmental stage in woody species, which can be categorized into two types: preformed and induced [36]. In preformed rooting, root primordia develop before cutting detachment from the mother plant, as documented in Carya illinoinensis and Acer truncatum, where differentiated root primordium cells are present [37]. Conversely, species of the genus Cinnamomum typically exhibit induced AR formation, wherein root initiation occurs through callus induction [7, 8, 38]. Although more complex than the preformed type, induced AR formation predominates in woody plants, including C. camphora and C. parthenoxylon [7, 38]. In this study, anatomical observations at CK0 showed no preformed root primordia, indicating that C. kanehirae follows the induced type. Root primordia may originate from various tissues, including the cambium, phloem, cortex, rays, pith, primary rays, vascular cambium, or callus tissues [39]. In the present study, abundant callus differentiation was observed in the cortical region, resulting in pronounced swelling at the basal portion of the stem segments. Thereafter, meristematic centers emerged within the callus and progressively developed into conical root primordia, which later elongated to give rise to ARs. Our results demonstrate that root primordia in C. kanehirae arise from callus tissue, aligning with previous reports on AR formation in Cinnamomum cuttings.

Regulation mechanism of nutrients during AR formation

Initial AR formation involves intensive cell division and differentiation, requiring substantial metabolic resources for energy support [40]. This study revealed a significant decrease in soluble sugar content during callus induction, indicating that sugars function as the primary energy source supporting AR formation through rapid cell proliferation [41]. Analysis of differentially expressed genes showed upregulation of Invertase (INV), which converts sucrose to glucose and fructose, thus enhancing direct energy supply. Conversely, downregulation of Sucrose synthase (SUS) indicated a temporary reduction in cell wall biosynthesis [42]. During root primordium formation, soluble sugar content increased significantly, indicating a coordinated supply of energy and carbon skeletons in the middle and late stages of AR development. Soluble proteins, primarily comprising enzymes and structural proteins, serve essential functions in AR development [43]. The early stage showed increased soluble protein content, indicating enhanced metabolic activity supporting rapid cell division and differentiation. This study demonstrated significant increases in soluble protein levels during callus formation, providing material support for metabolic activities and growth, thereby promoting root primordium initiation and AR elongation.

Regulation mechanism of oxidoreductases during AR formation

MDA, a lipid peroxidation product induced by ROS, serves as a reliable indicator of oxidative stress in plant tissues [44]. During callus induction at the basal ends of C. kanehirae cuttings, MDA levels remained low, while significant increases occurred during root primordia initiation and AR elongation. This trend suggests enhanced membrane lipid peroxidation, likely caused by the accumulation of ROS. Previous studies have shown that ROS can accumulate in epidermal cells at nodal regions, where they induce localized programmed cell death, thereby reducing the mechanical resistance during root primordia penetration and facilitating adventitious root emergence and elongation [45]. Consistent with this pattern, SOD activity declined markedly during the root primordium initiation stage, suggesting reduced ROS scavenging capacity. The down-regulation of SOD may facilitate a controlled increase in ROS, helping maintain an appropriate balance between O₂⁻ and H₂O₂ that is conducive to cellular differentiation [46]. The expression pattern of CkSOD1 corresponded with SOD enzymatic activity throughout AR formation, supporting this observation. Thus, the physiological changes observed at CK2 likely reflect a controlled ROS accumulation and moderate oxidative stress, which may act as a signaling cue promoting root primordia formation and subsequent adventitious root development [47]. POD, which eliminates H₂O₂ and contributes to cell wall strengthening, exhibited stage-specific regulation: CkPOD68, associated with lignin biosynthesis and cell wall reinforcement, showed significant downregulation during callus induction, suggesting reduced defense responses that may facilitate cellular dedifferentiation during initial AR formation [48]. PPO, involved in phenolic metabolism and tissue browning, showed marked suppression during callus formation, suggesting that browning inhibition supports callus differentiation and proliferation.

