Genome-wide analysis of the HbWOX gene family and HbWOX14-mediated enhancement of hairy root transformation in Hevea brasiliensis

Genome-wide analysis of the HbWOX gene family and HbWOX14-mediated enhancement of hairy root transformation in Hevea brasiliensis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide analysis of the HbWOX gene family and HbWOX14-mediated enhancement of hairy root transformation in Hevea brasiliensis Chunlei Zhang, Xin Yin, Junwei Ye, Keyi Cheng, Maoxian Hou, Yan Zheng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8197958/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version Abstract Background The WOX gene family, a plant-specific group of essential transcription factors, plays important regulatory roles in stem cell maintenance, organogenesis, and hormone response cascades. For rubber trees, genotype-dependent limitations in tissue culture and low genetic transformation efficiency have long posed bottlenecks to their molecular breeding progress, where optimizing root regeneration systems is a critical breakthrough point. Results To address these challenges, we identified 17 HbWOX family members from the GT1 genome of rubber trees using bioinformatic approaches, and systematically analyzed their gene structures, conserved motifs, cis-acting elements, and tissue-specific expression profiles. Our findings revealed obvious copy-number diversification within the HbWOX family. Most members harbor a conserved homeodomain (HD) and share the specific motif "PEVSSRWNPTPEQLR", reflecting evolutionary conservation and functional specialization of this gene family in rubber trees. Notably, HbWOX14 exhibited root-specific and high-level expression, with its promoter region significantly enriched for auxin-responsive cis-elements. Functional verification showed that HbWOX14 responds rapidly to auxins such as IAA, and its expression pattern is tightly associated with root development processes. Using an Agrobacterium rhizogenes -mediated transformation system, overexpression of HbWOX14 increased the induction rate of rubber tree hairy roots from 13.3% to 66.7%, while simultaneously significantly improving root length, number, and thickness. Transcriptome and Gene Ontology (GO) enrichment analyses further demonstrated that HbWOX14 exerts its regulatory function by activating the auxin signaling pathway and modulating the expression of genes involved in cell division and differentiation. Additionally, it suppresses abscisic acid and jasmonic acid-mediated stress response pathways while coordinating flavonoid glycosylation-related secondary metabolic processes, collectively creating a favorable molecular environment for hairy root development. Conclusion This study is the first to systematically clarify the evolutionary characteristics and functional divergence of the HbWOX gene family in rubber trees, with a specific focus on verifying the key role of HbWOX14 in enhancing hairy root regeneration efficiency. These findings not only enrich our understanding of the plant WOX gene family's functional diversity but also provide crucial theoretical basis and practical technical support for accelerating rubber tree molecular breeding and improving its genetic transformation system. Hevea brasiliensis HbWOX14 Hairy root transformation Transformation efficiency Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background The WUSCHEL -related homeobox ( WOX ) gene family, as a plant-specific family of key transcription factors, plays important regulatory roles in core life processes such as stem-cell maintenance, organogenesis, wound repair, and stress responses [ 1 , 2 ]. WOX genes belong to the homeodomain (HD) superfamily [ 3 , 4 ], with the proteins they encode featuring a highly conserved HD comprising approximately 60 amino acids that adopts a “helix–loop–helix–turn–helix” conformation. This unique structure enables specific binding to DNA sequences, facilitating precise recognition of key cis-acting elements in target gene promoters, such as the CAAT-box, TATA-box, CTCC motif, and TATTGTAGAC motif and regulating downstream gene-expression programs that finely control plant growth and development [ 5 ]. During stem-cell division, WOX genes contribute to maintaining the stability of stem-cell populations and balancing proliferation by regulating the expression of associated cyclin genes [ 6 , 7 ]. At the stage of root primordium initiation, these genes coordinate the timing of expression for cell-differentiation-related genes to ensure that root primordia develop along designated pathways [ 6 , 8 ]. Furthermore, WOX genes regulate activities within the root apical meristem and promote continuous growth of adventitious roots through synergistic interactions with the auxin signaling pathway [ 9 ]. In addition to their conserved homeodomain features, the number, type, and arrangement of these motifs will contribute to the molecular foundation for their functional diversity and specificity. For example, although CsFAR proteins across different phylogenetic groups (e.g., Class I, II, III) share certain fundamental motifs in cucumber ( Cucumis sativus L.), the significant differences existed in the quantity, sequential order, and relative positioning of these motifs directly contribute to the functional differentiation among CsFAR proteins from various classes [ 10 , 11 ]. Similarly, the differential distribution of motifs among FanSWEET proteins facilitates their roles in sugar transport and metabolic regulation across diverse strawberry tissues and organs [ 12 ]. The structural characteristics of these key gene family suggest that they have continuously expanded their regulatory scope through variation and recombination of auxiliary motifs during plant evolution process, while maintaining core DNA-binding capabilities essential for fundamental regulatory functions, which precisely contributed to adapt to the requirements of different stages of plant growth and development [ 5 , 13 , 14 ]. In classical model plants, such as Arabidopsis thaliana, Oryza sativa, and Zea mays, the genome-wide identification of the WOX gene family has been completed and functionally characterized. WOX5 has been demonstrated to maintain stem-cell homeostasis in the root apical meristem, with its expression being precisely regulated by the auxin concentration gradient. The absence of the WOX5 gene results in root growth arrest [ 4 , 15 ]. In the shoot apex, MdWUS-1 promotes the formation of leaf-derived buds in apple ( Malus pumila Mill.) and plays a significant role in maintenance [ 16 , 17 ]. In macadamia ( Macadamia integrifolia Maiden et Betche ), a homolog of WOX has been identified that participates in bud regeneration [ 18 ]. In jasmine (Jasminum sambac), WOX1 primarily influences root primordium initiation and differentiation [ 19 ], while WOX5 exhibits relatively high expression levels in roots of wheat (Triticum aestivum) [ 20 ]. WOX14 is mainly involved in plant root formation and elongation; overexpression of ZmWOX14 significantly enhances callus induction efficiency and improves plant regeneration capability in maize ( Zea mays L.), providing an important genetic resource for maize transformation [ 21 ]. Similarly, studies have shown that WOX14 is implicated in adventitious-root formation within Arabidopsis thaliana [ 5 ]. Plant root development and organ regeneration are biological processes that are intricately coordinated by transcriptional regulatory networks and plant hormone signaling. Within this network, WOX genes serve as core regulatory elements, playing crucial roles in stem cell maintenance, meristem establishment, and hormone responses [ 22 ]. Previous studies have demonstrated that WOX genes are influenced by upstream plant hormones such as auxin and cytokinin within the regulatory framework [ 22 ]. In conjunction with multiple hormone-regulated signaling pathways, these genes further regulate downstream hormone homeostasis and signal transduction, thereby facilitating the organogenesis of roots and other organs [ 15 ]. Auxin signaling has been shown to activate the expression of WOX11 , which subsequently activates RR2, promoting the initiation and development of the root cap meristem in Oryza sativa [ 23 ] LsWOX9 precisely regulates the initiation and emergence of lateral-root primordia by influencing the polar transport and distribution of auxin. This gene serves as a critical link between upstream auxin signaling and downstream lateral-root growth during lateral-root development in tomato ( Solanum lycopersicum ) [ 24 ]. In poplar species ( Populus spp. ), PtoWOX5 a primarily governs adventitious root development through auxin signal transduction [ 25 ]. The process of somatic embryogenesis in rapeseed ( Brassica napus ) is highly dependent on the ratio of exogenous to endogenous auxin/cytokinin levels [ 26 ]. As a key downstream effector, BnWOX5 significantly induces and transduces hormonal signals to initiate subsequent embryonic development [ 15 ]. Additionally, WOX14 participates in gibberellin biosynthesis and signaling; through its coordination with auxin and other hormones, it promotes hypocotyl formation as well as lateral root development in Arabidopsis thaliana [ 27 ]. These studies collectively underscore the critical role of WOX genes in regulating root regeneration under various hormonal conditions. However, the molecular mechanisms by which different members of the WOX family respond to specific hormone types exhibit significant species specificity across diverse plants. The rubber tree ( Hevea brasiliensis ), as the primary commercial source of natural rubber, possesses irreplaceable strategic significance across various sectors, including industry, agriculture, national defense, transportation, and machinery manufacturing. Tissue culture serves as an efficient method for vegetative propagation and represents a crucial pathway for cultivating high-yield varieties of rubber trees. Through tissue culture techniques, elite cultivars of rubber trees can be rapidly multiplied; furthermore, traits such as yield and stress resistance can be enhanced through approaches like somaclonal variation screening and gene editing. Currently, the application of tissue culture in H. brasiliensis encounters several limitations [ 12 ]. Only a limited number of cultivars can be propagated rapidly using this method; conversely, most elite cultivars that exhibit high yield and strong resistance are exceedingly challenging to propagate. Genotype dependence has emerged as a significant constraint on clonal propagation in H. brasiliensis [ 28 ]. Consequently, addressing the genotype barrier to enhance both the transformation efficiency of desirable traits and the regeneration capacity of plants has become a critical issue requiring urgent resolution within rubber tree breeding research [ 29 ]. Previous studies have indicated that employing regeneration-related genes such as WOX can effectively improve plant genetic transformation efficiency while broadening genotype adaptability [ 30 , 31 ]. In this study, we systematically identified the WOX gene family in rubber tree, determined the number and classification of its members, analyzed the gene structure and evolutionary characteristics of each member, and examined expression changes of different HbWOX genes in roots, stems, and leaves under various hormone treatments through RT-qPCR. Additionally, using hairy roots from rubber trees overexpressing the HbWOX14 gene as materials, we conducted transcriptome sequencing and GO enrichment analysis to elucidate the biological processes and signaling pathways involved in HbWOX14 -mediated root regeneration. Our findings indicate that HbWOX14 significantly enhances transgenic hairy-root induction. These results provide crucial theoretical and technical support for identifying key functional genes and addressing challenges related to genetic transformation in rubber trees. Materials and methods Identification of WOX gene family members in rubber tree The WOX genes of Arabidopsis were retrieved from the Arabidopsis database TAIR ( http://www.arabidopsis.org ) [ 24 , 25 ]. The Arabidopsis WOX gene sequences served as query files, and the GT1 genome sequence of rubber tree ( Hevea brasiliensis ) [ 32 ] was used as the reference file. Local BLAST protein alignments were performed with an E-value < 1e − 5 to obtain candidate rubber-tree WOX genes. Protein domain prediction was carried out using SMART (SMART: Mainpage (embl.de)) and Pfam, and members containing the HOX domain were retained. The preliminarily determined members were further aligned on the NCBI website, Pfam IDs were obtained from the literature (Zhao et al.,2009; [ 9 ] (ID: PF00046), yielding the candidate H. brasiliensis WOX gene family members [ 24 , 5 ]. Phylogenetic trees were constructed for the candidate H. brasiliensis WOX members together with Arabidopsis WOX genes using filtreebest and the iTOL website, thereby determining the WOX gene family members in the GT1 genome of rubber tree. The rubber-tree WOX genes ( HbWOX ) were then named following the nomenclature of Arabidopsis WOX family members ( AtWOX ). Gene structure and conserved motif analysis of HbWOX family members The intron–exon organizations of HbWOX family members in rubber tree were analyzed using the online Gene Structure Display Server (GSDS; http://gsds.cbi.pku.edu.cn/ ). Conserved domains and motifs of the HbWOX proteins were predicted online with NCBI Batch CD-Search and MEME ( https://meme-suite.org/meme/ ), respectively. Cis-acting element prediction of the promoters WOX genes Using TBtools, we extracted the 2,000-bp upstream promoter regions of HbWOX genes and submitted these sequences to PlantCare ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) for prediction. The results obtained from PlantCare were curated and simplified, and the final annotations were visualized using TBtools. RNA extraction and RT-qPCR Roots, stems, and leaves from the elite rubber tree clone ‘GT1’ were utilized as experimental materials. Immediately after collection, samples were snap-frozen in liquid nitrogen and subsequently stored at − 80°C until total RNA extraction. Total RNA was extracted using the Eastep® Super Total RNA Extraction Kit (Promega, Madison, WI, USA). The concentration of RNA was measured with a NanoDrop 1000 (NanoDrop Technologies, Inc.), while RNA integrity was assessed on 0.8% agarose gels. Approximately 5 µg RNA was reverse-transcribed using the GoScript® Reverse Transcription System (Promega, Madison, WI, USA) to generate cDNA. RT-qPCR was performed on cDNA from different tissues and treatments using FastStart™ Universal SYBR® Green Master (ROX; Roche, Indianapolis, IN, USA) on a 7500 Real-Time PCR System (Applied Biosystems, USA), with three technical replicates. The resulting cDNA products were stored at − 20°C as templates for qPCR. Using diluted cDNA as template, amplification was also carried out with TB Green® Premix Ex Taq™ II on a QuantStudio® 5 Real-Time PCR System (Applied Biosystems, USA). Each 20 µL reaction contained 10 µL TB Green Premix Ex Taq II, 0.8 µL each of forward and reverse primers, 2 µL cDNA template, and 6.4 µL nuclease-free water. The RT-qPCR cycling program was: 95°C for 10 min; then 40 cycles of 94°C for 5 s and 60°C for 15 s. Melting-curve analysis was performed to verify amplification specificity. Three technical replicates were