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Article

Hormone Fluctuation and Gene Expression During Early Stages of the Hickory Grafting Process

by
Qiaoyu Huang
1,2,†,
Haixia Liu
1,2,†,
Qinyuan Shen
1,2,†,
Huwei Yuan
1,2,
Fuqiang Cui
1,2,
Daoliang Yan
1,2,
Wona Ding
3,
Xiaofei Wang
1,2,* and
Bingsong Zheng
1,2,*
1
National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A&F University, Hangzhou 311300, China
2
Provincial Key Laboratory for Non-Wood Forest and Quality Control and Utilization of Its Products, Zhejiang A&F University, Hangzhou 311300, China
3
College of Science and Technology, Ningbo University, Ningbo 315300, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(14), 2229; https://doi.org/10.3390/plants14142229
Submission received: 6 May 2025 / Revised: 13 July 2025 / Accepted: 17 July 2025 / Published: 18 July 2025
(This article belongs to the Section Plant Development and Morphogenesis)
Browse Figures
Versions Notes

Abstract

Grafting involves complex hormonal interactions at graft interfaces that are not yet fully understood. In this study, we analyzed hormone fluctuations and gene expression during callus proliferation and vascular tissue differentiation in hickory (Carya cathayensis Sarg.) grafts. Cytokinin and ethylene precursor ACC levels steadily increased after grafting. The biosynthetic genes for these hormones (IPT3, ACS1, ACO1, and ACO5) exhibited heightened expression. Genes related to cytokinin signaling (RR3, ARR4, and ZFP5) and ethylene signaling (MKK9, ESE1, and ESE3) were similarly upregulated. Conversely, genes associated with jasmonic acid, abscisic acid, and strigolactone pathways were downregulated, including synthesis genes (AOC4 and AOS) and those involved in signal transduction (NAC3, WRKY51, and SMAX1). Correspondingly, JA-Ile and 5-deoxystrigol levels significantly decreased. Indole-3-acetic acid (IAA) levels also dropped during the early stages of graft union formation. These results suggest that low auxin concentrations may be essential in the initial stages after grafting to encourage callus proliferation, followed by an increase at later stages to facilitate vascular bundle differentiation. These findings imply that maintaining a balance between low auxin levels and elevated cytokinin and ethylene levels may be critical to support cell division and callus formation during the initial proliferation phase. Later, during the vascular differentiation phase, a gradual rise in auxin levels, accompanied by elevated ethylene, may facilitate the differentiation of vascular bundles in hickory grafts.

1. Introduction

Grafting represents a fundamental horticultural technique wherein a branch or bud is joined to the stem or root of another plant to generate a new, grafted individual [1]. This method is extensively utilized in fruit and vegetable seedling production and serves a vital role in agricultural research [2,3]. Successful grafting requires the formation of callus tissue connecting the initially separate scion and rootstock, with callus formation indicating compatibility between graft partners [4]. The reconstruction of vascular tissue at the graft site is essential for graft viability [5], and grafting triggers physiological responses related to wounding hormones and signaling pathways critical for vascular tissue regeneration and attachment [6,7]. This response enhances the activity of genes related to auxin, a key hormone in vascular development [8]. Auxin distribution is regulated by polar auxin transport controlled by PIN and ABCB proteins. Activation of PIN1 and ABCB1 genes is vital for graft development, as these genes facilitate auxin efflux and transport [9].
The results of a number of studies demonstrate the significance of additional phytohormones, including cytokinins (CK), ethylene (ETH), and abscisic acid (ABA), in the grafting process [10,11,12,13]. These hormones interact with auxins to regulate plant growth and development. Research has highlighted that CK influence auxin transport during vascularization by regulating PIN protein distribution within nascent vascular tissues in Arabidopsis [14]. In pecan (Carya illinoinensis (Wangenh.) K. Koch) grafting, a B-type Arabidopsis response regulator (ARR) functions as a primary regulator of callus proliferation at the graft junction, mediated by CK [15]. Incision of the grafted surface initiates a wounding stress response that is strongly regulated by the jasmonic acid (JA) signaling pathway. A substantial increase in JA levels occurs within 30 s of wounding in Arabidopsis, with changes in JA-Ile levels detected within 5 min of wounding [16]. JA additionally serves as a crucial long-distance signal in the wound signaling pathway and plays an essential role in promoting union at the graft interface [17].
Transcriptomic analyses have revealed that ETH biosynthesis genes are activated in grafts of Arabidopsis hypocotyls [18] and tobacco [19]. Application of the ETH precursor 1-aminocyclopropane-1-carboxylate (ACC) enhanced scion growth in tobacco grafts; in comparison, the ETH inhibitor aminoethoxyvinylglycine (AVG) impeded graft union and diminished scion growth [19], demonstrating the positive role of ETH in grafting. Synergistic effects between hormone classes have been observed, with IAA, GA (gibberellin), and CK promoting cell differentiation, vascular bundle formation, and xylem/phloem reconnection [20]. Similarly, strigolactones (SL) operate in conjunction with auxin and CK to regulate plant branching [21]. However, comprehensive quantification of hormones and related compounds in their biosynthetic pathways at the rootstock–scion graft junction remains an emerging research area.
Hickory is a valuable nut tree native to China. The hickory industry is constrained by numerous challenges, including slow plant growth, excessive tree height, restricted habitat, and seed cultivation complications. Grafting serves as a method to enhance the adaptability and production of hickory [22]. The authors of previous studies have explored the role of auxin in hickory grafting, examining auxin response factors (ARFs) [23], AUX/IAA [24], CcGH3 [25], TIR1/AFB [26], CcPIN [27], and CcABCB [28]. However, a comprehensive understanding of hormonal changes and molecular mechanisms supporting graft survival remains limited. Based on the results of our previous study [22], we analyzed hormone levels in hickory grafts at various time points using LC–MS/MS to elucidate their role in graft establishment. In addition, we utilized transcriptomic data to examine changes in hormone-related gene expression during early graft union formation.