Circadian Rhythm Involvement in early stage of AR formation

Enrichment analysis showed that the circadian rhythm pathway was significantly enriched during callus induction in C. kanehirae, consistent with previous studies reporting the involvement of circadian regulation in the early stages of AR formation in mulberry cuttings [49]. The circadian clock integrates environmental signals with internal developmental processes through the central oscillator, light signaling pathways, and hormonal regulation [50]. During the transition of stem cuttings from a shoot state to root formation, the circadian system may undergo adjustments to coordinate developmental reprogramming [51].

In this study, several core clock genes, including CkPRR, CkLHY, CkPRR7, and CkPRR5, were downregulated during callus induction, suggesting that early AR initiation may be associated with a temporary attenuation of circadian oscillatory activity, which may facilitate cell dedifferentiation and root primordium initiation [52]. Meanwhile, the upregulation of photoreceptor-related genes such as CkPHYA and CkPHYB may enhance light signal input into the circadian system, thereby contributing to clock regulation [53]. Studies have indicated a close relationship between circadian rhythms and auxin-regulated processes during root development [54]. Therefore, the potential interaction between circadian regulation and auxin during AR formation in C. kanehirae cuttings will be further discussed in the following section.

Regulation of polar auxin transport during early AR formation stage

Endogenous phytohormones are critical in AR formation, with auxin as a primary regulator of root induction through polar auxin transport (PAT) [55]. This study revealed significant upregulation of several PIN genes associated with PAT during callus induction, displaying expression patterns inverse to IAA content. This inverse relationship indicates a potential feedback mechanism where decreased auxin levels may enhance PIN transcription, facilitating local auxin redistribution to root primordium initiation sites.

Previous studies showed that circadian rhythms regulate auxin-related growth processes, potentially through PIN3-mediated auxin transport [56]. Light signaling is known to regulate auxin transport by affecting the polarity and localization of PIN proteins, thereby influencing auxin distribution during plant development [57]. In the present study, the upregulation of CkPHYA and CkPHYB suggests enhanced light signal perception, which may contribute to the redistribution of auxin through PIN-mediated transport. In addition, the marked upregulation of CHALCONE SYNTHASE (CHS), coupled with enrichment of the flavonoid biosynthesis pathway in STEM analysis, indicates activation of flavonoid metabolism during callus formation. Research has shown that flavonoids function as auxin transport modulators by affecting PIN protein activity and localization [58]. Thus, enhanced flavonoid biosynthesis during this stage may facilitate spatial regulation of auxin distribution and root primordium initiation. ROS, generated by RBOH enzymes, have been shown to influence cytoskeletal dynamics and vesicle trafficking, potentially affecting cellular auxin distribution [59]. In the present study, the hub gene CkRbohA identified through WGCNA demonstrated negative correlation with IAA levels, suggesting that RBOHA-mediated ROS production may participate in a redox-associated regulatory network affecting local auxin accumulation during callus induction. [47]. Previous studies have indicated that ROS can modulate auxin transport by influencing the activity or localization of PIN transporters, thereby contributing to the establishment of local auxin gradients required for root primordium initiation [47]. In addition, flavonoids, which are known modulators of auxin transport, may interact with ROS signaling to further refine auxin distribution patterns within the rooting zone.

Regulation mechanism of auxin-cytokinin signaling during AR formation

In contrast to IAA’s dynamic changes, ZR exhibited an opposite pattern, while DEGs involved in cytokinin signaling showed stage-specific expression patterns. The auxin/cytokinin ratio maintains crucial importance in AR formation: moderate ratios promote callus induction, while higher ratios facilitate root regeneration [60]. Auxin-responsive transcription factors (ARFs) are considered key mediators linking hormonal dynamics with developmental reprogramming. Correlation analysis revealed that CkARF3 showed a positive association with both IAA levels and the IAA/ZR ratio, suggesting that it rapidly responds to changes in auxin signaling and hormonal balance. As downstream targets of ARFs, Lateral Organ Boundaries Domain (LBD) transcription factors facilitate auxin-responsive transcriptional regulation in this process. The significant upregulation of CkLBD16 during callus formation aligns with the conserved function of LBD genes, as their poplar homologs similarly promote callus initiation under low auxin conditions [61].