set for each sample. Relative expression levels were calculated using the 2^ −ΔΔCt method. Statistical comparisons between treatment and control were conducted with independent-samples t-tests. The primers used in this study are listed in Table S1 . Acquisition and treatment of experimental materials One-year-old tender stems and current-year seedlings of the elite rubber-tree clone ‘GT1’ were used as experimental materials. After seedlings were collected, roots were removed to facilitate infection by Agrobacterium rhizogenes . Seedlings were then planted in a 3:1 (v/v) mixture of vermiculite and nutrient soil and grown for 30–45 d under a 16 h light/8 h dark photoperiod, a light intensity of 100 µmol·m⁻²·s⁻¹, and a temperature of 30 ± 2°C. Once growth stabilized, vigorously growing seedlings with a height of 20–25 cm and tender stems were selected as explants. All ‘GT1’ materials used in this study were maintained and provided by Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. For hormone treatment, four plant growth regulators were used: α-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 6-benzylaminopurine (6-BA). Each hormone was prepared as a stock solution and diluted with sterile water to working solutions of 100 mg·L⁻¹ and 200 mg·L⁻¹. Seedlings were randomly grouped, and the working solutions were evenly sprayed onto the leaves. Samples were collected at 0.5 h, 1 h, 3 h, and 6 h after treatment. At each sampling time, surface liquid was blotted with sterile filter paper, and samples were immediately snap-frozen in liquid nitrogen. For each treatment combination (hormone × concentration × time point), three biological replicates were set. All samples were stored at − 80°C for subsequent total RNA extraction and gene-expression analysis by RT-qPCR. Establishment of a hairy-root transformation system in rubber tree A. rhizogenes strains K599 and LBA9402 carrying the pR101 plasmid were cultured with shaking. When the cultures reached OD₆₀₀ = 0.5, 0.8, or were used undiluted (original culture), explants were inoculated. After co-cultivation and selection, three inoculation methods were applied: Injection, CDB (Cut–Dip–Budding), and Flat placement. Hairy roots were preliminarily induced after about 30 d. Genomic DNA was extracted from the obtained hairy roots, with three biological replicates. Seedlings measuring 20–25 cm in height and one-year-old tender stems were used as explants. Two to three superficial wounds were gently made on each explant, followed by inoculation using one of the three methods, (1) Injection method: the A. rhizogenes inoculum was loaded into a 10 mL syringe and injected into the explants. (2) CDB method: the prepared A. rhizogenes suspension was directly smeared onto the wounds after 30 min, explants were inserted into a moistened mixed substrate. (3) Flat-placement method: explants were immersed in the A. rhizogenes inoculum for 30 min with gentle shaking every 5 min; explants were then removed, blotted with sterile filter paper to remove excess inoculum, and placed flat on the surface of the moistened mixed substrate. Co-cultivation was conducted in darkness for 72 h at a temperature of 30 ± 2°C. After this period, explants were then transferred to the corresponding substrate and cultured for 30–45 d under a 16 h·d⁻¹ photoperiod, light intensity of 100 µmol·m⁻²·s⁻¹, and temperature of 30 ± 2°C. The hairy-root induction rate was calculated as: Induction rate (%) = (number of explants producing hairy roots / total number of explants) × 100%. Detection of transgenic hairy roots Genomic DNA was extracted from transgenic hairy roots using the CTAB method. PCR amplification was conducted with universal GFP primers, and the resulting amplification bands were analyzed by agarose gel electrophoresis to ascertain whether the reporter gene had been stably integrated into the hairy roots. Wild-type roots served as a negative control. The positive transformation rate was calculated as follows: Positive transformation rate (%) = (number of hairy roots with a GFP band / total number examined) × 100%. The primers used in this study are listed in Table S1 . Data statistics and analysis Statistical analysis was performed using Ordinary one-way ANOVA and Student’s t-test (*P < 0.05, **P < 0.01 and ***P < 0.001). Data analysis was conducted using Microsoft Excel 2019 and GraphPad Prism 8.0. Results Identification and gene structure analysis of WOX family members in rubber tree A total of 17 WOX gene members in rubber tree have been identified. Based on sequence similarity to Arabidopsis WOX genes (Fig. S1 ), the WOX genes in rubber-tree were renamed accordingly (Table S2 , S3). Notably, WOX7, WOX9, WOX10 , and WOX12 are absent in rubber tree, HbWOX8 and HbWOX6 exhibited three copies, HbWOX2 , HbWOX5 , HbWOX11 , HbWOX13 , and HbWOX14 are present as single-copy genes within the rubber tree. Otherwise, the remaining members possess two copies each, indicating an expansion of the WOX family specific to this species. Chromosome localization and synteny analysis of the HbWOX gene revealed that the 17 WOX gene members of the rubber tree were distributed on different contigs, each member having a unique position in the genome with no tandem gene duplication. Five pairs of homologous genes were identified with fragment duplications, involving 10 HbWOX genes, including HbWOX1.1/HbWOX1.2 , HbWOX3.1/HbWOX3.2 , HbWOX4.1/HbWOX4.2 , HbWOX6.1/HbWOX6.2 , and HbWOX8.1/HbWOX8.3 . The extensive fragment duplications among family members on different chromosomes may be the main reason for the expansion of the WOX gene family in rubber trees (Table S4 ; Fig. S2 ; S3). Conserved motif analysis can enhance our understanding of specific gene functions and their evolutionary conservation [ 33 ]. Utilizing protein sequences from the identified HbWOX members for motif analysis. we observed that each gene contains at least one conserved motif with a maximum of eleven motifs per gene (Fig. 1 , A). Among these proteins, HbWOX6.1, HbWOX6.2, HbWOX6.3, HbWOX1.1, HbWOX1.2, HbWOX3.1, HbWOX3.2, HbWOX4.1, and HbWOX4.2 share two identical conserved motifs, HbWOX2 and HbWOX5 share one identical conserved motif, HbWOX11, HbWOX8.1, HbWOX8.2, and HbWOX8.3 share two identical conserved motifs, and all of these contain motif 1. In addition, with the exception of HbWOX13, the “PEVSSRWNPTPEQLR” motif is present in the other 16 HbWOX proteins, indicating very high degree of conservation. This suggests that this motif may represent a signature functional domain capable of specifically regulating processes such as wound responses, cell proliferation, and organ regeneration. Consequently, it exhibits marked functional divergence from proteins primarily involved in stem-cell maintenance. The homeodomain found in WOX proteins of rubber tree is conserved across all 17 proteins, including HbWOX14, highlighting significant conservation at the amino acid level (Fig. S4 ). Its characteristic three-dimensional architecture follows a “helix–turn–helix” pattern (Fig. S5). The two α-helices are highly conserved while the loop and turn regions connecting them exhibit comparatively less conservation. This pronounced sequence and structural conservation underscore the central role of the homeodomain (HD) in WOX protein function and its stability throughout plant evolution. To explore the structural features related to HbWOX gene function, introns and exons were analyzed for all 17 family members (Fig. 1 , B). The number of exons varied from 2 to 5, while the number of introns ranged from 1 to 5. Notably, HbWOX13 and HbWOX4.1 each possess five exons, HbWOX8.2 , HbWOX11 , HbWOX2 , HbWOX6.1 , and HbWOX3.2 contain two exons each, HbWOX1.1, HbWOX1.2, HbWOX6.3 , and HbWOX8.3 each contain four exons, and the remaining genes, including HbWOX14 , each contain three exons, showing a high degree of consistency. Gene structures exhibit marked differences among various genes, however, variations within homologous pairs are relatively minor, reflecting functional distinctions among the different genes involved. Cis-element analysis of the promoters of WOX genes in rubber-tree The 2,000-bp upstream promoter sequences of HbWOX genes were extracted, and cis-acting elements were analyzed using PlantCare to predict putative biological functions and visualized by TBtools (Fig. 2 ). The analysis identified a total of 20 distinct cis-elements, including numerous light-responsive elements, auxin-related elements, and those induced by abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin (GA), and salicylic acid (SA). Additionally, elements associated with stress defenses against low temperature and anaerobiosis were detected. Cis-element compositions varied among homologous genes. Notably, HbWOX14 contained a relatively high number of auxin-related elements, while HbWOX1.1 harbored more drought-responsive elements. HbWOX1.2 exhibited the greatest abundance of light-responsive elements. These findings suggest that HbWOX14 may function as a key downstream gene in the auxin signaling pathway, implying that HbWOX14 could perceive endogenous or exogenous auxin signals to activate downstream organogenesis-related gene networks. Furthermore, these results underscore the significant role of HbWOX14 in regulating regeneration during rubber tree tissue culture. Effects of different hormones treatments on HbWOX gene expression level Furthermore, we profiled the spatiotemporal expression of 17 HbWOX genes in rubber tree leaves following treatments with four types of hormones at two concentrations over different time points: IBA (100 mg, 200 mg) ( Fig. 3 , A), IAA (100 mg, 200 mg) (Fig. 3 , B), 6-BA (100 mg, 200 mg) (Fig. 3 , C), and NAA (100 mg, 200 mg) (Fig. 3 , D) using RT-qPCR. The expression levels of the 17 HbWOX genes exhibited significant variations across the four hormone treatments. Under IBA treatment, HbWOX14 demonstrated elevated transcript levels at 6 hours for both concentrations (100 mg and 200 mg) compared to those observed at 1 hour and 3 hours. In contrast, HbWOX13 showed high expression exclusively at the earlier time points of 1 hour and 3 hours under the lower concentration of IBA (100 mg). Following IAA treatment, HbWOX14 displayed a peak in transcript abundance at the half-hour mark for both concentrations (100 mg and 200 mg), which positively correlated with increasing IAA concentration. However, its expression declined over time and approached minimal levels by the three-hour mark. Under treatment with 6-BA, both HbWOX6.1 and HbWOX6.2 were expressed more highly at half an hour than at six hours. Notably, after six hours under a concentration of 100 mg for HbWOX14 was significantly lower than that observed under the higher concentration of 200 mg. Under NAA treatment, HbWOX1.1 exhibited higher expression levels at 0.5 h and 1 h with a concentration of 100 mg, but its expression was suppressed at 200 mg. In contrast, HbWOX14 maintained low expression across all NAA treatments, indicating lower sensitivity and potential specificity to this auxin. The transcriptional patterns suggest that these genes respond differentially to distinct auxins. Overall expression levels at 0.5-1 h were greater than those observed at 3 h and 6 h, implying that these genes are responsive within a short time frame and may primarily be involved in early hormone responses as well as cell differentiation processes. At elevated auxin concentrations, several genes were significantly up-regulated, indicating their involvement in hormone responses and plant cell differentiation. Notably, HbWOX14 demonstrated high expression under IAA treatment at both concentrations, considering role of IAA in root apex synthesis and transport as well as its influence on root development. Conversely, its low responsiveness to NAA suggests a certain specificity towards different types of auxins, thereby supporting the notion of signal recognition and transduction for HbWOX14 within the auxin signaling network of the rubber tree. Spatiotemporal expression analysis of the HbWOX gene family Using RT-qPCR, we profiled the spatiotemporal expression of 17 members of the HbWOX gene family across various rubber tree tissues, including roots, stems, and leaves (Fig. 4 ). Several genes exhibited high expression levels in specific tissues, indicating a degree of tissue specificity. Notably, HbWOX14 demonstrated nearly the highest transcript level in roots among these genes. In stems, HbWOX3.1, HbWOX4.2 , HbWOX8.2 , and HbWOX11 were highly transcribed. Conversely, in leaves, significant transcription was observed for HbWOX4.1 and HbWOX1.1 . These transcriptional patterns suggest that these genes may play roles in organ growth and development. While some homologs also showed detectable expression in roots, their levels were considerably lower than that of HbWOX14 . The peak expression observed for HbWOX14 indicates its potential role as a key regulator of root growth and development; it appears to have retained a core function related to root morphogenesis throughout evolution. In contrast to this pattern, certain genes, particularly HbWOX5 and HbWOX6.1 , showed no detectable transcription across any tissue types examined. Furthermore, pronounced differences or even opposing expression patterns were noted among homologous genes within the same tissue type. This suggests possible functional divergence among them. For instance, while HbWOX8.2 was highly expressed in roots, both HbWOX8.1 and HbWOX8.3 exhibited minimal transcription levels. Similarly, marked differences between HbWOX4 .1 and HbWO4 .2 were evident in leaves, which may indicate evolutionary functional divergence. In summary, our findings reveal that HbWOX14 displays significant tissue specificity with transcript levels substantially higher than those of the other 16 gene family members in roots, implying that HbWOX14 may play a key role in regulating root growth and development in rubber tree. Establishment of an Agrobacterium rhizogenes –mediated hairy-root transformation system in rubber tree To develop a rapid and efficient gene-function validation system for the rubber tree, we established an Agrobacterium rhizogenes –mediated hairy-root transformation system (HRTS). A. rhizogenes strains K599 and LBA9402 (Table S5), both carrying the GFP reporter (plasmid pR101), were used to inoculate the basal region of one-year-old seedlings through three methods: injection (Fig. 5 , A), flat placement methods (Fig. 5 , B) and CDB (Cut–Dip–Budding) (Fig. 5 , C). Among these approaches, CDB yielded the highest transformation efficiency (Fig. 5 , F), nine plants developed transgenic roots via this method, whereas only three plants exhibited transgenic roots with the injection method and none with flat placement (Fig. 5 , D). Notably, LBA9402 demonstrated a significant advantage over K599, out of 30 inoculated seedlings, 23 produced transgenic hairy roots using LBA9402 compared to just three with K599, indicating a higher infection efficiency associated with LBA9402(Fig. 5 , E). Hairy roots first appeared 15 days post-inoculation (Fig. 5 , G), fibrous roots became visible by day 30(Fig. 5 , H), while fully developed hairy roots formed by day 45 (Fig. 5 , I). This timeline illustrates that the LBA9402-CDB pipeline reliably produces transgenic hairy roots (Fig. 5 , J). We hypothesize that the superior efficiency observed with CDB is attributable to its moderate