2. Results

2.1. Variations in Endogenous Hormone Levels During Different Phases of Hickory Grafting

In previous studies on hickory grafting, researchers have identified 7 and 14 days after grafting (DAG) as crucial stages for morphological changes, including necrotic layer formation, callus growth, and new vascular tissue development [22,29]. In this study, we quantified phytohormone concentrations in hickory rootstocks and scions from 0 to 14 DAG. During the initial phase following hickory grafting, 7 and 14 DAG, endogenous auxin levels exhibited a marked decrease (Figure 1A). Similarly, SL and ABA concentrations significantly decreased during this period (Figure 1B,C). JA content changes demonstrated tissue specificity; in the rootstock, there was a rapid and substantial decrease immediately after grafting; in the scion, in comparison, a notable reduction was observed only at 14 days post-grafting (Figure 1D). Salicylic acid (SA) level fluctuations differed markedly between the rootstocks and scions. Post-grafting, rootstock SA content remained relatively stable; in the scion, in comparison, a significant decrease was recorded (Figure 1E). In contrast, trans-Zeatin riboside (tZ) levels increased significantly in the rootstock after grafting, with the scion exhibiting a substantial increase at 14 DAG (Figure 1F). In addition, the pattern of change in 1-aminocyclopropane-1-carboxylic acid (ACC) paralleled that of CK. Post-grafting, rootstock ACC content increased substantially, with that of the scion showing significant elevation 14 days thereafter (Figure 1G).

2.2. Modifications of Auxin Metabolism and Signaling Pathways

Auxin plays a crucial role in vascular tissue development, which is essential for successful graft union formation [13]. We analyzed auxin and its associated metabolites concentrations in rootstocks and scions at 0, 7, and 14 DAG to understand their contribution to grafting and vascular bundles integration in hickory (Figure 2A–G). Among IAA amino acid conjugates, IAA-alanine (IAA-Ala) demonstrated initial accumulation (0 DAG) followed by a gradual decline in the scion, with IAA-aspartate (IAA-Asp) exhibiting an increase (Figure 2A,B). IAA-Glu levels remained stable in the scion and exceeded rootstock levels (Figure 2C). Indole-3-acetonitrile (IAN) concentrations remained relatively constant in both the rootstocks and scions (Figure 2D). Indole-3-carboxylic acid (ICA) and indole-3-carbaldehyde (ICAld), byproducts of tryptophan (TRP) metabolism, exhibited continuous downregulation in scion tissues with temporal coordination to the declining TRP pool (Figure 2E,F,I). Indole-3-pyruvic acid (IPA) content exhibited significant increases at both 7 DAG and 14 DAG compared to 0 DAG in the scions (Figure 2G). Methoxy-indole-3-acetic acid (MEIAA), a methionine derivative of IAA, demonstrated a negative correlation with TRP, suggesting potential involvement in IAA signal termination or transport regulation (Figure 2H). TRP, an IAA synthesis precursor, showed high initial expression in the rootstock followed by gradual decline (Figure 2I).
Analysis of gene expression patterns provided further insights into auxin dynamics during graft formation (Figure 3A). Consistent with decreased IAA levels, the auxin biosynthesis gene tryptophan aminotransferase related 2 (TAR2) exhibited downregulation in rootstock from 0 to 14 DAG. In contrast, methylesterase 17 (MES17), which encodes a protein that converts inactive MeIAA to active IAA, exhibited upregulation during graft formation in both scions and rootstocks (Figure 2C). The genes encoding F-box auxin receptors AFB5 and AFB2, which are essential regulators of auxin perception, showed upregulation in rootstocks and scions throughout graft union formation. In addition, nucleoside diphosphate kinase 2 (NDPK2), encoding a protein that modulates auxin transport, exhibited upregulation during graft formation, notably in rootstocks. Activation of auxin signaling pathways involves complex regulatory cascades. Accordingly, the expression levels of auxin-activated signaling pathway genes COV1 and ETA3 exhibited significant downregulation at 7 and 14 DAG. qRT-PCR validation confirmed these expression patterns across the examined genes, revealing that TAR2, ETA3, and COV1 were upregulated after grafting in both rootstocks and scions at 7 and 14 DAG; in comparison, MES17, AFB2, AFB5, and NDPK2 exhibited corresponding downregulation (Figure 3B–H). These expression trends closely align with transcriptomic data.

2.3. Modifications of CK Metabolism and Signaling

CK are plant hormones derived from adenine. They are involved in various aspects of plant development, including cell division, lateral root formation, and meristem maintenance. The wound healing process during grafting is similar to callus formation in wounded plants. Our analysis results highlight an increased cytokinin response during wound healing in hickory grafting (Figure 1F). Quantitative analysis demonstrated coordinated induction of multiple CK metabolites including: cis-zeatin (cZ), cis-zeatin riboside (cZR), cZ-riboside dihydrozeatin (DZ), dihydrozeatin ribonucleoside (DZHR), dihydrozeatin-7-glucoside (DHZ7G), dihydrozeatin-O-glucoside riboside (DHZROG), N6-isopentenyladenosine (IPR), trans-zeatin-O-glucoside (tZOG), and trans-zeatin riboside (tZR), all exhibiting significant accumulation compared to 0 DAG (Figure 4A–I). DZ exhibited the most pronounced change, with it being undetectable at 0 DAG but showing progressive accumulation at 7 and 14 DAG in both rootstocks and scions (Figure 4C). In contrast, meta-Topolin-9-glucoside (mT9G) levels remained constant throughout the experimental period in both the scions and rootstocks (Figure 4J).
Our RNA-seq analysis results revealed significant upregulation of IPT3, which encodes key enzymes catalyzing the rate-limiting step in CK biosynthesis. Notably, IPT3 expression exhibited upregulation at 7 and 14 DAG in both scions and rootstocks (Figure 5A). CK transport exhibited enhancement through elevated ABCG14 expression in rootstocks after grafting. Furthermore, components of CK signaling pathways demonstrated coordinated induction. Specifically, type-A response regulators RR3 and ARR4 exhibited upregulation in both scions and rootstocks; in comparison, zinc finger protein ZFP5 exhibited similar expression patterns. Multiple LOG genes (LOG1, LOG4, and LOG5) encoding enzymes essential for CK activation through riboside hydrolysis displayed dynamic expression changes during grafting in rootstocks and scions [30]. qRT-PCR analysis validated these expression profiles, confirming that IPT3, LOG5, RR3, and ABCG14 demonstrated sustained upregulation after grafting in both rootstocks and scions at 7 and 14 DAG; in comparison, RR4 exhibited reciprocal downregulation. Notably, LOG1 and LOG4 displayed upregulation in rootstocks contrasting with downregulation observed in scions (Figure 5B–H). These expression patterns demonstrated concordance with transcriptomic data.