As development progressed to the root primordium formation stage, endogenous IAA levels increased markedly while cytokinin levels declined. Research has shown that ARF3 promotes primordium development by directly suppressing cytokinin biosynthesis, response genes, and key differentiation-repressing genes [62]. In addition, ARR genes act as important regulators in cytokinin signaling and contribute to root primordium and root meristem formation by modulating cytokinin responses and maintaining hormonal balance [63]. In Arabidopsis thaliana, auxin regulates cytokinin responses through ARF5/MONOPTEROS’s negative regulation of ARR7 and ARR15, enabling precise hormonal crosstalk [64]. Interestingly, several A-type cytokinin-responsive genes were significantly upregulated at this stage. As negative regulators of cytokinin signaling, the significant induction of CkARRs may represent a feedback regulatory mechanism within the cytokinin pathway and may also suggest a potential antagonistic interaction with ARF3, thereby contributing to the initiation of root primordia.

Regulation mechanism of ABA-responsive networks during AR formation

ABA is a crucial phytohormone that regulates cell division and elongation, thereby influencing root elongation and overall root system architecture [65]. In this investigation, endogenous ABA content increased progressively during AR formation. The hub genes CkPYL4 and CkMYB306, identified through WGCNA, exhibited a strong positive correlation with ABA concentration. The PYL genes encode a family of soluble receptors that serve a central role in ABA signal perception and transduction [66]. In the canonical ABA signaling pathway, PYL receptors interact with PP2Cs to relieve the inhibition of SnRK2 kinases, thereby activating downstream signaling cascades involved in stress responses and developmental regulation [67]. Elevated expression of PYL genes during AR development may indicate activation of ABA signaling, which contributes to stress adaptation and the precise regulation of hormonal interactions necessary for efficient root initiation [68]. In Triticum aestivum, the PYL4 gene demonstrates predominant expression in roots, with its transcription significantly induced under stress conditions in response to elevated ABA levels [69]. A comparable expression pattern of CkPYL4 was observed in this study, indicating that CkPYL4 may participate in ABA-mediated regulation of plant growth and osmotic stress responses during AR formation in C. kanehirae cuttings. Correlation analysis further revealed that the downstream ABA signaling gene CkSnRK2 displayed a similar expression trend to CkPYL4, supporting the activation of the ABA signaling pathway during AR formation. In Arabidopsis thaliana, MYB transcription factors function as positive regulators of ABA responses, enhancing plant adaptation to developmental cues, suggesting that CkMYB306 may similarly engage in ABA-mediated signaling during AR formation [70]. Notably, AtMYB96 has been reported to induce GH3 expression, thereby repressing auxin signaling [71]. This is consistent with our observation of upregulated CkGH3 expression and the concomitant decline in IAA concentration during the root elongation stage in C. kanehirae. Although direct functional evidence in C. kanehirae is lacking, these patterns support a model in which ABA may indirectly modulate IAA homeostasis via MYB-mediated regulation of GH3, facilitating orderly adventitious root elongation.

The OVATE Family Protein (OFP) family comprises transcriptional repressors that influence plant growth and development through participation in feedback regulation of phytohormone signaling and biosynthetic pathways [72]. For example, early seedling root growth in A. thaliana is regulated by AtOFP1 in an ABA-dependent manner [73]. Similarly, CkOFP7 demonstrated a significant positive correlation with ABA concentration, suggesting its potential role as a transcriptional repressor during AR formation in C. kanehirae and its participation in growth regulation through ABA-mediated signaling pathways.

During AR regeneration from cuttings in soil, explants inevitably encounter various environmental stresses. Previous research has shown that in poplar cuttings, the JA and ABA pathways operate cooperatively to enhance stress tolerance, where JA amplifies ABA signaling through a positive feedback-like mechanism, facilitating AR regeneration under adverse conditions [24]. In this study, genes involved in JA biosynthesis showed significant enrichment in the purple module, which demonstrated a strong positive correlation with ABA concentration, suggesting potential JA and ABA interaction during AR formation in C. kanehirae cuttings. In the JA biosynthetic pathway, OPR catalyzes 12-oxo-phytodienoic acid (OPDA) reduction and functions as a key enzyme [74]. The identified hub gene, CkOPR1, showed marked upregulation during the callus induction stage, suggesting activation and accumulation of JA biosynthesis at this stage, potentially contributing to wound stress protection and creating favorable conditions for subsequent AR development.