wounding effect which facilitates close contact between bacteria and wound sites while minimizing excessive damage and maintaining suitable local humidity conditions. The emergence of hairy roots in rubber tree seedlings occurred within 15 days following infection and reached full development by day 45. These findings demonstrate that our transformation system exhibits high efficiency and practicality. Improving the efficiency of Agrobacterium -mediated hairy root transformation in rubber trees by utilizing HbWOX14 in rubber tree Based on the experimental results presented, we concluded that HbWOX14 most significantly promotes root growth and development in rubber tree. Furthermore, the combination of A. rhizogenes strain LBA9402 with the CDB method maximizes transformation efficiency. To further optimize this protocol, we infected rubber tree seedlings with LBA9402 carrying an overexpression construct for HbWOX14 (Fig. 6 , A-D) and verified the presence of hairy roots harboring both the GFP vector and the GFP–HbWOX14 vector through RT-qPCR analysis (Fig. S6, B). Continuous observation and quantification of the transformation process revealed that plants overexpressing HbWOX14 achieved a hairy root induction rate of 66.7%, while seedlings containing only the GFP vector exhibited a mere 13.3% induction rate (Fig. S6, A). This indicates that overexpression of HbWOX14 significantly enhances both hairy root induction and growth. As illustrated in our findings, HbWOX14 overexpression not only induced hairy roots at an earlier stage but also resulted in a greater number and more robust roots. We quantified four phenotypic traits, length (Fig. 6 , E), diameter (Fig. 6 , F), root number (Fig. 6 , G), and newly formed bud count (Fig. 6 , H) and found that hairy roots expressing HbWOX14 averaged 59 mm in length, approximately 2.5 times longer than those from the GFP control (24 mm). In terms of root quantity, lines expressing HbWOX14 produced an average of four transgenic hairy roots per plant (Fig. 6 , C) compared to fewer than two from controls (Fig. 6 , B, D). Additionally, roots carrying HbWOX14 were thicker as evidenced by an average diameter of ~ 1.2 mm versus ~ 0.5 mm for controls. Thus, our findings suggest that overexpressing HbWOX14 markedly enhances both growth and development of rubber tree hairy roots likely by sustaining activity within apical meristems while promoting cell division and differentiation, hereby improving developmental capacity within root primordia. GO enrichment analysis of differentially expressed genes in HbWOX14 -overexpressed lines Using samples overexpressing PRI101: GFP as the control group, we identified genes with significantly different expression level and conducted a detailed analysis in HbWOX14 -overexpressed lines (Fig. S6, A), resulting in the identification of 77 upregulated genes and 28 downregulated genes. Principal component analysis (PCA) (Fig. 7 , A) and Gene Ontology (GO) functional enrichment analysis (Fig. 7 , B) were subsequently performed. The results revealed that differentially expressed genes (DEGs) in hairy roots induced by overexpressing HbWOX14 , were significantly enriched in pathways related to development and hormone signaling (Fig. 7 , C). This suggests that HbWOX14 may serve as a key gene promoting hairy-root formation. In terms of hormone responses, DEGs exhibited marked enrichment for cytokinin response, ABA response, and associated signal transduction pathways. These findings indicate that HbWOX14 shapes a hormonal microenvironment conducive to the initiation and development of hairy roots by modulating the plant hormone signaling network. Notably, multiple genes involved in auxin polar transport and signaling were found to be upregulated, providing an essential hormonal regulatory basis for efficient hairy-root induction. Regarding development-related processes, DEGs were primarily enriched in cell development (GO:0048468), establishment of organ organization (GO:0048589), and cell–cell junction organization (GO:0005911). Genes closely associated with cell division and differentiation, such as GT014369 , were significantly upregulated. These include those involved in cell-cycle regulation (GO:0005515), microtubule-based movement (GO:0016887), and cell-wall biogenesis (GO:0009506). Collectively, these changes create prerequisites for hairy-root morphogenesis. The enrichment of genes associated with cell junctions (GT014369) and plasmodesmata (GT031829) further suggests that HbWOX14 may coordinate hairy-root development by enhancing intercellular communication. Additionally, pathways related to glycosyltransferase activity (GO:0102132), particularly flavonoid glycosylation (GO:0102132), were significantly enriched. This finding is consistent with the roles of these pathways in hormone homeostasis and developmental signal regulation, as flavonoid glycosylation influences secondary metabolism and modulates the distribution and activity of hormones such as auxin. Principal Component Analysis (PCA) also indicated a prominent enrichment of differentially expressed genes (DEGs) in flavonoid glycosylation and related glycosyltransferase activities, implies that HbWOX14 affects hairy-root development by regulating modifications of secondary metabolites. Furthermore, upregulated genes exhibited significant enrichment in hormone-response pathways, particularly those responding to cytokinin (GO:0009691) and abscisic acid (GO:0051603), indicating that HbWOX14 promotes hairy-root growth through remodeling the hormonal regulatory network. Further, we validated 16 genes that exhibited significant changes following the introduction of HbWOX14 through RT-qPCR. The results indicated that 12 genes were significantly upregulated, while 4 showed slight downregulation (Fig. 8 , A-P), largely consistent with the RNA-seq findings. Among these, ERF4 ( GT005774 ), WOX11 ( GT031829 ), WOX13 ( GT005626 ), RHA2B ( GT014369 ), and MYB62 ( GT005678 ) are involved in regulating the auxin signaling pathway and displayed markedly increased transcript levels in HbWOX14 -overexpressing hairy roots. Additionally, ERF4 ( GT005774 ), RHA2B ( GT014369 ), and MYB108 ( GT012677 ) primarily participate in the jasmonic acid signaling pathway by promoting the expression of MYB75 and MYB111 , thereby activating downstream anthocyanin biosynthetic pathways. Five upregulated genes, like MYB73 ( GT001820 ), MYB74 ( GT034650 ), HB-21 ( GT024700 ), HB-40 ( GT037945 ), and CIPK7 ( GT005901 ), are jointly regulated by auxin and abscisic acid, playing a crucial role in balancing and modulating plant growth and development. ANAC083 ( GT033271 ) is regulated by auxin, abscisic acid, and jasmonic acid pathways, with abscisic acid and jasmonic acid serving as core stress signals. This gene is typically induced to suppress auxin signaling to coordinate growth arrest with stress defense mechanisms. However, in HbWOX14 -overexpressing hairy roots, we observed a notable decrease in ANAC083 expression. This finding suggests that HbWOX14 can inhibit ABA- and JA-mediated stress-response signals while enhancing auxin signaling transduction to promote the growth and proliferation of rubber-tree hairy roots. These results indicate that HbWOX14 serves as a core regulatory gene that enhances the efficient initiation and subsequent growth of rubber-tree hairy roots by coordinating the plant hormone signaling network, particularly in responses to auxin, cytokinin, and abscisic acid (Fig. 9 ). This coordination plays a crucial role in root cell division, differentiation, and organogenesis. Furthermore, in conjunction with the GO enrichment results mentioned above, HbWOX14 may also regulate secondary metabolic processes such as flavonoid glycosylation, thereby indirectly influencing hormone signal transduction, a critical factor for the growth and development of rubber-tree roots. Accordingly, we concentrated on genes involved in hormone signal-transduction pathways and developed a schematic representation of their potential roles in rubber-tree root development. These genes are implicated in auxin signaling, abscisic acid signaling, and jasmonic acid signaling. Notably, most cluster within the auxin pathway highlighted its central importance for rubber-tree root growth and development. Additionally, upregulated expression of MYB -family genes is believed to facilitate the development of rubber-tree hairy roots. These genes may integrate key components from the auxin ( ARF7/PIN1 ), jasmonic acid ( MYC2 ), and abscisic acid ( ABF2 ) signaling pathways to synergistically promote both initiation and elongation of hairy roots in rubber trees. In summary, these findings suggest that auxin-, jasmonic-acid-, and abscisic-acid-mediated signaling pathways play pivotal regulatory roles in inducing proliferation among rubber-tree hairy roots. The HbWOX14 gene establishes a molecular network that effectively promotes high-efficiency hairy-root formation through coordinated regulation of hormone-response pathways alongside cellular developmental processes, thus occupying an essential regulatory position within the context of growth and development for rubber-tree hairy roots. Discussion Through genome-wide identification, we identified 17 HbWOX members, all of which encode proteins containing the conserved homeodomain (HD). The “helix–turn–helix” three-dimensional conformation formed by this domain is highly consistent with that of WOX proteins in Arabidopsis thaliana, Zea mays, and other species, confirming the core supporting role of the HD for DNA binding during plant evolution. With the exception of HbWOX13, the remaining 16 HbWOX proteins possess the conserved motif “PEVSSRWNPTPEQLR.” This feature resembles the diversification of conserved motifs observed in cucumber CsFARs proteins and in the strawberry SWEET gene family, indicating that retention of core WOX motifs preserves basal functions while variations in these motifs may confer functional specificity. Gene structure analysis revealed that HbWOX genes contain between 2 to 5 exons and 1 to 5 introns. Notably, HbWOX14 has three exons, the most common configuration within this family, and this streamlined yet conserved structure closely resembles that of AtWOX14 and OsWOX11 . Such structural similarities may facilitate rapid assembly of mRNA during transcription and enhance efficient expression of functional proteins. In comparison to the Arabidopsis WOX family, rubber tree lacks four members, namely WOX7 , WOX9 , WOX10 , and WOX12 , and exhibits copy number variations for certain genes. There are three copies each for HbWOX6 and HbWOX8 but only one copy for both HbWOX2 and HbWOX14 . This divergence in copy number and gene structure may reflect evolutionary optimization within developmental regulatory networks. Furthermore, tissue-specific expression analyses further elucidated functional differentiation among HbWOX genes. Notably, HbWOX14 displays significantly higher transcript levels in roots compared to stems or leaves, far exceeding those observed for other family members, which is similar to AtWOX14 in A. thaliana and OsWOX11 in O. sativa. In Arabidopsis thaliana, AtWOX14 primarily plays a role in lateral root development and post-wounding cellular reprogramming [ 34 , 27 ]. In contrast, ZmWOX14 in Zea mays is predominantly expressed in the root apical meristem and regulates cell division within the root cap [ 3 ]. The notably high expression of HbWOX14 in rubber tree roots, alongside its sustained expression throughout both root primordium formation and adventitious root elongation, suggests that it has a core regulatory function in the construction of the rubber tree's root system [ 35 ]. Furthermore, expression divergence among certain homologs in the rubber tree, such as the elevated levels of HbWOX8.2 expression in roots compared to nearly undetectable levels of HbWOX8.1 and HbWOX8.3 , which reflects a pattern of functional diversification observed among WOX family members in A. thaliana. This indicates that the WOX gene family in rubber trees has achieved developmental specialization through evolutionary changes in their expression patterns. Genetic improvement and molecular breeding of rubber-tree are pivotal for the sustainable development of the global natural rubber industry. However, challenges, such as, low transformation efficiency, prolonged and unstable hairy-root induction, and inadequate stability have historically hindered gene-function validation, secondary metabolism regulation, and stress-resilience breeding. In this study, we firstly demonstrate that overexpression of HbWOX14 significantly enhances the efficiency in Agrobacterium-mediated hairy-root transformation. Within the hairy-root transformation system established herein, HbWOX14 overexpression increased the induction rate from 13.3% to 66.7%, accompanied by notable improvements in root length, number, and thickness compared to controls. This enhancement is similar to the increased callus-induction efficiency observed upon ZmWOX14 overexpression in maize and supports the genotype-barrier-breaking function of WOX5 in Arabidopsis thaliana. These findings confirm a distinct role for WOX genes in enhancing plant genetic-transformation efficiency. The improved induction rates associated with HbWOX14 overexpression suggest that HbWOX14 functions not only as a regulatory switch for hairy-root transformation efficiency in rubber trees but also serves as an optimizer of both hairy-root developmental quality and genetic stability. From an evolutionary and functional perspective, WOX genes are plant-specific transcription factors that play crucial roles in regulating cell division and differentiation, organ primordium formation, and stress responses [ 36 ]. AtWOX14 has been shown to regulate vascular development through interactions with cell-wall biosynthesis genes, this function is also observed in pumpkin [ 37 ]. Additionally, PtWOX14 is involved in the cell proliferation associated with wood formation in poplar [ 15 ]. Consistently, the promotion of hairy-root transformation by HbWOX14 demonstrated here underscores the conserved role of the WOX family in controlling plant organogenesis. Furthermore, the hairy-root system based on HbWOX14 overexpression facilitates rapid target-gene transformation within approximately one month, enhancing validation efficiency by more than an order of magnitude. Compared to fairy roots overexpressing PRI101-GFP , hairy roots overexpressing HbWOX14 -OE exhibited differentially expressed genes, which significantly enriched in pathways related to hormone response, cell development, and flavonoid glycosylation, which is similar to Arabidopsis WOX genes in promoting organ regeneration through multiple pathways. In the hormone-response pathways, genes associated with auxin polar transport and signaling, such as ERF4 , WOX11 , and RHA2B (GT014369), were notably upregulated. This pattern is conserved in Oryza sativa where OsWOX11 enhances crown-root development by activating auxin signaling [ 38 , 39 ]. Conversely, in rubber trees, cytokinin-response genes were simultaneously upregulated, forming an auxin–cytokinin co-regulatory network, similarity to Brassica napus BnWOX5 that promotes somatic embryogenesis by modulating hormone ratios. The pathway-enrichment characteristics of the DEGs underscore the central regulatory role of HbWOX14 . Notably, the prominent enrichment of