2.4. Dynamics of JA Metabolism and Signaling

JA serves as a crucial long-distance signaling mediator in response to both biotic and abiotic stresses [31], in addition to wound signaling pathways [16]. Quantitative analysis of JA metabolites revealed a systemic reduction in JA levels across both rootstocks and scions post-grafting. Conjugated forms such as JA-Phe, JA-Val, and JA-Ile exhibited coordinated downregulation, with JA-Val becoming undetectable after grafting (Figure 6A–C). Notably, levels of H2JA, a bioactive conjugate involved in JA homeostasis, also decreased after grafting (Figure 6D). Oxophytodienoic acid (OPDA) exhibited a slight reduction in rootstocks but remained stable in scions, corresponding to the unchanged levels of methyl jasmonate (MEJA) (Figure 6E,F). Of note, oxidative JA derivatives OPC-4 and OPC-6 demonstrated time-dependent accumulation, peaking at later grafting stages (Figure 6G,H).
Consistent with the measured JA levels, our transcriptome analysis results revealed significant downregulation of the JA biosynthesis gene allene oxide cyclase 4 (AOC4), particularly in rootstocks during the callus formation stage (Figure 7A). Genes encoding allene oxide synthase (AOS), which catalyzes the initial step in JA biosynthesis, exhibited downregulation expression profiles. Several transcription factors associated with JA signaling pathways also downregulated downregulation. Notably, NAC3 (a known JA-responsive gene [32]) exhibited marked downregulation in rootstocks during 0–7 DAG. Similarly, WRKY51, which mediates JA-induced defense responses [33], exhibited significant repression in both tissues throughout the 14-DAG (Figure 7A). This coordinated downregulation of JA biosynthesis and signaling components likely reflects an adaptive mechanism that attenuates defense responses to facilitate successful graft union formation and tissue repair during early developmental stages. Our qRT-PCR analysis results confirmed these expression trends, confirming that AOC4, AOS, and WRKY51 showed sustained downregulation after grafting at both 7 DAG and 14 DAG; in comparison, NAC3 exhibited downregulation in rootstocks, contrasting with upregulation in scions (Figure 7B–E). These expression patterns demonstrate agreement with transcriptomic data.

2.5. Dynamics of ETH Metabolism and Signaling

Consistent with its established role in plant wound responses and cell expansion, exogenous ETH application enhances callus formation and cell proliferation [34]. Analysis of ACC levels, the immediate ETH precursor, revealed progressive accumulation in rootstock tissues across the grafting timeline. The ACC concentration reached 75 ng·g−1 at 14 DAG, representing a two-fold increase relative to day 0 levels (Figure 1G). Scion tissues exhibited a parallel temporal increase in ETH content.
These biochemical changes were correlated with the transcriptomic upregulation of the ETH biosynthesis gene 1-amino-cyclopropane-1-carboxylic acid synthase 1 (ACS1) in both graft tissues. ACS catalyzes the rate-limiting step in ETH synthesis by producing ACC, which is subsequently converted to ETH by ACC oxidase (ACO). Notably, ACO1 expression increased in rootstocks during graft formation, with ACO5 exhibiting upregulation in scions (Figure 8A). Key regulators of ETH signaling also demonstrated significant induction. The MAPKK family member MKK9, which modulates ETH biosynthesis and calcineurin signaling, demonstrated substantial upregulation, as did the ETH-responsive ERF/AP2 transcription factors ESE1 and ESE3 (Figure 8A). MKK9 activation triggers the expression of multiple stress-related genes including ACS2, ACS6, and cytochrome P450 enzymes involved in phytoalexin biosynthesis [35]. Our qRT-PCR analysis results validated these expression dynamics by confirming that ACS1, MKK9, and ESE1 exhibited sustained upregulation in both rootstocks and scions at 7 and 14 DAG (Figure 8B,D,E). In contrast, while ACO1, and ESE3 showed rootstock predominant upregulation (Figure 8C,F). These expression patterns correspond closely with the transcriptomic data.

2.6. Modifications of SL Metabolism and Signaling

SL have been shown to modulate cambial activity via an auxin-dependent mechanism, suggesting their potential involvement in the grafting process [36]. A pronounced decrease in SL concentration was observed after grafting, particularly at 7 DAG, when SL levels decreased by approximately 4.5- and 8.4-fold in the rootstock and scion, respectively (Figure 1B). Transcriptomic profiling revealed coordinated changes in SL pathway genes. DWARF14 (D14), which encodes an α/β hydrolase essential for SL perception, exhibited significant upregulation at both 7 and 14 DAG. Concurrently, the SL-responsive suppressor gene SMAX1 displayed marked downregulation during the same period (Figure 9A). The expression patterns of D14 and SMAX1 were further validated by qRT-PCR analysis (Figure 9B,C). These molecular changes align with physiological observations. Reduced SL levels appear to alleviate signaling repression, thereby promoting cell proliferation and callus formation at the graft interface [37]. This regulatory mechanism may facilitate vascular reconnection and tissue integration during early graft union establishment.