In summary, AR formation in C. kanehirae is governed by an integrated network of metabolic, redox, and hormonal signals. Callus induction is fueled by metabolic reprogramming, including dynamic soluble sugar and protein changes, while moderate ROS accumulation and stage-specific oxidoreductase activity promote cellular dedifferentiation and primordium initiation. Polar auxin transport, coordinated with flavonoid metabolism and ROS signaling, establishes local IAA gradients critical for root primordium formation. Hormonal crosstalk further modulates AR development: ABA signaling, mediated by hub genes CkMYB306, may indirectly regulate IAA homeostasis via GH3, facilitating orderly root elongation; cytokinin dynamics align with auxin to support callus induction and differentiation; JA biosynthesis, represented by CkOPR1, cooperates with ABA to enhance stress adaptation. Collectively, these findings depict a unified regulatory framework in which hub genes integrate metabolic, redox, and hormonal cues to orchestrate callus formation, root primordium initiation, and adventitious root elongation, providing insights into optimization of rooting conditions.

Conclusions

In summary, AR formation in Cinnamomum kanehirae cuttings involves a precisely coordinated network integrating hormonal regulation, redox balance, and metabolic activity. The initial formation of callus tissue and subsequent root primordia differentiation coincide with dynamic changes in soluble sugar and protein contents, indicating high energy demand and biosynthetic activity. The temporal patterns of MDA, POD, and SOD suggest oxidative stress functions as a critical signaling and regulatory factor during root induction. Transcriptomic profiling revealed auxin signaling as a central component, while cytokinin acts synergistically with IAA and ABA contributes to stress adaptation, collectively promoting root initiation. Additionally, the involvement of circadian rhythm in regulating polar auxin transport emphasizes the temporal coordination underlying root development. WGCNA identified key hub genes, including CkPYL4, CkOPR2, CkOFP7, CkMYB306, and CkRbohA, associated with hormone signaling and stress responses. These findings provide new insights into the physiological and molecular mechanisms underlying AR formation in C. kanehirae and offer potential targets for improving cutting propagation efficiency. Future studies should focus on the functional validation of these candidate hub genes. Integrating physiological analyses with molecular approaches will further clarify the regulatory networks governing AR formation and support the development of more efficient propagation strategies for C. kanehirae and other woody species.

Supplementary Information

Supplementary Material 1 (18.4KB, docx)

Acknowledgements

We thank the reviewers for their critical review of the manuscript.

Authors’ contributions

T.Z. Conceptualization; Y.Z. (Yueting Zhang). methodology; X.L.software; Y.Z. (Yuntong Zhang) and Y.Z.(Yongjie Zheng) validation; Y.Z. (Yuntong Zhang), X.L. and Y.Z.(Yongjie Zheng) formal analysis; X.D. and Y.Z.(Yongjie Zheng) investigation; X.D. and T.Z resources; Y.Z. (Yueting Zhang) data curation; Y.Z. (Yuntong Zhang)writing—original draft preparation; X.L. and Y.Z.(Yongjie Zheng) writing—review and editing; Y.Z. (Yuntong Zhang) and X.D. visualization; J.W. supervision; J.W. and T.Z. project administration; T.Z. funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the the Basic Research Project of the Jiangxi Academy of Forestry (Project No. 2025522001 and 2025522704).

Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA029237) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/browse/CRA029237.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

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.

Yuntong Zhang and Xinliang Liu contributed equally to this work.

Contributor Information

Yongjie Zheng, Email: zyj_bio2015@163.com.

Ting Zhang, Email: zhangtycx@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (18.4KB, docx)

Data Availability Statement

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA029237) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/browse/CRA029237.

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