flavonoid glycosylation appears unique to rubber trees, while flavonoid glycosylation is known to participate in hormone homeostasis, as reported in Arabidopsis, its stronger enrichment observed here may be attributed to a heightened demand for phenolic-metabolism buffering within rubber trees [ 40 , 41 ]. Glycosylation can mitigate phenolic toxicity and reduce explant browning that inhibits hairy-root initiation while also modulating auxin distribution and activity [ 42 ]. Thereby, this further supports the notion that HbWOX14 integrates secondary metabolism with hormone signaling to provide dual safeguards for hairy-root development. In cell-development pathways, genes associated with cell-cycle regulation, microtubule movement, and cell-wall biogenesis (e.g., GT014369) were significantly upregulated, as AtWOX14 promotes vascular differentiation through the regulation of cell-division genes. In contrast, in rubber tree, the enrichment of genes related to cell junctions and plasmodesmata suggests a coordination of overall hairy-root development via enhanced intercellular communication, an adaptive regulation likely necessary for maintaining structural stability during woody-root growth. Moreover, in roots overexpressing HbWOX14 , key negative regulators within ABA and JA signaling pathways (e.g., ANAC083 ; GT033271 ) were downregulated. These stress hormones are known to suppress growth under adverse conditions [ 43 , 44 ], similar to the inhibitory effect observed with gibberellin on Asiatic lily [ 34 ]. Thus, HbWOX14 appears to repress ABA/JA stress signaling while enhancing auxin signal transmission [ 45 ]. Additionally, lower ABA levels under heavy-metal stress have been shown to enhance plant growth in Arabidopsis [ 46 ]. Accordingly, HbWOX14 can coordinate these pathways by positively regulating auxin signaling [ 22 ] while simultaneously repressing stress-related hormone pathways [ 39 ], thereby achieving regulatory control over rubber-tree root development [ 47 , 48 ]. Conclusions This study systematically investigated the function and evolution of WOX gene family in rubber trees, focusing on resolving low genetic transformation efficiency and unstable hairy root induction, providing a robust support for molecular breeding in rubber tree. We identified 17 HbWOX gene family members, which all harbor a conserved homeodomain (HD) with a "helix–turn–helix" conformation consistent with WOX proteins in Arabidopsis and maize. Most of HbWOX genes retain the motif "PEVSSRWNPTPEQLR", while HbWOX13 is absent, implying functional differentiation of HbWOX . HbWOX14 is a root-specific regulator, and overexpressing HbWOX14 raised Agrobacterium -mediated hairy root induction rate from 13.3% to 66.7%, and improved root length, number, and thickness, breaking genotype limitations. Further transcriptome analysis indicated that HbWOX14 acts via a coordinated network, activating auxin/cytokinin pathways, repressing ABA/JA stress signaling, upregulating cell cycle/wall genes, and enriching flavonoid glycosylation. This study clarifies the characteristics of HbWOX family and the role of HbWOX14 in improving transgenic efficiency in rubber tree study, enriching plant WOX research and providing technical support for rubber tree breeding, facilitating the natural rubber industry’s sustainability. Abbreviations HD homeodomain GO Gene Ontology RR2 Response Regulator 2 PCA Principal component analysis HRTS hairy-root transformation system CDB Cut–Dip–Budding DEGs Differentially expressed genes. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB1080000), the Project for Major Science and Technology Planning in Yunnan (Grant No. 202402AE09001904), Special Program for Technological Innovation of Xishuangbanna Science and Technology Bureau (2025kjcx003), Yunnan Revitalization Talent Support Program Yunling Scholar Project to Yongping Yang. Author Contribution YPY, YQY and YZ conceived and designed the experiments. CLZ, KYC and MXH participated in the sample collecting and interpretation of data. CLZ, KYC and MXH performed the experiments and analysed the sequencing data. CLZ, XY and JWY wrote the manuscript. YZ, YQY and YYQ contributed with valuable discussions and revised the paper. All authors have read and approved the final manuscript. Acknowledgement The authors are grateful the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences for providing the plant materials. 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09:39:24","extension":"xml","order_by":52,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":174973,"visible":true,"origin":"","legend":"","description":"","filename":"8718e9a45834479682e0d5d3a04c9dbc1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/2ab9fd09dcbffbac4b5598db.xml"},{"id":97333851,"identity":"59f75b8f-3c00-4989-b385-270845851ede","added_by":"auto","created_at":"2025-12-03 09:39:31","extension":"html","order_by":53,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":198195,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/030fef8a6ec31eb820065e8c.html"},{"id":97333789,"identity":"e563d570-ad47-458c-b0c1-0fe663d61844","added_by":"auto","created_at":"2025-12-03 09:39:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5138793,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConserved motif analysis of the HbWOX proteins and Structural analysis of the\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e HbWOX\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Conserved protein domain and motif prediction by NCBI Batch CD-search with the MEME website. (B) The distribution of introns and exons for each gene was visualized on TBtools using genome annotation gff3 files and genomic fasta sequence files. The green boxes indicate exons, black lines indicate introns.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/0542fdafc06f1f4dfc006c5a.png"},{"id":97333848,"identity":"40ea977e-0ed1-4c8d-8bbd-6cc1aec7fff3","added_by":"auto","created_at":"2025-12-03 09:39:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4981795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCis-element analysis of the\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e HbWOX\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2000 bp upstream of the \u003cem\u003eHbWOX\u003c/em\u003e gene as the promoter region, predicted using the PlantCare website and visualized by TBtools.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/d58c7fcefdfb40b5e1c7978f.png"},{"id":97333733,"identity":"afb8d20c-0633-4bdb-a7f1-725246cf8182","added_by":"auto","created_at":"2025-12-03 09:39:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4842474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression pattern of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHbWOX\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes under different hormone treatments based on RT-qPCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression patterns of \u003cem\u003eHbWOX\u003c/em\u003e genes detected by qRT-PCR under four hormone treatments with NAA (A), IAA (B), IBA (C), and 6-BA (D). Total RNA was extracted and reverse-transcribed into cDNA for qRT-PCR analysis of gene expression after IBA treatment (A), IAA treatment (B), 6-BA treatment (C), and NAA treatment (D). The working concentration for each hormone was 100 mg·L⁻¹ and 200 mg·L⁻¹, with three biological replicates for each treatment. Leaf samples were collected at 0.5, 1, 3, and 6 hours after treatment used for total RNA extraction and gene expression analysis by RT-qPCR.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/764bf0b7ac3a91612764a29f.png"},{"id":97333834,"identity":"1167c20b-4d79-49e3-aa5e-47a1151708be","added_by":"auto","created_at":"2025-12-03 09:39:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1778556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTissue-specific expression analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHbWOX\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phenotype of the rubber tree GT1 seedling. The orange arrows indicate different tissue parts. (B) Expression levels of \u003cem\u003eHbWOX\u003c/em\u003egenes in root, stem, and leaf tissues in rubber tree GT1 analyzed by RT-qPCR. Data indicates the mean ± SD, n=3.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/0fe80b166744540d91e2a14e.png"},{"id":97333842,"identity":"92ada9fc-1f78-440c-a783-8508e44d76a5","added_by":"auto","created_at":"2025-12-03 09:39:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5725114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment of transgenic hairy root induction system in rubber tree\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of injection method, \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e strain LBA9402 carrying the GFP vector was injected into the stem-leaf junction of the plant. (B) flat placement method, explants were immersed in \u003cem\u003eA. rhizogenes\u003c/em\u003e LBA9402 carrying the GFP vector and then placed flat on moist substrate. (C) Schematic diagram of CDB method, after removing the roots of rubber tree seedlings, the hypocotyl wound was directly coated with \u003cem\u003eA. rhizogenes\u003c/em\u003e LBA9402 suspension. (D) Number of hairy roots induced by the three infection methods: Injection, Flat placement, and CDB. Data represent the mean ± SD, n=3. *, indicates P \u0026lt; 0.05. (E) Statistics analysis of hairy root numbers induced by two \u003cem\u003eA. rhizogenes\u003c/em\u003e strains, LBA9402 and K599, in rubber tree seedlings. Data represent the mean ± SD, n=3. *, indicates P \u0026lt; 0.05. (F-I) The growth process of hairy root induction with CDB method. Phenotype of transgenic hairy roots induced by \u003cem\u003eA. rhizogenes\u003c/em\u003e LBA9402 carrying the GFP vector at 0 days (F), 15 days (G), 30 days (H), and 45 days (I) post-infection. (J) PCR products of genomic DNA of hairy roots from wild-type (WT) and PRI101-GFP overexpressed line. DNA was extracted from the induced hairy roots, and the obtained products were identified by Polymerase Chain Reaction.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/5f805151deaeda65b12789d3.png"},{"id":97333785,"identity":"3038d020-81f6-4252-9428-5f636d632d2c","added_by":"auto","created_at":"2025-12-03 09:39:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8199554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of PRI101: GFP-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHbWOX14 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand PRI101: GFP overexpressed lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Scheme of the T-DNA region of \u003cem\u003eHbWOX14\u003c/em\u003eoverexpression vector. In the gene schematic, light green represents the \u003cem\u003eHbWOX14\u003c/em\u003egene, orange parts indicate the right border (RB) and left border (LB), grass green represents the promoter. Purple represents neomycin phosphotransferase II, yellow represents the terminator. (B) Phenotype of hairy roots obtained by overexpressing PRI101: GFP-\u003cem\u003eHbWOX14\u003c/em\u003e. (C) Phenotype of hairy roots obtained by overexpressing PRI101: GFP. (D) PCR analysis and identification of wild-type (WT), PRI101: GFP-\u003cem\u003eHbWOX14\u003c/em\u003e and PRI101: GFP overexpressed lines. (E-H) Statistics analysis of root length (E), root diameter (F), root number (G), and newly formed bud number (H) for hairy roots overexpressing PRI101: GFP-\u003cem\u003eHbWOX14\u003c/em\u003eand PRI101: GFP vectors. Data represent the mean ± SD, n=3. ***, indicates p \u0026lt; 0.001; **, indicates p \u0026lt; 0.01; ns, indicates no significance.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/5c4c3e01c76922a7ae44c2ec.png"},{"id":97333849,"identity":"e6746bb0-8db1-44fc-b52f-8f6052fee070","added_by":"auto","created_at":"2025-12-03 09:39:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9030802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRI101: GFP-HbWOX14\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRI101: GFP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoverexpressed lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) PCA plot of \u003cem\u003ePRI101: GFP-HbWOX14\u003c/em\u003e and \u003cem\u003ePRI101: GFP\u003c/em\u003e overexpressed lines. (B) GO enrichment analysis of differentially expressed genes (DEGs). (C) Volcano plot of DEGs between \u003cem\u003ePRI101: GFP-HbWOX14\u003c/em\u003eand \u003cem\u003ePRI101: GFP\u003c/em\u003e overexpressed lines.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/ea59b37f40139dcfdde675c1.png"},{"id":97370874,"identity":"d2f70445-6ecd-42a6-91fd-e974e1f388ad","added_by":"auto","created_at":"2025-12-03 16:28:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3934190,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRT-qPCR validation of significantly differentially expressed genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRI101: GFP-HbWOX14\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRI101: GFP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoverexpressed lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-H) qRT-PCR validation of DEGs related to plant growth. \u003cem\u003eAtERF4 \u003c/em\u003e(A), \u003cem\u003eAtCIPK7\u003c/em\u003e (B), \u003cem\u003eAtHB-21\u003c/em\u003e (C), \u003cem\u003eAtHB-40\u003c/em\u003e(D), \u003cem\u003eAtMYB63 \u003c/em\u003e(E), \u003cem\u003eAtMYB108\u003c/em\u003e (F), \u003cem\u003eAtMYB75\u003c/em\u003e (G), \u003cem\u003eAtMYB111\u003c/em\u003e(H). (I-P) qRT-PCR validation of DEGs related to hormones. \u003cem\u003eAtANAC083 \u003c/em\u003e(I), \u003cem\u003eAtWOX11\u003c/em\u003e (J), \u003cem\u003eAtWOX13 \u003c/em\u003e(K), \u003cem\u003eAtRHA2B\u003c/em\u003e (L), \u003cem\u003eAtMYB59\u003c/em\u003e (M), \u003cem\u003eAtMYB73\u003c/em\u003e (N), \u003cem\u003eAtMYB74\u003c/em\u003e (O), \u003cem\u003eAtMYB62\u003c/em\u003e (P). Data represent the mean ± SD, n=3.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/c1d305a29dccc46cabe43b66.png"},{"id":97333831,"identity":"de6c81a0-1b1c-483c-be52-104fb7c47ba4","added_by":"auto","created_at":"2025-12-03 09:39:29","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":768659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignaling pathway network of significantly differentially expressed genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRI101: GFP-HbWOX14\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e overexpressed lines.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/9a6359c521e9fd1e50fbbd54.png"},{"id":97665259,"identity":"107baa96-4769-4fe5-89a1-c105e092e05b","added_by":"auto","created_at":"2025-12-08 09:17:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":43146650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/91b4beb6-d21c-41f7-a164-9ba4eef59e22.pdf"},{"id":97333850,"identity":"b04dd951-693f-41be-8a74-6ad709d7b650","added_by":"auto","created_at":"2025-12-03 09:39:31","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3375962,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/dd10fcd87e2c5858a452fd07.docx"},{"id":97333856,"identity":"eebe8d0d-4803-41bf-bf19-b859848f4c3c","added_by":"auto","created_at":"2025-12-03 09:39:31","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20706,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/76c89abf1e2638924275fbc9.xlsx"},{"id":97333853,"identity":"1b54083d-6ec6-42a3-b8d1-293949df29b1","added_by":"auto","created_at":"2025-12-03 09:39:31","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":108135,"visible":true,"origin":"","legend":"","description":"","filename":"Gelimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/8d5d5e2eb14e9223435dd3ec.png"},{"id":97370006,"identity":"2bca5959-98d8-442a-80d5-166d7c61f30c","added_by":"auto","created_at":"2025-12-03 16:26:22","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":41183,"visible":true,"origin":"","legend":"","description":"","filename":"Gelimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8197958/v1/773592e7e57acf3db416b288.