3. Discussion

3.1. Auxin-Induced Vascular Tissue Formation and the Graft Union Process

Auxins play essential roles in vascular tissue formation and wound union processes, particularly in the differentiation of cambium into secondary phloem and secondary xylem, which requires high auxin concentrations [13,38]. During the initial stage of hickory grafting, the concentration of IAA, the principal form of auxin, decreased by 78% in rootstocks and 24% in scions, accompanied by significant reductions in the auxin precursor Trp. This decline in IAA levels aligns with transcriptomic evidence showing decreased expression of auxin synthesis pathway genes, demonstrating that the graft site had not yet progressed to vascular bundle differentiation. This observation aligns with anatomical studies that have identified callus formation as a preliminary phase preceding vascular reconnection [22]. The observed downregulation of TAR2 (Figure 3B) in rootstocks further confirms reduced auxin biosynthesis during early grafting. However, the concurrent upregulation of MES17 (Figure 3C)—which converts inactive MeIAA to active IAA—suggests a compensatory mechanism. This two-part regulatory strategy involves TAR2 suppression to lower new auxin production and MES17 activation to recycle stored auxin, ensuring sufficient auxin for early wound responses while maintaining homeostasis.
The wounding phase, characterized by isolation layer formation at the rootstock-scion junction, precedes callus proliferation and vascular differentiation. Similar reductions in IAA at the graft unions have been documented in pecan [15] and litchi (Litchi chinensis Sonn.) [39], with low IAA concentrations predominantly inducing phloem differentiation, whereas high concentrations promote both phloem and xylem [9]. Notably, IAA levels recover, coinciding with the peak of xylem differentiation [9]. Early auxin responsive genes, including AUX/IAA, GH3, and PIN families, significantly impact signaling pathways [24,25,27]. The GH3 gene family regulates auxin homeostasis, potentially through suppression during the early grafting stages, which may result from inhibited amino acid synthesis (e.g., IAA-Asp) and subsequent free IAA release [25]. P The SCF-type E3 ubiquitin ligase-proteasome pathway, involving TIR1/AFB receptors, is central to auxin signaling [20,40]. In this study, decreased auxin levels between 7 and 14 DAG coincided with significant upregulation AFB5 and AFB2 (Figure 3E,F), genes implicated in SCF-dependent proteolytic metabolism. Elevated AFB expression likely primes the system for rapid auxin signaling despite low IAA levels, facilitating the transition from wound healing to cell proliferation.
Rootstock-specific upregulation of NDPK2 (Figure 3H)—a modulator of auxin transport—suggests localized reprogramming of auxin flux. By regulating PIN1 phosphorylation (as in Arabidopsis), increased NDPK2 expression may redirect auxin toward the graft interface, creating gradients essential for cambium activation [41,42]. Concurrently, downregulation of COV1 and ETA3 (Figure 3D,G)—genes involved in auxin-activated signaling—likely reduces auxin efflux, concentrating residual IAA to promote localized cell division rather than long-distance transport. Together, these dynamics illustrate auxin’s dual role: early depletion facilitates CK-dominant callus proliferation, whereas later rebound (coinciding with AFB upregulation) primes vascular differentiation, underscoring the hormone’s pivotal role in orchestrating graft union phases.

3.2. Role of CK in Callus Formation After Grafting in Hickory

CK are fundamental to wound union and subsequent differentiation processes [8]. Evidence suggests that initial post-injury stages are characterized by increased concentrations of various CK derivatives, which are integral to the healing process [43,44,45]. In hickory, tZ levels peaked at 7 DAG before declining at 14 DAG, with tZ and its precursor tZR showing maximum accumulation in both rootstocks and scions at 7 DAG. This pattern aligns with observations in oil tea (Camellia oleifera Abel.) [34] and Arabidopsis [46], where CK facilitate callus initiation and proliferation. Anatomical investigations have revealed that callus formation in hickory occurs mainly between 7 and 14 DAG, coinciding with isolation layer breakdown and increased cell division [22]. The elevated tZ levels during this period aligned with a role for CK in promoting cell division and callus formation, thus enhancing graft survival. Our transcriptomic analysis results revealed enhanced expression of CK-related genes, including the IPT3 gene for CK synthesis at 7 and 14 DAG [45]. Additionally, the ABCG14 root-to-shoot CK transporter showed significant upregulation at 14 DAG in rootstocks [46]. The rice homolog OsABCG18 similarly regulates long-range CK transport [47], suggesting that CK movement from roots to shoots may be important for successful grafting.
IPT3 upregulation (Figure 5B) directly correlated with elevated CK levels (Figure 1F), confirming its role as the rate-limiting enzyme in biosynthesis. Sustained IPT3 expression in both tissues likely reflects wound-induced CK activation, consistent with Arabidopsis and tobacco studies linking IPT genes to CK-driven cell proliferation [48,49]. Rootstock-specific ABCG14 upregulation (Figure 5H) underscores its function in CK transport, generating gradients to direct CK toward the graft interface for localized cell division. Coordinated induction of type-A response regulators (RR3 and ARR4) and ZFP5 (Figure 5A,F,G) reflects active CK signaling, with type-A RRs forming feedback loops to balance proliferation and differentiation. Tissue-specific LOG gene expression (Figure 5C–E) reveals nuanced CK regulation: systemic LOG5 upregulation supports broad CK activation, whereas LOG1/LOG4 divergence (root-up/scion-down) suggests localized control of CK riboside hydrolysis. Elevated LOG1/LOG4 in rootstocks may enhance CK bioavailability for callus initiation; in comparison, their suppression in scions prevents hyperplasia from excessive CK accumulation. Together, these dynamics highlight CK’s pivotal role in coordinating wound healing and vascular differentiation, thereby prioritizing meristematic growth during critical graft union phases.

3.3. Interaction Between ETH and Auxins During Hickory Grafting

The gaseous phytohormone ETH serves a vital role in plant growth, development, and stress responses. Its biosynthesis is activated at wound sites of plant grafts, aiding in the healing and union processes [50,51,52]. In this study, the results of biochemical analyses revealed a progressive increase in ACC concentration, reaching twice the initial level by 14 DAG, which aligns with transcriptomic upregulation of the ETH biosynthesis gene ACS1 in both rootstocks and scions. ACS1, encoding the rate-limiting enzyme in ETH synthesis, converts S-adenosyl-L-methionine to ACC, which is subsequently oxidized to ETH by ACC oxidase. Notably, ACO1 expression increased in rootstocks, while whereas ACO5 showed scion-specific upregulation (Figure 8A,C), indicating tissue-specific modulation of ETH production. These findings corroborate grafting studies in tobacco, wherein elevated ACC levels at the wound site promoted isolation layer formation and union establishment [19].
ETH signaling was further activated through the coordinated induction of key regulatory components. The MAPKK family member MKK9, which links ETH biosynthesis to stress responses [35], demonstrated substantial upregulation in both tissues. Concurrently, ETH-responsive ERF/AP2 transcription factors ESE1 and ESE3 (ERF B-6 subfamily) were induced at 14 DAG (Figure 8E,F), paralleling observations in Arabidopsis wherein ERF1 promotes auxin biosynthesis by activating WEAK ETHYLENE INSENSITIVE2 (WEI2) [23]. This regulatory cascade is consistent with the well-established role of ETH acting upstream of auxin signaling. Previous studies have indicated that ETH enhances auxin biosynthesis and root growth regulation in Arabidopsis by directly promoting the expression of auxin-related genes [53,54]. In hickory, ESE upregulation may similarly regulate auxin-related processes, facilitating vascular bundle differentiation during later graft stages.