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide analysis of the HbWOX gene family and HbWOX14-mediated enhancement of hairy root transformation in Hevea brasiliensis","fulltext":[{"header":"Background","content":"\u003cp\u003eThe \u003cem\u003eWUSCHEL\u003c/em\u003e-related homeobox (\u003cem\u003eWOX\u003c/em\u003e) gene family, as a plant-specific family of key transcription factors, plays important regulatory roles in core life processes such as stem-cell maintenance, organogenesis, wound repair, and stress responses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eWOX\u003c/em\u003e genes belong to the homeodomain (HD) superfamily [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], with the proteins they encode featuring a highly conserved HD comprising approximately 60 amino acids that adopts a \u0026ldquo;helix\u0026ndash;loop\u0026ndash;helix\u0026ndash;turn\u0026ndash;helix\u0026rdquo; conformation. This unique structure enables specific binding to DNA sequences, facilitating precise recognition of key cis-acting elements in target gene promoters, such as the CAAT-box, TATA-box, CTCC motif, and TATTGTAGAC motif and regulating downstream gene-expression programs that finely control plant growth and development [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. During stem-cell division, \u003cem\u003eWOX\u003c/em\u003e genes contribute to maintaining the stability of stem-cell populations and balancing proliferation by regulating the expression of associated cyclin genes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. At the stage of root primordium initiation, these genes coordinate the timing of expression for cell-differentiation-related genes to ensure that root primordia develop along designated pathways [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, \u003cem\u003eWOX\u003c/em\u003e genes regulate activities within the root apical meristem and promote continuous growth of adventitious roots through synergistic interactions with the auxin signaling pathway [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition to their conserved homeodomain features, the number, type, and arrangement of these motifs will contribute to the molecular foundation for their functional diversity and specificity. For example, although CsFAR proteins across different phylogenetic groups (e.g., Class I, II, III) share certain fundamental motifs in cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.), the significant differences existed in the quantity, sequential order, and relative positioning of these motifs directly contribute to the functional differentiation among CsFAR proteins from various classes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Similarly, the differential distribution of motifs among FanSWEET proteins facilitates their roles in sugar transport and metabolic regulation across diverse strawberry tissues and organs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The structural characteristics of these key gene family suggest that they have continuously expanded their regulatory scope through variation and recombination of auxiliary motifs during plant evolution process, while maintaining core DNA-binding capabilities essential for fundamental regulatory functions, which precisely contributed to adapt to the requirements of different stages of plant growth and development [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn classical model plants, such as Arabidopsis thaliana, Oryza sativa, and Zea mays, the genome-wide identification of the \u003cem\u003eWOX\u003c/em\u003e gene family has been completed and functionally characterized. \u003cem\u003eWOX5\u003c/em\u003e has been demonstrated to maintain stem-cell homeostasis in the root apical meristem, with its expression being precisely regulated by the auxin concentration gradient. The absence of the \u003cem\u003eWOX5\u003c/em\u003e gene results in root growth arrest [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the shoot apex, \u003cem\u003eMdWUS-1\u003c/em\u003e promotes the formation of leaf-derived buds in apple (\u003cem\u003eMalus pumila\u003c/em\u003e Mill.) and plays a significant role in maintenance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In macadamia (\u003cem\u003eMacadamia integrifolia Maiden et Betche\u003c/em\u003e), a homolog of \u003cem\u003eWOX\u003c/em\u003e has been identified that participates in bud regeneration [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In jasmine (Jasminum sambac), \u003cem\u003eWOX1\u003c/em\u003e primarily influences root primordium initiation and differentiation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], while \u003cem\u003eWOX5\u003c/em\u003e exhibits relatively high expression levels in roots of wheat (Triticum aestivum) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. \u003cem\u003eWOX14\u003c/em\u003e is mainly involved in plant root formation and elongation; overexpression of \u003cem\u003eZmWOX14\u003c/em\u003e significantly enhances callus induction efficiency and improves plant regeneration capability in maize (\u003cem\u003eZea mays\u003c/em\u003e L.), providing an important genetic resource for maize transformation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Similarly, studies have shown that \u003cem\u003eWOX14\u003c/em\u003e is implicated in adventitious-root formation within Arabidopsis thaliana [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePlant root development and organ regeneration are biological processes that are intricately coordinated by transcriptional regulatory networks and plant hormone signaling. Within this network, \u003cem\u003eWOX\u003c/em\u003e genes serve as core regulatory elements, playing crucial roles in stem cell maintenance, meristem establishment, and hormone responses [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Previous studies have demonstrated that \u003cem\u003eWOX\u003c/em\u003e genes are influenced by upstream plant hormones such as auxin and cytokinin within the regulatory framework [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In conjunction with multiple hormone-regulated signaling pathways, these genes further regulate downstream hormone homeostasis and signal transduction, thereby facilitating the organogenesis of roots and other organs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Auxin signaling has been shown to activate the expression of \u003cem\u003eWOX11\u003c/em\u003e, which subsequently activates RR2, promoting the initiation and development of the root cap meristem in Oryza sativa [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] \u003cem\u003eLsWOX9\u003c/em\u003e precisely regulates the initiation and emergence of lateral-root primordia by influencing the polar transport and distribution of auxin. This gene serves as a critical link between upstream auxin signaling and downstream lateral-root growth during lateral-root development in tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In poplar species (\u003cem\u003ePopulus spp.\u003c/em\u003e), \u003cem\u003ePtoWOX5\u003c/em\u003ea primarily governs adventitious root development through auxin signal transduction [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The process of somatic embryogenesis in rapeseed (\u003cem\u003eBrassica napus\u003c/em\u003e) is highly dependent on the ratio of exogenous to endogenous auxin/cytokinin levels [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As a key downstream effector, \u003cem\u003eBnWOX5\u003c/em\u003e significantly induces and transduces hormonal signals to initiate subsequent embryonic development [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, \u003cem\u003eWOX14\u003c/em\u003e participates in gibberellin biosynthesis and signaling; through its coordination with auxin and other hormones, it promotes hypocotyl formation as well as lateral root development in Arabidopsis thaliana [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These studies collectively underscore the critical role of \u003cem\u003eWOX\u003c/em\u003e genes in regulating root regeneration under various hormonal conditions. However, the molecular mechanisms by which different members of the \u003cem\u003eWOX\u003c/em\u003e family respond to specific hormone types exhibit significant species specificity across diverse plants.\u003c/p\u003e\u003cp\u003eThe rubber tree (\u003cem\u003eHevea brasiliensis\u003c/em\u003e), as the primary commercial source of natural rubber, possesses irreplaceable strategic significance across various sectors, including industry, agriculture, national defense, transportation, and machinery manufacturing. Tissue culture serves as an efficient method for vegetative propagation and represents a crucial pathway for cultivating high-yield varieties of rubber trees. Through tissue culture techniques, elite cultivars of rubber trees can be rapidly multiplied; furthermore, traits such as yield and stress resistance can be enhanced through approaches like somaclonal variation screening and gene editing. Currently, the application of tissue culture in \u003cem\u003eH. brasiliensis\u003c/em\u003e encounters several limitations [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Only a limited number of cultivars can be propagated rapidly using this method; conversely, most elite cultivars that exhibit high yield and strong resistance are exceedingly challenging to propagate. Genotype dependence has emerged as a significant constraint on clonal propagation in \u003cem\u003eH. brasiliensis\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Consequently, addressing the genotype barrier to enhance both the transformation efficiency of desirable traits and the regeneration capacity of plants has become a critical issue requiring urgent resolution within rubber tree breeding research [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous studies have indicated that employing regeneration-related genes such as \u003cem\u003eWOX\u003c/em\u003e can effectively improve plant genetic transformation efficiency while broadening genotype adaptability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we systematically identified the \u003cem\u003eWOX\u003c/em\u003e gene family in rubber tree, determined the number and classification of its members, analyzed the gene structure and evolutionary characteristics of each member, and examined expression changes of different \u003cem\u003eHbWOX\u003c/em\u003e genes in roots, stems, and leaves under various hormone treatments through RT-qPCR. Additionally, using hairy roots from rubber trees overexpressing the \u003cem\u003eHbWOX14\u003c/em\u003e gene as materials, we conducted transcriptome sequencing and GO enrichment analysis to elucidate the biological processes and signaling pathways involved in \u003cem\u003eHbWOX14\u003c/em\u003e-mediated root regeneration. Our findings indicate that \u003cem\u003eHbWOX14\u003c/em\u003e significantly enhances transgenic hairy-root induction. These results provide crucial theoretical and technical support for identifying key functional genes and addressing challenges related to genetic transformation in rubber trees.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003eWOX\u003c/b\u003e \u003cb\u003egene family members in rubber tree\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eWOX\u003c/em\u003e genes of \u003cem\u003eArabidopsis\u003c/em\u003e were retrieved from the \u003cem\u003eArabidopsis\u003c/em\u003e database TAIR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.arabidopsis.org\u003c/span\u003e\u003cspan address=\"http://www.arabidopsis.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The \u003cem\u003eArabidopsis WOX\u003c/em\u003e gene sequences served as query files, and the GT1 genome sequence of rubber tree (\u003cem\u003eHevea brasiliensis\u003c/em\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] was used as the reference file. Local BLAST protein alignments were performed with an E-value\u0026thinsp;\u0026lt;\u0026thinsp;1e\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e to obtain candidate rubber-tree \u003cem\u003eWOX\u003c/em\u003e genes. Protein domain prediction was carried out using SMART (SMART: Mainpage (embl.de)) and Pfam, and members containing the HOX domain were retained. The preliminarily determined members were further aligned on the NCBI website, Pfam IDs were obtained from the literature (Zhao et al.,2009; [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] (ID: PF00046), yielding the candidate \u003cem\u003eH. brasiliensis WOX\u003c/em\u003e gene family members [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Phylogenetic trees were constructed for the candidate \u003cem\u003eH. brasiliensis WOX\u003c/em\u003e members together with \u003cem\u003eArabidopsis WOX\u003c/em\u003e genes using filtreebest and the iTOL website, thereby determining the \u003cem\u003eWOX\u003c/em\u003e gene family members in the GT1 genome of rubber tree. The rubber-tree \u003cem\u003eWOX\u003c/em\u003e genes (\u003cem\u003eHbWOX\u003c/em\u003e) were then named following the nomenclature of \u003cem\u003eArabidopsis WOX\u003c/em\u003e family members (\u003cem\u003eAtWOX\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene structure and conserved motif analysis of\u003c/b\u003e \u003cb\u003eHbWOX\u003c/b\u003e \u003cb\u003efamily members\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe intron\u0026ndash;exon organizations of \u003cem\u003eHbWOX\u003c/em\u003e family members in rubber tree were analyzed using the online Gene Structure Display Server (GSDS; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Conserved domains and motifs of the \u003cem\u003eHbWOX\u003c/em\u003e proteins were predicted online with NCBI Batch CD-Search and MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), respectively.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCis-acting element prediction of the promoters\u003c/b\u003e \u003cb\u003eWOX\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing TBtools, we extracted the 2,000-bp upstream promoter regions of \u003cem\u003eHbWOX\u003c/em\u003e genes and submitted these sequences to PlantCare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for prediction. The results obtained from PlantCare were curated and simplified, and the final annotations were visualized using TBtools.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eRNA extraction and RT-qPCR\u003c/h2\u003e\u003cp\u003eRoots, stems, and leaves from the elite rubber tree clone \u0026lsquo;GT1\u0026rsquo; were utilized as experimental materials. Immediately after collection, samples were snap-frozen in liquid nitrogen and subsequently stored at \u0026minus;\u0026thinsp;80\u0026deg;C until total RNA extraction. Total RNA was extracted using the Eastep\u0026reg; Super Total RNA Extraction Kit (Promega, Madison, WI, USA). The concentration of RNA was measured with a NanoDrop 1000 (NanoDrop Technologies, Inc.), while RNA integrity was assessed on 0.8% agarose gels.\u003c/p\u003e\u003cp\u003eApproximately 5 \u0026micro;g RNA was reverse-transcribed using the GoScript\u0026reg; Reverse Transcription System (Promega, Madison, WI, USA) to generate cDNA. RT-qPCR was performed on cDNA from different tissues and treatments using FastStart\u0026trade; Universal SYBR\u0026reg; Green Master (ROX; Roche, Indianapolis, IN, USA) on a 7500 Real-Time PCR System (Applied Biosystems, USA), with three technical replicates. The resulting cDNA products were stored at \u0026minus;\u0026thinsp;20\u0026deg;C as templates for qPCR. Using diluted cDNA as template, amplification was also carried out with TB Green\u0026reg; Premix Ex Taq\u0026trade; II on a QuantStudio\u0026reg; 5 Real-Time PCR System (Applied Biosystems, USA). Each 20 \u0026micro;L reaction contained 10 \u0026micro;L TB Green Premix Ex Taq II, 0.8 \u0026micro;L each of forward and reverse primers, 2 \u0026micro;L cDNA template, and 6.4 \u0026micro;L nuclease-free water. The RT-qPCR cycling program was: 95\u0026deg;C for 10 min; then 40 cycles of 94\u0026deg;C for 5 s and 60\u0026deg;C for 15 s. Melting-curve analysis was performed to verify amplification specificity. Three technical replicates were set for each sample. Relative expression levels were calculated using the 2^\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. Statistical comparisons between treatment and control were conducted with independent-samples t-tests. The primers used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAcquisition and treatment of experimental materials\u003c/h3\u003e\n\u003cp\u003eOne-year-old tender stems and current-year seedlings of the elite rubber-tree clone \u0026lsquo;GT1\u0026rsquo; were used as experimental materials. After seedlings were collected, roots were removed to facilitate infection by \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e. Seedlings were then planted in a 3:1 (v/v) mixture of vermiculite and nutrient soil and grown for 30\u0026ndash;45 d under a 16 h light/8 h dark photoperiod, a light intensity of 100 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1;, and a temperature of 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Once growth stabilized, vigorously growing seedlings with a height of 20\u0026ndash;25 cm and tender stems were selected as explants. All \u0026lsquo;GT1\u0026rsquo; materials used in this study were maintained and provided by Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.\u003c/p\u003e\u003cp\u003eFor hormone treatment, four plant growth regulators were used: α-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 6-benzylaminopurine (6-BA). Each hormone was prepared as a stock solution and diluted with sterile water to working solutions of 100 mg\u0026middot;L⁻\u0026sup1; and 200 mg\u0026middot;L⁻\u0026sup1;. Seedlings were randomly grouped, and the working solutions were evenly sprayed onto the leaves. Samples were collected at 0.5 h, 1 h, 3 h, and 6 h after treatment. At each sampling time, surface liquid was blotted with sterile filter paper, and samples were immediately snap-frozen in liquid nitrogen. For each treatment combination (hormone \u0026times; concentration \u0026times; time point), three biological replicates were set. All samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent total RNA extraction and gene-expression analysis by RT-qPCR.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstablishment of a hairy-root transformation system in rubber tree\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. rhizogenes\u003c/em\u003e strains K599 and LBA9402 carrying the pR101 plasmid were cultured with shaking. When the cultures reached OD₆₀₀ = 0.5, 0.8, or were used undiluted (original culture), explants were inoculated. After co-cultivation and selection, three inoculation methods were applied: Injection, CDB (Cut\u0026ndash;Dip\u0026ndash;Budding), and Flat placement. Hairy roots were preliminarily induced after about 30 d. Genomic DNA was extracted from the obtained hairy roots, with three biological replicates.\u003c/p\u003e\u003cp\u003eSeedlings measuring 20\u0026ndash;25 cm in height and one-year-old tender stems were used as explants. Two to three superficial wounds were gently made on each explant, followed by inoculation using one of the three methods, (1) Injection method: the \u003cem\u003eA. rhizogenes\u003c/em\u003e inoculum was loaded into a 10 mL syringe and injected into the explants. (2) CDB method: the prepared \u003cem\u003eA. rhizogenes\u003c/em\u003e suspension was directly smeared onto the wounds after 30 min, explants were inserted into a moistened mixed substrate. (3) Flat-placement method: explants were immersed in the \u003cem\u003eA. rhizogenes\u003c/em\u003e inoculum for 30 min with gentle shaking every 5 min; explants were then removed, blotted with sterile filter paper to remove excess inoculum, and placed flat on the surface of the moistened mixed substrate.\u003c/p\u003e\u003cp\u003eCo-cultivation was conducted in darkness for 72 h at a temperature of 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. After this period, explants were then transferred to the corresponding substrate and cultured for 30\u0026ndash;45 d under a 16 h\u0026middot;d⁻\u0026sup1; photoperiod, light intensity of 100 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1;, and temperature of 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. The hairy-root induction rate was calculated as:\u003c/p\u003e\u003cp\u003eInduction rate (%) = (number of explants producing hairy roots / total number of explants) \u0026times; 100%.\u003c/p\u003e\n\u003ch3\u003eDetection of transgenic hairy roots\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from transgenic hairy roots using the CTAB method. PCR amplification was conducted with universal GFP primers, and the resulting amplification bands were analyzed by agarose gel electrophoresis to ascertain whether the reporter gene had been stably integrated into the hairy roots. Wild-type roots served as a negative control. The positive transformation rate was calculated as follows: Positive transformation rate (%) = (number of hairy roots with a GFP band / total number examined) \u0026times; 100%. The primers used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eData statistics and analysis\u003c/h3\u003e\n\u003cp\u003eStatistical analysis was performed using Ordinary one-way ANOVA and Student\u0026rsquo;s t-test (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Data analysis was conducted using Microsoft Excel 2019 and GraphPad Prism 8.0.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification and gene structure analysis of\u003c/strong\u003e \u003cstrong\u003eWOX\u003c/strong\u003e \u003cstrong\u003efamily members in rubber tree\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 17 \u003cem\u003eWOX\u003c/em\u003e gene members in rubber tree have been identified. Based on sequence similarity to \u003cem\u003eArabidopsis WOX\u003c/em\u003e genes (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e), the \u003cem\u003eWOX\u003c/em\u003e genes in rubber-tree were renamed accordingly (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e, S3). Notably, \u003cem\u003eWOX7, WOX9, WOX10\u003c/em\u003e, and \u003cem\u003eWOX12\u003c/em\u003e are absent in rubber tree, \u003cem\u003eHbWOX8\u003c/em\u003e and \u003cem\u003eHbWOX6\u003c/em\u003e exhibited three copies, \u003cem\u003eHbWOX2\u003c/em\u003e, \u003cem\u003eHbWOX5\u003c/em\u003e, \u003cem\u003eHbWOX11\u003c/em\u003e, \u003cem\u003eHbWOX13\u003c/em\u003e, and \u003cem\u003eHbWOX14\u003c/em\u003e are present as single-copy genes within the rubber tree. Otherwise, the remaining members possess two copies each, indicating an expansion of the \u003cem\u003eWOX\u003c/em\u003e family specific to this species. Chromosome localization and synteny analysis of the \u003cem\u003eHbWOX\u003c/em\u003e gene revealed that the 17 WOX gene members of the rubber tree were distributed on different contigs, each member having a unique position in the genome with no tandem gene duplication. Five pairs of homologous genes were identified with fragment duplications, involving 10 \u003cem\u003eHbWOX\u003c/em\u003e genes, including \u003cem\u003eHbWOX1.1/HbWOX1.2\u003c/em\u003e, \u003cem\u003eHbWOX3.1/HbWOX3.2\u003c/em\u003e, \u003cem\u003eHbWOX4.1/HbWOX4.2\u003c/em\u003e, \u003cem\u003eHbWOX6.1/HbWOX6.2\u003c/em\u003e, and \u003cem\u003eHbWOX8.1/HbWOX8.3\u003c/em\u003e. The extensive fragment duplications among family members on different chromosomes may be the main reason for the expansion of the WOX gene family in rubber trees (Table \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e; S3).\u003c/p\u003e\n\u003cp\u003eConserved motif analysis can enhance our understanding of specific gene functions and their evolutionary conservation [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Utilizing protein sequences from the identified HbWOX members for motif analysis. we observed that each gene contains at least one conserved motif with a maximum of eleven motifs per gene (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, A). Among these proteins, HbWOX6.1, HbWOX6.2, HbWOX6.3, HbWOX1.1, HbWOX1.2, HbWOX3.1, HbWOX3.2, HbWOX4.1, and HbWOX4.2 share two identical conserved motifs, HbWOX2 and HbWOX5 share one identical conserved motif, HbWOX11, HbWOX8.1, HbWOX8.2, and HbWOX8.3 share two identical conserved motifs, and all of these contain motif 1. In addition, with the exception of HbWOX13, the \u0026ldquo;PEVSSRWNPTPEQLR\u0026rdquo; motif is present in the other 16 HbWOX proteins, indicating very high degree of conservation. This suggests that this motif may represent a signature functional domain capable of specifically regulating processes such as wound responses, cell proliferation, and organ regeneration. Consequently, it exhibits marked functional divergence from proteins primarily involved in stem-cell maintenance. The homeodomain found in WOX proteins of rubber tree is conserved across all 17 proteins, including HbWOX14, highlighting significant conservation at the amino acid level (Fig. \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003e). Its characteristic three-dimensional architecture follows a \u0026ldquo;helix\u0026ndash;turn\u0026ndash;helix\u0026rdquo; pattern (Fig. S5). The two \u0026alpha;-helices are highly conserved while the loop and turn regions connecting them exhibit comparatively less conservation. This pronounced sequence and structural conservation underscore the central role of the homeodomain (HD) in WOX protein function and its stability throughout plant evolution.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo explore the structural features related to \u003cem\u003eHbWOX\u003c/em\u003e gene function, introns and exons were analyzed for all 17 family members (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, B). The number of exons varied from 2 to 5, while the number of introns ranged from 1 to 5. Notably, \u003cem\u003eHbWOX13\u003c/em\u003e and \u003cem\u003eHbWOX4.1\u003c/em\u003e each possess five exons, \u003cem\u003eHbWOX8.2\u003c/em\u003e, \u003cem\u003eHbWOX11\u003c/em\u003e, \u003cem\u003eHbWOX2\u003c/em\u003e, \u003cem\u003eHbWOX6.1\u003c/em\u003e, and \u003cem\u003eHbWOX3.2\u003c/em\u003e contain two exons each, \u003cem\u003eHbWOX1.1, HbWOX1.2, HbWOX6.3\u003c/em\u003e, and \u003cem\u003eHbWOX8.3\u003c/em\u003e each contain four exons, and the remaining genes, including \u003cem\u003eHbWOX14\u003c/em\u003e, each contain three exons, showing a high degree of consistency. Gene structures exhibit marked differences among various genes, however, variations within homologous pairs are relatively minor, reflecting functional distinctions among the different genes involved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCis-element analysis of the promoters of\u003c/strong\u003e \u003cstrong\u003eWOX\u003c/strong\u003e \u003cstrong\u003egenes in rubber-tree\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 2,000-bp upstream promoter sequences of \u003cem\u003eHbWOX\u003c/em\u003e genes were extracted, and cis-acting elements were analyzed using PlantCare to predict putative biological functions and visualized by TBtools (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The analysis identified a total of 20 distinct cis-elements, including numerous light-responsive elements, auxin-related elements, and those induced by abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin (GA), and salicylic acid (SA). Additionally, elements associated with stress defenses against low temperature and anaerobiosis were detected. Cis-element compositions varied among homologous genes. Notably, \u003cem\u003eHbWOX14\u003c/em\u003e contained a relatively high number of auxin-related elements, while \u003cem\u003eHbWOX1.1\u003c/em\u003e harbored more drought-responsive elements. \u003cem\u003eHbWOX1.2\u003c/em\u003e exhibited the greatest abundance of light-responsive elements. These findings suggest that \u003cem\u003eHbWOX14\u003c/em\u003e may function as a key downstream gene in the auxin signaling pathway, implying that \u003cem\u003eHbWOX14\u003c/em\u003e could perceive endogenous or exogenous auxin signals to activate downstream organogenesis-related gene networks. Furthermore, these results underscore the significant role of \u003cem\u003eHbWOX14\u003c/em\u003e in regulating regeneration during rubber tree tissue culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of different hormones treatments on\u003c/strong\u003e \u003cstrong\u003eHbWOX\u003c/strong\u003e \u003cstrong\u003egene expression level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, we profiled the spatiotemporal expression of 17 \u003cem\u003eHbWOX\u003c/em\u003e genes in rubber tree leaves following treatments with four types of hormones at two concentrations over different time points: IBA (100 mg, 200 mg) ( Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, A), IAA (100 mg, 200 mg) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, B), 6-BA (100 mg, 200 mg) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, C), and NAA (100 mg, 200 mg) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, D) using RT-qPCR. The expression levels of the 17 \u003cem\u003eHbWOX\u003c/em\u003e genes exhibited significant variations across the four hormone treatments. Under IBA treatment, \u003cem\u003eHbWOX14\u003c/em\u003e demonstrated elevated transcript levels at 6 hours for both concentrations (100 mg and 200 mg) compared to those observed at 1 hour and 3 hours. In contrast, \u003cem\u003eHbWOX13\u003c/em\u003e showed high expression exclusively at the earlier time points of 1 hour and 3 hours under the lower concentration of IBA (100 mg). Following IAA treatment, \u003cem\u003eHbWOX14\u003c/em\u003e displayed a peak in transcript abundance at the half-hour mark for both concentrations (100 mg and 200 mg), which positively correlated with increasing IAA concentration. However, its expression declined over time and approached minimal levels by the three-hour mark. Under treatment with 6-BA, both \u003cem\u003eHbWOX6.1\u003c/em\u003e and \u003cem\u003eHbWOX6.2\u003c/em\u003e were expressed more highly at half an hour than at six hours. Notably, after six hours under a concentration of 100 mg for \u003cem\u003eHbWOX14\u003c/em\u003e was significantly lower than that observed under the higher concentration of 200 mg. Under NAA treatment, \u003cem\u003eHbWOX1.1\u003c/em\u003e exhibited higher expression levels at 0.5 h and 1 h with a concentration of 100 mg, but its expression was suppressed at 200 mg. In contrast, \u003cem\u003eHbWOX14\u003c/em\u003e maintained low expression across all NAA treatments, indicating lower sensitivity and potential specificity to this auxin. The transcriptional patterns suggest that these genes respond differentially to distinct auxins. Overall expression levels at 0.5-1 h were greater than those observed at 3 h and 6 h, implying that these genes are responsive within a short time frame and may primarily be involved in early hormone responses as well as cell differentiation processes. At elevated auxin concentrations, several genes were significantly up-regulated, indicating their involvement in hormone responses and plant cell differentiation. Notably, \u003cem\u003eHbWOX14\u003c/em\u003e demonstrated high expression under IAA treatment at both concentrations, considering role of IAA in root apex synthesis and transport as well as its influence on root development. Conversely, its low responsiveness to NAA suggests a certain specificity towards different types of auxins, thereby supporting the notion of signal recognition and transduction for \u003cem\u003eHbWOX14\u003c/em\u003e within the auxin signaling network of the rubber tree.