3.4. Necessity of Low SL Concentrations for Hickory Grafting

SL, carotenoid-derived molecules, are instrumental in the regulation of plant development and architecture [55]. In hickory, a pronounced reduction in SL concentration was observed post-grafting, with levels decreasing by approximately 4.5-fold in rootstocks and 8.4-fold in scions at 7 DAG (Figure 1B). This dramatic decline aligns with transcriptomic and qRT-PCR data showing demonstrating coordinated modulation of SL pathway genes. The α/β hydrolase receptor D14, essential for SL perception, was significantly upregulated at both 7 and 14 DAG, while whereas the SL-responsive suppressor gene SMAX1 exhibited marked downregulation during the same period (Figure 9A–C). These molecular dynamics suggest a negative feedback mechanism in which reduced SL bioavailability triggers compensatory D14 induction to enhance tissue sensitivity to residual SL, thereby optimizing signaling efficacy.
Research on SL-deficient mutants in Arabidopsis has shown reduced formation layer activity, which SL application could restore, indicating the influence of SL on cell division within the formation layer [36]. Despite these findings, the specific role of SL in the wound response remains unclear. Grafting experiments in pea (Pisum sativum L.) have highlighted the role of SL in suppressing branch development, serving as secondary messengers for auxin in the maintenance of apical dominance [56]. This interaction suggested a regulatory mechanism by which auxin modulates SL and CK biosynthesis to control stem branching [55].
Of note, our study revealed that SL levels and SMAX1 expression at hickory graft interfaces progressively declined below pre-grafting (0 DAG) levels during the observation period. These findings imply that the proliferation of hickory calluses and the differentiation of vascular bundles require low concentrations of SL. The successful use of SL-deficient mutants in grafting suggests that SL may exert a negative regulatory effect on graft union processes [57,58], highlighting the complex interplay of hormonal interactions necessary for successful grafting.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The experiment was conducted in a controlled environment greenhouse maintained at 25 ± 3 °C, with relative humidity of 60–70%, a 12 h light/12 h dark photoperiod, and a photosynthetic photon flux density of 500–700 µmol·m−2·s−1. Two-year-old hickory plants (var. ‘Zhelinshan 1’, Figure S1) of uniform size (height: 40–50 cm; stem diameter: 8–10 mm) were selected as rootstocks and cultivated in pots containing 10 kg of soil mixture (3:1 peat:perlite (v/v) with 2 kg/m3 controlled-release fertilizer (STANLEY®, Shanghai, China)). The seedlings were irrigated every five days.

4.2. Hickory Grafting and Sample Collection

Grafting was performed in April using two-year-old plants as rootstocks and 7–8 cm annual branches with buds from a 15-year-old hickory tree (var. ‘Zhelinshan 1’) as scions, ensuring genetic uniformity within the clone population. Skilled technicians pruned the rootstocks to a length of 8–10 cm and grafted them with annual branch scions, wrapping the joints in polyethylene film to promote successful union (see Figure S2). For each time point (0, 7, and 14 DAG), five grafts were randomly selected for sampling after grafting. Samples were collected from the graft union interface, encompassing slices from both the rootstocks and scions, which were excised using a sharp, sterile single-sided blade to a thickness of approximately 1 mm. Samples from rootstocks and scions were collected separately. Immediately following collection, the samples were immersed in liquid nitrogen and stored at −80 °C for subsequent analyses.

4.3. Determination of Hormone Concentrations

4.3.1. Sample Preparation and Extraction

For extraction, 50 mg of ground sample was mixed with 1 mL of a solvent mixture (methanol (Sigma-Aldrich®, St. Louis, MO, USA), water, and formic acid (Sigma-Aldrich®, St. Louis, MO, USA); 15:4:1) and 10 μL of an internal standard (100 ng·mL−1) (Table S3). The mixture was centrifuged at 12,000 rpm for 5 min at 4 °C to separate the soluble fraction. After centrifugation, the supernatant was evaporated to remove the solvent, reconstituted in 80% methanol, and filtered through a 0.22 μm membrane for LC–MS/MS analysis [59,60].

4.3.2. Ultra-Performance Liquid Chromatography (UPLC)

During our analysis, we employed a UPLC–Electrospray ionization–tandem mass spectrometry (ESI–MS/MS) system (QTRAP 6500+, SCIEX, Framingham, MA, USA) with a Waters ACQUITY UPLC HSS T3 C18 column (Waters, Milford, MA, USA). The solvent system included water with 0.04% acetic acid (solvent A) and acetonitrile with 0.04% acetic acid (solvent B). The gradient program began with 5% solvent B for the first minute, increased linearly to 95% solvent B from 1 to 8 min, maintained at 95% solvent B for 8 to 9 min, and then returned to 5% solvent B for a total of 12 min. The analysis involved the use of a 2 μL injection volume, as supported by evidence from a previous work [61,62,63].

4.3.3. ESI–MS/MS Conditions

Phytohormones were detected using a QTRAP 6500+ LC–MS/MS system in positive and negative ionization modes. Parameters included a source temperature of 550 °C and ion spray voltages of 5500 V for positive mode and −4500 V for negative mode. Phytohormones were identified using a customized multiple reaction monitoring (MRM) program. Data were collected using Analyst software version 1.6.3, and metabolites were analyzed with Multiquant software version 3.0.3, enabling precise measurement of phytohormone levels in the samples [64,65,66].