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpatiotemporal expression analysis of the\u003c/strong\u003e \u003cstrong\u003eHbWOX\u003c/strong\u003e \u003cstrong\u003egene family\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing RT-qPCR, we profiled the spatiotemporal expression of 17 members of the \u003cem\u003eHbWOX\u003c/em\u003e gene family across various rubber tree tissues, including roots, stems, and leaves (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Several genes exhibited high expression levels in specific tissues, indicating a degree of tissue specificity. Notably, \u003cem\u003eHbWOX14\u003c/em\u003e demonstrated nearly the highest transcript level in roots among these genes. In stems, \u003cem\u003eHbWOX3.1, HbWOX4.2\u003c/em\u003e, \u003cem\u003eHbWOX8.2\u003c/em\u003e, and \u003cem\u003eHbWOX11\u003c/em\u003e were highly transcribed. Conversely, in leaves, significant transcription was observed for \u003cem\u003eHbWOX4.1\u003c/em\u003e and \u003cem\u003eHbWOX1.1\u003c/em\u003e. These transcriptional patterns suggest that these genes may play roles in organ growth and development. While some homologs also showed detectable expression in roots, their levels were considerably lower than that of \u003cem\u003eHbWOX14\u003c/em\u003e. The peak expression observed for \u003cem\u003eHbWOX14\u003c/em\u003e indicates its potential role as a key regulator of root growth and development; it appears to have retained a core function related to root morphogenesis throughout evolution. In contrast to this pattern, certain genes, particularly \u003cem\u003eHbWOX5\u003c/em\u003e and \u003cem\u003eHbWOX6.1\u003c/em\u003e, showed no detectable transcription across any tissue types examined. Furthermore, pronounced differences or even opposing expression patterns were noted among homologous genes within the same tissue type. This suggests possible functional divergence among them. For instance, while \u003cem\u003eHbWOX8.2\u003c/em\u003e was highly expressed in roots, both \u003cem\u003eHbWOX8.1\u003c/em\u003e and \u003cem\u003eHbWOX8.3\u003c/em\u003e exhibited minimal transcription levels. Similarly, marked differences between \u003cem\u003eHbWOX4 .1\u003c/em\u003e and \u003cem\u003eHbWO4 .2\u003c/em\u003e were evident in leaves, which may indicate evolutionary functional divergence. In summary, our findings reveal that \u003cem\u003eHbWOX14\u003c/em\u003e displays significant tissue specificity with transcript levels substantially higher than those of the other 16 gene family members in roots, implying that \u003cem\u003eHbWOX14\u003c/em\u003e may play a key role in regulating root growth and development in rubber tree.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of an\u003c/strong\u003e \u003cstrong\u003eAgrobacterium rhizogenes\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;mediated hairy-root transformation system in rubber tree\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo develop a rapid and efficient gene-function validation system for the rubber tree, we established an \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e\u0026ndash;mediated hairy-root transformation system (HRTS). \u003cem\u003eA. rhizogenes\u003c/em\u003e strains K599 and LBA9402 (Table S5), both carrying the GFP reporter (plasmid pR101), were used to inoculate the basal region of one-year-old seedlings through three methods: injection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, A), flat placement methods (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, B) and CDB (Cut\u0026ndash;Dip\u0026ndash;Budding) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, C). Among these approaches, CDB yielded the highest transformation efficiency (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, F), nine plants developed transgenic roots via this method, whereas only three plants exhibited transgenic roots with the injection method and none with flat placement (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, D). Notably, LBA9402 demonstrated a significant advantage over K599, out of 30 inoculated seedlings, 23 produced transgenic hairy roots using LBA9402 compared to just three with K599, indicating a higher infection efficiency associated with LBA9402(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, E). Hairy roots first appeared 15 days post-inoculation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, G), fibrous roots became visible by day 30(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, H), while fully developed hairy roots formed by day 45 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, I). This timeline illustrates that the LBA9402-CDB pipeline reliably produces transgenic hairy roots (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, J). We hypothesize that the superior efficiency observed with CDB is attributable to its moderate wounding effect which facilitates close contact between bacteria and wound sites while minimizing excessive damage and maintaining suitable local humidity conditions. The emergence of hairy roots in rubber tree seedlings occurred within 15 days following infection and reached full development by day 45. These findings demonstrate that our transformation system exhibits high efficiency and practicality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImproving the efficiency of\u003c/strong\u003e \u003cstrong\u003eAgrobacterium\u003c/strong\u003e\u003cstrong\u003e-mediated hairy root transformation in rubber trees by utilizing\u003c/strong\u003e \u003cstrong\u003eHbWOX14\u003c/strong\u003e \u003cstrong\u003ein rubber tree\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the experimental results presented, we concluded that \u003cem\u003eHbWOX14\u003c/em\u003e most significantly promotes root growth and development in rubber tree. Furthermore, the combination of \u003cem\u003eA. rhizogenes\u003c/em\u003e strain LBA9402 with the CDB method maximizes transformation efficiency. To further optimize this protocol, we infected rubber tree seedlings with LBA9402 carrying an overexpression construct for \u003cem\u003eHbWOX14\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, A-D) and verified the presence of hairy roots harboring both the \u003cem\u003eGFP\u003c/em\u003e vector and the \u003cem\u003eGFP\u0026ndash;HbWOX14\u003c/em\u003e vector through RT-qPCR analysis (Fig. S6, B). Continuous observation and quantification of the transformation process revealed that plants overexpressing \u003cem\u003eHbWOX14\u003c/em\u003e achieved a hairy root induction rate of 66.7%, while seedlings containing only the \u003cem\u003eGFP\u003c/em\u003e vector exhibited a mere 13.3% induction rate (Fig. S6, A). This indicates that overexpression of \u003cem\u003eHbWOX14\u003c/em\u003e significantly enhances both hairy root induction and growth. As illustrated in our findings, \u003cem\u003eHbWOX14\u003c/em\u003e overexpression not only induced hairy roots at an earlier stage but also resulted in a greater number and more robust roots. We quantified four phenotypic traits, length (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, E), diameter (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, F), root number (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, G), and newly formed bud count (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, H) and found that hairy roots expressing \u003cem\u003eHbWOX14\u003c/em\u003e averaged 59 mm in length, approximately 2.5 times longer than those from the \u003cem\u003eGFP\u003c/em\u003e control (24 mm). In terms of root quantity, lines expressing \u003cem\u003eHbWOX14\u003c/em\u003e produced an average of four transgenic hairy roots per plant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, C) compared to fewer than two from controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, B, D). Additionally, roots carrying \u003cem\u003eHbWOX14\u003c/em\u003e were thicker as evidenced by an average diameter of ~\u0026thinsp;1.2 mm versus ~\u0026thinsp;0.5 mm for controls. Thus, our findings suggest that overexpressing \u003cem\u003eHbWOX14\u003c/em\u003e markedly enhances both growth and development of rubber tree hairy roots likely by sustaining activity within apical meristems while promoting cell division and differentiation, hereby improving developmental capacity within root primordia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGO enrichment analysis of differentially expressed genes in\u003c/strong\u003e \u003cstrong\u003eHbWOX14\u003c/strong\u003e\u003cstrong\u003e-overexpressed lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing samples overexpressing \u003cem\u003ePRI101: GFP\u003c/em\u003e as the control group, we identified genes with significantly different expression level and conducted a detailed analysis in \u003cem\u003eHbWOX14\u003c/em\u003e-overexpressed lines (Fig. S6, A), resulting in the identification of 77 upregulated genes and 28 downregulated genes. Principal component analysis (PCA) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, A) and Gene Ontology (GO) functional enrichment analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, B) were subsequently performed. The results revealed that differentially expressed genes (DEGs) in hairy roots induced by overexpressing \u003cem\u003eHbWOX14\u003c/em\u003e, were significantly enriched in pathways related to development and hormone signaling (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, C). This suggests that \u003cem\u003eHbWOX14\u003c/em\u003e may serve as a key gene promoting hairy-root formation. In terms of hormone responses, DEGs exhibited marked enrichment for cytokinin response, ABA response, and associated signal transduction pathways. These findings indicate that \u003cem\u003eHbWOX14\u003c/em\u003e shapes a hormonal microenvironment conducive to the initiation and development of hairy roots by modulating the plant hormone signaling network. Notably, multiple genes involved in auxin polar transport and signaling were found to be upregulated, providing an essential hormonal regulatory basis for efficient hairy-root induction. Regarding development-related processes, DEGs were primarily enriched in cell development (GO:0048468), establishment of organ organization (GO:0048589), and cell\u0026ndash;cell junction organization (GO:0005911). Genes closely associated with cell division and differentiation, such as \u003cem\u003eGT014369\u003c/em\u003e, were significantly upregulated. These include those involved in cell-cycle regulation (GO:0005515), microtubule-based movement (GO:0016887), and cell-wall biogenesis (GO:0009506). Collectively, these changes create prerequisites for hairy-root morphogenesis. The enrichment of genes associated with cell junctions (GT014369) and plasmodesmata (GT031829) further suggests that \u003cem\u003eHbWOX14\u003c/em\u003e may coordinate hairy-root development by enhancing intercellular communication. Additionally, pathways related to glycosyltransferase activity (GO:0102132), particularly flavonoid glycosylation (GO:0102132), were significantly enriched. This finding is consistent with the roles of these pathways in hormone homeostasis and developmental signal regulation, as flavonoid glycosylation influences secondary metabolism and modulates the distribution and activity of hormones such as auxin. Principal Component Analysis (PCA) also indicated a prominent enrichment of differentially expressed genes (DEGs) in flavonoid glycosylation and related glycosyltransferase activities, implies that \u003cem\u003eHbWOX14\u003c/em\u003e affects hairy-root development by regulating modifications of secondary metabolites. Furthermore, upregulated genes exhibited significant enrichment in hormone-response pathways, particularly those responding to cytokinin (GO:0009691) and abscisic acid (GO:0051603), indicating that \u003cem\u003eHbWOX14\u003c/em\u003e promotes hairy-root growth through remodeling the hormonal regulatory network.\u003c/p\u003e\n\u003cp\u003eFurther, we validated 16 genes that exhibited significant changes following the introduction of \u003cem\u003eHbWOX14\u003c/em\u003e through RT-qPCR. The results indicated that 12 genes were significantly upregulated, while 4 showed slight downregulation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, A-P), largely consistent with the RNA-seq findings. Among these, \u003cem\u003eERF4\u003c/em\u003e (\u003cem\u003eGT005774\u003c/em\u003e), \u003cem\u003eWOX11\u003c/em\u003e (\u003cem\u003eGT031829\u003c/em\u003e), \u003cem\u003eWOX13\u003c/em\u003e (\u003cem\u003eGT005626\u003c/em\u003e), \u003cem\u003eRHA2B\u003c/em\u003e (\u003cem\u003eGT014369\u003c/em\u003e), and \u003cem\u003eMYB62\u003c/em\u003e (\u003cem\u003eGT005678\u003c/em\u003e) are involved in regulating the auxin signaling pathway and displayed markedly increased transcript levels in \u003cem\u003eHbWOX14\u003c/em\u003e-overexpressing hairy roots. Additionally, \u003cem\u003eERF4\u003c/em\u003e (\u003cem\u003eGT005774\u003c/em\u003e), \u003cem\u003eRHA2B\u003c/em\u003e (\u003cem\u003eGT014369\u003c/em\u003e), and \u003cem\u003eMYB108\u003c/em\u003e (\u003cem\u003eGT012677\u003c/em\u003e) primarily participate in the jasmonic acid signaling pathway by promoting the expression of \u003cem\u003eMYB75\u003c/em\u003e and \u003cem\u003eMYB111\u003c/em\u003e, thereby activating downstream anthocyanin biosynthetic pathways. Five upregulated genes, like \u003cem\u003eMYB73\u003c/em\u003e (\u003cem\u003eGT001820\u003c/em\u003e), \u003cem\u003eMYB74\u003c/em\u003e (\u003cem\u003eGT034650\u003c/em\u003e), \u003cem\u003eHB-21\u003c/em\u003e (\u003cem\u003eGT024700\u003c/em\u003e), \u003cem\u003eHB-40\u003c/em\u003e (\u003cem\u003eGT037945\u003c/em\u003e), and \u003cem\u003eCIPK7\u003c/em\u003e (\u003cem\u003eGT005901\u003c/em\u003e), are jointly regulated by auxin and abscisic acid, playing a crucial role in balancing and modulating plant growth and development. \u003cem\u003eANAC083\u003c/em\u003e (\u003cem\u003eGT033271\u003c/em\u003e) is regulated by auxin, abscisic acid, and jasmonic acid pathways, with abscisic acid and jasmonic acid serving as core stress signals. This gene is typically induced to suppress auxin signaling to coordinate growth arrest with stress defense mechanisms. However, in \u003cem\u003eHbWOX14\u003c/em\u003e-overexpressing hairy roots, we observed a notable decrease in \u003cem\u003eANAC083\u003c/em\u003e expression. This finding suggests that \u003cem\u003eHbWOX14\u003c/em\u003e can inhibit ABA- and JA-mediated stress-response signals while enhancing auxin signaling transduction to promote the growth and proliferation of rubber-tree hairy roots.