4.4. RNA-Sequencing

For sequencing, total RNA was extracted from the sample using an mRNA Mini Kit (Qiagen, Hilden, Germany). RNA was digested using Turbo-DNase (Ambion, Foster City, CA, USA). RNA quality was evaluated on a NanoPhotometer® spectrophotometer (IMPLEN, München, Germany). The integrity and quantity of total RNA were assessed using a BioAnalyzer (Agilent Technologies, La Jolla, CA, USA). RNA-Seq was performed by LC-bio (Hangzhou, China) on the Illumina HiSeq 4000 platform. Raw reads were trimmed with Trimmomatic (v0.39; ILLUMINACLIP:2:30:10, LEADING:20, TRAILING:20, SLIDINGWINDOW:4:15). Q20, Q30 and GC contents of the clean data were calculated. For alignment to the hickory reference genome (http://www.juglandaceae.net/ (accessed on 5 March 2024)), we used HISAT2 (v2.2.1; --dta --rna-strandness RF) with E-value < 1 × 10−5. Differential expression analysis involved the use of DESeq2 (v1.36.0; adjusted p-value < 0.05, |log2FC| > 1). Functional annotation of genes was performed using two public databases: KEGG (The Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology). Gene expression level was evaluated based on fragments per kilobase of exon model per million mapped reads (FPKM) (FDR < 0.05). The heat map representing the transcriptomic analysis was created by using TBtools (v2.310) to gain insights into gene expression dynamics in these pathways [67].

4.5. qRT-PCR Analysis

Total RNA was extracted from rootstock and scion tissues using a MiniBEST Plant RNA Extraction Kit (Takara, Dalian, China). Reverse transcription reactions were performed using the PrimeScriptTM RT Reagent Kit (Takara, Dalian, China) following the manufacturer’s protocol. Target gene expression levels were quantified via qRT-PCR with TB Green® Premix Ex Taq ™ II FAST qPCR (Takara, Dalian, China). Primer sequences for qRT-PCR analysis were designed using Primer Premier 5.0 software, with β -actin serving as the internal control (Table S2). The thermal cycling parameters were as follows: 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 10 s. All assays included three independent biological replicates, each with three technical replicates. Relative gene expression levels were calculated using the 2 −∆∆Ct method.

4.6. Statistical Analysis

Statistical analyses were performed using IBM SPSS Statistics 26.0, and graphs were created with GraphPad Prism 8.0. Significant differences were identified though analysis of variance (ANOVA) with a significance threshold of p < 0.05. Data are shown as the mean ± standard error (SE).

5. Conclusions

In summary, in this study, we have elucidated the dynamic hormonal interactions occurring during the early stages of hickory graft union formation. The hormones auxin, CK, ETH, JA, and SLs exhibit spatially and temporally regulated changes that orchestrate defense responses and tissue regeneration. An early reduction in auxin levels is associated with altered expression of biosynthesis genes (TAR2 and MES17), indicating a transition from vascular development to wound-induced cell proliferation. The accumulation of CK, driven by the activation of IPT3 and LOG, corresponds with meristem activation and callus growth. The biosynthesis (ACS1 and ACO) and signaling (MKK9) pathways of ETH facilitate stress adaptation and tissue fusion. Systemic suppression of JA (via downregulation of AOC4 and AOS) and alterations in SL biosynthesis (D14) and SL signaling (SMAX1) reflect a trade-off between reduced defense and enhanced regenerative growth. These hormonal interactions promote vascular reconnection, as evidenced by increases in tZ and ACC levels and the induction of ABCG14. Tissue-specific responses, such as the reduction in JA in the scion and the decline of SL in the rootstocks, underscore the complex communication occurring at the graft interface. These findings advance the understanding of hormone-mediated mechanisms underlying the early stages of graft union formation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14142229/s1. Table S1. Differentially expressed genes involved in hormone biosynthesis and signaling pathways in hickory rootstocks and scions at 0, 7, 14 DAG with corresponding FPKM Values. Table S2. Primers used in this study. Table S3. Chemical information for internal standard solution preparation. Figure S1. Phenotype of hickory ‘Zhelinshan 1’. Figure S2. Schematic diagram of hickory grafting procedure.

Author Contributions

Methodology, and formal analysis, Q.H. and H.L.; validation, Q.H. and Q.S.; investigation, Q.H.; resources, Q.H. and Q.S.; data curation, H.L. and W.D.; writing—original draft preparation, Q.H.; writing—review and editing, H.Y., F.C. and D.Y.; visualization, H.Y.; conceptualization, supervision, project administration, and funding acquisition, X.W. and B.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by grants from the National Natural Science Foundation of China (32071807), the Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties, China (2021C02066-12), the Scientific Research Development Fund Project of Zhejiang A&F University (2022LFR001, 2023LFR066), the Open Foundation of State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University (SKLSS-KF2023-07), “The Higher Education Discipline Innovation and Talent Introduction Project (111 Project)” Zhejiang A&F University (No. D18008).

Data Availability Statement

Data will be made available on request.

Acknowledgments

We are sincerely grateful to Guohua Xia for his invaluable help preparing the figures for ‘Zhelinshan 1’. We also thank Meng Liu for creating the schematic diagram of the hickory grafting method.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CKcytokinins
ETHethylene
ABAabscisic acid
JAjasmonic acid
ACC1-aminocyclopropane-1-carboxylate
ARRArabidopsis response regulator
AVGaminoethoxyvinylglycine
GAgibberellin
ARFauxin response factor
DAGdays after grafting
tZtrans-Zeatin riboside
IAA-AspIAA-aspartate
IAA-AlaIAA-alanine
IANIndole-3-acetonitrile
ICAIndole-3-carboxylic acid
ICAldindole-3-carbaldehyde
TRPtryptophan
IPAIndole-3-pyruvic acid
MEIAAMethoxy-indole-3-acetic acid
TAR2tryptophan aminotransferase related 2
MES17methylesterase 17
NDPK2nucleoside diphosphate kinase 2
cZcis-zeatin
cZRcis-zeatin riboside
DZcZ-riboside dihydrozeatin
DZHRdihydrozeatin ribonucleoside
DHZ7Gdihydrozeatin-7-glucoside
DHZROGdihydrozeatin-O-glucoside riboside
IPRN6-isopentenyladenosine
tZOGtrans-zeatin-O-glucoside
tZRtrans-zeatin riboside
mT9Gmeta-Topolin-9-glucoside
OPDAOxophytodienoic acid
MEJAmethyl jasmonate
AOSoxide synthase
AOC4allene oxide cyclase 4
ACOACC oxidase
UPLCUltra-performance liquid chromatography
ESI–MS/MSElectrospray ionization–tandem mass spectrometry

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Plants 14 02229 g001
Figure 1. Temporal dynamics of phytohormone accumulation in the rootstocks and scions during graft healing. Seven phytohormones (A–G) measured in the rootstocks and scions at three key stages of graft development: 0 DAG (white bars), 7 DAG (gray bars), and 14 DAG (black bars). Each panel represents a specific hormone: (A) IAA, (B) tZ, (C) ACC, (D) JA, (E) SA, (F) SL, and (G) ABA. Values are expressed as ng·g−1 fresh weight (FW). The data is presented as means ± SE (the standard error) (n = 3 biological replicates). Different lowercase letters above bars indicate statistically significant differences between time points within each tissue type (one-way analysis of variance ANOVA, Tukey’s test, p < 0.05).
Figure 1. Temporal dynamics of phytohormone accumulation in the rootstocks and scions during graft healing. Seven phytohormones (A–G) measured in the rootstocks and scions at three key stages of graft development: 0 DAG (white bars), 7 DAG (gray bars), and 14 DAG (black bars). Each panel represents a specific hormone: (A) IAA, (B) tZ, (C) ACC, (D) JA, (E) SA, (F) SL, and (G) ABA. Values are expressed as ng·g−1 fresh weight (FW). The data is presented as means ± SE (the standard error) (n = 3 biological replicates). Different lowercase letters above bars indicate statistically significant differences between time points within each tissue type (one-way analysis of variance ANOVA, Tukey’s test, p < 0.05).
Plants 14 02229 g001
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Figure 2. Temporal dynamics of auxin metabolites accumulation in the rootstocks and scions during graft healing. Nine auxin metabolites (A–I) measured in the rootstocks and scions at three key stages of graft development: 0 DAG (white bars), 7 DAG (gray bars), and 14 DAG (black bars). Each panel represents an auxin metabolite: (A) IAA-Ala, (B) IAA-Asp, (C) IAA-Glu, (D) IAN, (E) ICA, (F) ICAld, (G) IPA, (H) MEIAA, and (I) TRP. Values are expressed as ng·g−1 FW. The data is presented as means ± SE (the standard error). Different lowercase letters above bars indicate statistically significant differences between time points within each tissue type (one-way ANOVA, Tukey’s test, p < 0.05).
Figure 2. Temporal dynamics of auxin metabolites accumulation in the rootstocks and scions during graft healing. Nine auxin metabolites (A–I) measured in the rootstocks and scions at three key stages of graft development: 0 DAG (white bars), 7 DAG (gray bars), and 14 DAG (black bars). Each panel represents an auxin metabolite: (A) IAA-Ala, (B) IAA-Asp, (C) IAA-Glu, (D) IAN, (E) ICA, (F) ICAld, (G) IPA, (H) MEIAA, and (I) TRP. Values are expressed as ng·g−1 FW. The data is presented as means ± SE (the standard error). Different lowercase letters above bars indicate statistically significant differences between time points within each tissue type (one-way ANOVA, Tukey’s test, p < 0.05).
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Plants 14 02229 g003
Figure 3. Differential expression genes related to auxin biosynthesis and signal transduction during graft union development. (A) Expression heatmap of differential expression genes related to auxin biosynthesis and signal transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7, and 14 represent different time points (0, 7, and 14 DAG). The letters R and S represent the rootstocks and scions. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of TAR2 (B), MES17 (C), ETA3 (D), AFB2 (E), AFB5 (F), COV1 (G), and NDPK2 (H) in hickory rootstocks and scions. The expression level of hickory ACTIN gene was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
Figure 3. Differential expression genes related to auxin biosynthesis and signal transduction during graft union development. (A) Expression heatmap of differential expression genes related to auxin biosynthesis and signal transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7, and 14 represent different time points (0, 7, and 14 DAG). The letters R and S represent the rootstocks and scions. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of TAR2 (B), MES17 (C), ETA3 (D), AFB2 (E), AFB5 (F), COV1 (G), and NDPK2 (H) in hickory rootstocks and scions. The expression level of hickory ACTIN gene was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
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Plants 14 02229 g004
Figure 4. Temporal dynamics of CK metabolite accumulation in rootstocks and scions during graft healing. Ten CK metabolites (A–J) measured in rootstocks and scions at three key stages of graft development: 0 DAG (white bars), 7 DAG (gray bars), and 14 DAG (black bars). Each panel represents a CK metabolite: (A) cZ, (B) cZR, (C) DZ, (D) DHZ7G, (E) DHZR, (F) DHZROG, (G) tZOG, (H) tZR, (I) IPR, and (J) mT9G. Values are expressed as ng·g−1 FW. The data is presented as means ± SE. Different lowercase letters above bars indicate statistically significant differences between time points within each tissue type (one-way ANOVA, Tukey’s test, p < 0.05).
Figure 4. Temporal dynamics of CK metabolite accumulation in rootstocks and scions during graft healing. Ten CK metabolites (A–J) measured in rootstocks and scions at three key stages of graft development: 0 DAG (white bars), 7 DAG (gray bars), and 14 DAG (black bars). Each panel represents a CK metabolite: (A) cZ, (B) cZR, (C) DZ, (D) DHZ7G, (E) DHZR, (F) DHZROG, (G) tZOG, (H) tZR, (I) IPR, and (J) mT9G. Values are expressed as ng·g−1 FW. The data is presented as means ± SE. Different lowercase letters above bars indicate statistically significant differences between time points within each tissue type (one-way ANOVA, Tukey’s test, p < 0.05).
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Plants 14 02229 g005
Figure 5. Differential expression genes related to CK biosynthesis and signal transduction during graft union development. (A) Expression heatmap of differential expression genes related to CK biosynthesis and signal transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7, and 14 represent different time points (0, 7, and 14 DAG). The letters R and S represent rootstocks and scions. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of IPT3 (B), LOG1 (C), LOG4 (D), LOG5 (E), ARR4 (F), RR3 (G), and ABCG14 (H) in hickory rootstocks and scions. The expression level of hickory ACTIN was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
Figure 5. Differential expression genes related to CK biosynthesis and signal transduction during graft union development. (A) Expression heatmap of differential expression genes related to CK biosynthesis and signal transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7, and 14 represent different time points (0, 7, and 14 DAG). The letters R and S represent rootstocks and scions. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of IPT3 (B), LOG1 (C), LOG4 (D), LOG5 (E), ARR4 (F), RR3 (G), and ABCG14 (H) in hickory rootstocks and scions. The expression level of hickory ACTIN was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
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Plants 14 02229 g006
Figure 6. Temporal dynamics of JA metabolites accumulation in rootstocks and scions during graft healing. Eight JA metabolites (A–H) measured in rootstocks and scions at three key stages of graft development: 0 DAG (white bars), 7 DAG (gray bars), and 14 DAG (black bars). Each panel represents a CK metabolite: (A) JA-Phe, (B) JA-Val, (C) JA-Ile, (D) H2JA, (E) MEJA, (F) OPC-4, (G) OPC-6, and (H) OPDA. Values are expressed as ng·g−1 FW. The data is presented as means ± SE. Different lowercase letters above bars indicate statistically significant differences between time points within each tissue type (one-way ANOVA, Tukey’s test, p < 0.05).
Figure 6. Temporal dynamics of JA metabolites accumulation in rootstocks and scions during graft healing. Eight JA metabolites (A–H) measured in rootstocks and scions at three key stages of graft development: 0 DAG (white bars), 7 DAG (gray bars), and 14 DAG (black bars). Each panel represents a CK metabolite: (A) JA-Phe, (B) JA-Val, (C) JA-Ile, (D) H2JA, (E) MEJA, (F) OPC-4, (G) OPC-6, and (H) OPDA. Values are expressed as ng·g−1 FW. The data is presented as means ± SE. Different lowercase letters above bars indicate statistically significant differences between time points within each tissue type (one-way ANOVA, Tukey’s test, p < 0.05).
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Plants 14 02229 g007
Figure 7. Differential expression genes related to JA biosynthesis and signal transduction during graft union development. (A) Expression heatmap of differential expression genes related to JA biosynthesis and signal transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7, and 14 represent different time points (0, 7, and 14 DAG). The letters R and S represent rootstocks and scions. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of AOC4 (B), AOS (C), NAC3 (D), and WRKY51 (E) in hickory rootstocks and scions. The expression level of hickory ACTIN was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
Figure 7. Differential expression genes related to JA biosynthesis and signal transduction during graft union development. (A) Expression heatmap of differential expression genes related to JA biosynthesis and signal transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7, and 14 represent different time points (0, 7, and 14 DAG). The letters R and S represent rootstocks and scions. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of AOC4 (B), AOS (C), NAC3 (D), and WRKY51 (E) in hickory rootstocks and scions. The expression level of hickory ACTIN was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
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Plants 14 02229 g008
Figure 8. Differential expression genes related to ETH biosynthesis and signaling transduction during graft union development. (A) Expression heatmap of differential expression genes related to JA biosynthesis and signaling transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7, and 14 represent different time points (0, 7, and 14 DAG). The letters R and S represent rootstocks and scions. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of ACS1 (B), ACO1 (C), MKK9 (D), ESE1 (E), and ESE3 (F) in hickory rootstocks and scions. The expression level of hickory ACTIN was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
Figure 8. Differential expression genes related to ETH biosynthesis and signaling transduction during graft union development. (A) Expression heatmap of differential expression genes related to JA biosynthesis and signaling transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7, and 14 represent different time points (0, 7, and 14 DAG). The letters R and S represent rootstocks and scions. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of ACS1 (B), ACO1 (C), MKK9 (D), ESE1 (E), and ESE3 (F) in hickory rootstocks and scions. The expression level of hickory ACTIN was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
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Plants 14 02229 g009
Figure 9. Differential expression genes related to SL biosynthesis and signal transduction during graft union development. (A) Expression heatmap of differential expression genes related to SL biosynthesis and signal transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and the heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7 and 14 represent different time points (0, 7, and 14 DAG). The letter R and S represent rootstock and scion. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of D14 (B) and SMAX1 (C) in hickory rootstocks and scions. The expression level of hickory ACTIN was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
Figure 9. Differential expression genes related to SL biosynthesis and signal transduction during graft union development. (A) Expression heatmap of differential expression genes related to SL biosynthesis and signal transduction during graft union development. Log2 and row scale normalization were applied to the RNA-seq-based FPKM values, and the heatmap visualization was performed using TBtools (v2.310). The numbers 0, 7 and 14 represent different time points (0, 7, and 14 DAG). The letter R and S represent rootstock and scion. Color intensity reflects normalized expression values, with a red-to-blue gradient indicating high-to-low transcriptional activity. Expression of D14 (B) and SMAX1 (C) in hickory rootstocks and scions. The expression level of hickory ACTIN was used as the internal control to standardize the RNA samples for each reaction. The data is presented as means ± SE. Different lowercase letters indicate significant differences at p < 0.05.
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MDPI and ACS Style

Huang, Q.; Liu, H.; Shen, Q.; Yuan, H.; Cui, F.; Yan, D.; Ding, W.; Wang, X.; Zheng, B. Hormone Fluctuation and Gene Expression During Early Stages of the Hickory Grafting Process. Plants 2025, 14, 2229. https://doi.org/10.3390/plants14142229

AMA Style

Huang Q, Liu H, Shen Q, Yuan H, Cui F, Yan D, Ding W, Wang X, Zheng B. Hormone Fluctuation and Gene Expression During Early Stages of the Hickory Grafting Process. Plants. 2025; 14(14):2229. https://doi.org/10.3390/plants14142229

Chicago/Turabian Style

Huang, Qiaoyu, Haixia Liu, Qinyuan Shen, Huwei Yuan, Fuqiang Cui, Daoliang Yan, Wona Ding, Xiaofei Wang, and Bingsong Zheng. 2025. "Hormone Fluctuation and Gene Expression During Early Stages of the Hickory Grafting Process" Plants 14, no. 14: 2229. https://doi.org/10.3390/plants14142229

APA Style

Huang, Q., Liu, H., Shen, Q., Yuan, H., Cui, F., Yan, D., Ding, W., Wang, X., & Zheng, B. (2025). Hormone Fluctuation and Gene Expression During Early Stages of the Hickory Grafting Process. Plants, 14(14), 2229. https://doi.org/10.3390/plants14142229

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