\u003c/p\u003e\n\u003cp\u003eThese results indicate that \u003cem\u003eHbWOX14\u003c/em\u003e serves as a core regulatory gene that enhances the efficient initiation and subsequent growth of rubber-tree hairy roots by coordinating the plant hormone signaling network, particularly in responses to auxin, cytokinin, and abscisic acid (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). This coordination plays a crucial role in root cell division, differentiation, and organogenesis. Furthermore, in conjunction with the GO enrichment results mentioned above, \u003cem\u003eHbWOX14\u003c/em\u003e may also regulate secondary metabolic processes such as flavonoid glycosylation, thereby indirectly influencing hormone signal transduction, a critical factor for the growth and development of rubber-tree roots. Accordingly, we concentrated on genes involved in hormone signal-transduction pathways and developed a schematic representation of their potential roles in rubber-tree root development. These genes are implicated in auxin signaling, abscisic acid signaling, and jasmonic acid signaling. Notably, most cluster within the auxin pathway highlighted its central importance for rubber-tree root growth and development. Additionally, upregulated expression of \u003cem\u003eMYB\u003c/em\u003e-family genes is believed to facilitate the development of rubber-tree hairy roots. These genes may integrate key components from the auxin (\u003cem\u003eARF7/PIN1\u003c/em\u003e), jasmonic acid (\u003cem\u003eMYC2\u003c/em\u003e), and abscisic acid (\u003cem\u003eABF2\u003c/em\u003e) signaling pathways to synergistically promote both initiation and elongation of hairy roots in rubber trees. In summary, these findings suggest that auxin-, jasmonic-acid-, and abscisic-acid-mediated signaling pathways play pivotal regulatory roles in inducing proliferation among rubber-tree hairy roots. The \u003cem\u003eHbWOX14\u003c/em\u003e gene establishes a molecular network that effectively promotes high-efficiency hairy-root formation through coordinated regulation of hormone-response pathways alongside cellular developmental processes, thus occupying an essential regulatory position within the context of growth and development for rubber-tree hairy roots.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThrough genome-wide identification, we identified 17 \u003cem\u003eHbWOX\u003c/em\u003e members, all of which encode proteins containing the conserved homeodomain (HD). The \u0026ldquo;helix\u0026ndash;turn\u0026ndash;helix\u0026rdquo; three-dimensional conformation formed by this domain is highly consistent with that of WOX proteins in Arabidopsis thaliana, Zea mays, and other species, confirming the core supporting role of the HD for DNA binding during plant evolution. With the exception of HbWOX13, the remaining 16 HbWOX proteins possess the conserved motif \u0026ldquo;PEVSSRWNPTPEQLR.\u0026rdquo; This feature resembles the diversification of conserved motifs observed in cucumber CsFARs proteins and in the strawberry SWEET gene family, indicating that retention of core WOX motifs preserves basal functions while variations in these motifs may confer functional specificity. Gene structure analysis revealed that \u003cem\u003eHbWOX\u003c/em\u003e genes contain between 2 to 5 exons and 1 to 5 introns. Notably, \u003cem\u003eHbWOX14\u003c/em\u003e has three exons, the most common configuration within this family, and this streamlined yet conserved structure closely resembles that of \u003cem\u003eAtWOX14\u003c/em\u003e and \u003cem\u003eOsWOX11\u003c/em\u003e. Such structural similarities may facilitate rapid assembly of mRNA during transcription and enhance efficient expression of functional proteins. In comparison to the \u003cem\u003eArabidopsis WOX\u003c/em\u003e family, rubber tree lacks four members, namely \u003cem\u003eWOX7\u003c/em\u003e, \u003cem\u003eWOX9\u003c/em\u003e, \u003cem\u003eWOX10\u003c/em\u003e, and \u003cem\u003eWOX12\u003c/em\u003e, and exhibits copy number variations for certain genes. There are three copies each for \u003cem\u003eHbWOX6\u003c/em\u003e and \u003cem\u003eHbWOX8\u003c/em\u003e but only one copy for both \u003cem\u003eHbWOX2\u003c/em\u003e and \u003cem\u003eHbWOX14\u003c/em\u003e. This divergence in copy number and gene structure may reflect evolutionary optimization within developmental regulatory networks. Furthermore, tissue-specific expression analyses further elucidated functional differentiation among \u003cem\u003eHbWOX\u003c/em\u003e genes. Notably, \u003cem\u003eHbWOX14\u003c/em\u003e displays significantly higher transcript levels in roots compared to stems or leaves, far exceeding those observed for other family members, which is similar to \u003cem\u003eAtWOX14\u003c/em\u003e in A. thaliana and \u003cem\u003eOsWOX11\u003c/em\u003e in O. sativa. In Arabidopsis thaliana, \u003cem\u003eAtWOX14\u003c/em\u003e primarily plays a role in lateral root development and post-wounding cellular reprogramming [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In contrast, \u003cem\u003eZmWOX14\u003c/em\u003e in Zea mays is predominantly expressed in the root apical meristem and regulates cell division within the root cap [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The notably high expression of \u003cem\u003eHbWOX14\u003c/em\u003e in rubber tree roots, alongside its sustained expression throughout both root primordium formation and adventitious root elongation, suggests that it has a core regulatory function in the construction of the rubber tree's root system [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, expression divergence among certain homologs in the rubber tree, such as the elevated levels of \u003cem\u003eHbWOX8.2\u003c/em\u003e expression in roots compared to nearly undetectable levels of \u003cem\u003eHbWOX8.1\u003c/em\u003e and \u003cem\u003eHbWOX8.3\u003c/em\u003e, which reflects a pattern of functional diversification observed among \u003cem\u003eWOX\u003c/em\u003e family members in A. thaliana. This indicates that the \u003cem\u003eWOX\u003c/em\u003e gene family in rubber trees has achieved developmental specialization through evolutionary changes in their expression patterns.\u003c/p\u003e\u003cp\u003eGenetic improvement and molecular breeding of rubber-tree are pivotal for the sustainable development of the global natural rubber industry. However, challenges, such as, low transformation efficiency, prolonged and unstable hairy-root induction, and inadequate stability have historically hindered gene-function validation, secondary metabolism regulation, and stress-resilience breeding. In this study, we firstly demonstrate that overexpression of \u003cem\u003eHbWOX14\u003c/em\u003e significantly enhances the efficiency in Agrobacterium-mediated hairy-root transformation. Within the hairy-root transformation system established herein, \u003cem\u003eHbWOX14\u003c/em\u003e overexpression increased the induction rate from 13.3% to 66.7%, accompanied by notable improvements in root length, number, and thickness compared to controls. This enhancement is similar to the increased callus-induction efficiency observed upon \u003cem\u003eZmWOX14\u003c/em\u003e overexpression in maize and supports the genotype-barrier-breaking function of \u003cem\u003eWOX5\u003c/em\u003e in Arabidopsis thaliana. These findings confirm a distinct role for \u003cem\u003eWOX\u003c/em\u003e genes in enhancing plant genetic-transformation efficiency. The improved induction rates associated with \u003cem\u003eHbWOX14\u003c/em\u003e overexpression suggest that \u003cem\u003eHbWOX14\u003c/em\u003e functions not only as a regulatory switch for hairy-root transformation efficiency in rubber trees but also serves as an optimizer of both hairy-root developmental quality and genetic stability. From an evolutionary and functional perspective, \u003cem\u003eWOX\u003c/em\u003e genes are plant-specific transcription factors that play crucial roles in regulating cell division and differentiation, organ primordium formation, and stress responses [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. \u003cem\u003eAtWOX14\u003c/em\u003e has been shown to regulate vascular development through interactions with cell-wall biosynthesis genes, this function is also observed in pumpkin [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Additionally, \u003cem\u003ePtWOX14\u003c/em\u003e is involved in the cell proliferation associated with wood formation in poplar [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Consistently, the promotion of hairy-root transformation by \u003cem\u003eHbWOX14\u003c/em\u003e demonstrated here underscores the conserved role of the \u003cem\u003eWOX\u003c/em\u003e family in controlling plant organogenesis. Furthermore, the hairy-root system based on \u003cem\u003eHbWOX14\u003c/em\u003e overexpression facilitates rapid target-gene transformation within approximately one month, enhancing validation efficiency by more than an order of magnitude.\u003c/p\u003e\u003cp\u003eCompared to fairy roots overexpressing \u003cem\u003ePRI101-GFP\u003c/em\u003e, hairy roots overexpressing \u003cem\u003eHbWOX14\u003c/em\u003e-OE exhibited differentially expressed genes, which significantly enriched in pathways related to hormone response, cell development, and flavonoid glycosylation, which is similar to Arabidopsis \u003cem\u003eWOX\u003c/em\u003e genes in promoting organ regeneration through multiple pathways. In the hormone-response pathways, genes associated with auxin polar transport and signaling, such as \u003cem\u003eERF4\u003c/em\u003e, \u003cem\u003eWOX11\u003c/em\u003e, and \u003cem\u003eRHA2B\u003c/em\u003e (GT014369), were notably upregulated. This pattern is conserved in Oryza sativa where \u003cem\u003eOsWOX11\u003c/em\u003e enhances crown-root development by activating auxin signaling [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Conversely, in rubber trees, cytokinin-response genes were simultaneously upregulated, forming an auxin\u0026ndash;cytokinin co-regulatory network, similarity to Brassica napus \u003cem\u003eBnWOX5\u003c/em\u003e that promotes somatic embryogenesis by modulating hormone ratios. The pathway-enrichment characteristics of the DEGs underscore the central regulatory role of \u003cem\u003eHbWOX14\u003c/em\u003e. Notably, the prominent enrichment of flavonoid glycosylation appears unique to rubber trees, while flavonoid glycosylation is known to participate in hormone homeostasis, as reported in Arabidopsis, its stronger enrichment observed here may be attributed to a heightened demand for phenolic-metabolism buffering within rubber trees [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Glycosylation can mitigate phenolic toxicity and reduce explant browning that inhibits hairy-root initiation while also modulating auxin distribution and activity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Thereby, this further supports the notion that \u003cem\u003eHbWOX14\u003c/em\u003e integrates secondary metabolism with hormone signaling to provide dual safeguards for hairy-root development. In cell-development pathways, genes associated with cell-cycle regulation, microtubule movement, and cell-wall biogenesis (e.g., GT014369) were significantly upregulated, as \u003cem\u003eAtWOX14\u003c/em\u003e promotes vascular differentiation through the regulation of cell-division genes. In contrast, in rubber tree, the enrichment of genes related to cell junctions and plasmodesmata suggests a coordination of overall hairy-root development via enhanced intercellular communication, an adaptive regulation likely necessary for maintaining structural stability during woody-root growth. Moreover, in roots overexpressing \u003cem\u003eHbWOX14\u003c/em\u003e, key negative regulators within ABA and JA signaling pathways (e.g., \u003cem\u003eANAC083\u003c/em\u003e; \u003cem\u003eGT033271\u003c/em\u003e) were downregulated. These stress hormones are known to suppress growth under adverse conditions [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], similar to the inhibitory effect observed with gibberellin on Asiatic lily [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Thus, \u003cem\u003eHbWOX14\u003c/em\u003e appears to repress ABA/JA stress signaling while enhancing auxin signal transmission [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Additionally, lower ABA levels under heavy-metal stress have been shown to enhance plant growth in Arabidopsis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Accordingly, \u003cem\u003eHbWOX14\u003c/em\u003e can coordinate these pathways by positively regulating auxin signaling [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] while simultaneously repressing stress-related hormone pathways [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], thereby achieving regulatory control over rubber-tree root development [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study systematically investigated the function and evolution of \u003cem\u003eWOX\u003c/em\u003e gene family in rubber trees, focusing on resolving low genetic transformation efficiency and unstable hairy root induction, providing a robust support for molecular breeding in rubber tree. We identified 17 \u003cem\u003eHbWOX\u003c/em\u003e gene family members, which all harbor a conserved homeodomain (HD) with a \"helix\u0026ndash;turn\u0026ndash;helix\" conformation consistent with WOX proteins in \u003cem\u003eArabidopsis\u003c/em\u003e and maize. Most of \u003cem\u003eHbWOX\u003c/em\u003e genes retain the motif \"PEVSSRWNPTPEQLR\", while \u003cem\u003eHbWOX13\u003c/em\u003e is absent, implying functional differentiation of \u003cem\u003eHbWOX\u003c/em\u003e. \u003cem\u003eHbWOX14\u003c/em\u003e is a root-specific regulator, and overexpressing \u003cem\u003eHbWOX14\u003c/em\u003e raised \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated hairy root induction rate from 13.3% to 66.7%, and improved root length, number, and thickness, breaking genotype limitations. Further transcriptome analysis indicated that \u003cem\u003eHbWOX14\u003c/em\u003e acts via a coordinated network, activating auxin/cytokinin pathways, repressing ABA/JA stress signaling, upregulating cell cycle/wall genes, and enriching flavonoid glycosylation. This study clarifies the characteristics of \u003cem\u003eHbWOX\u003c/em\u003e family and the role of \u003cem\u003eHbWOX14\u003c/em\u003e in improving transgenic efficiency in rubber tree study, enriching plant \u003cem\u003eWOX\u003c/em\u003e research and providing technical support for rubber tree breeding, facilitating the natural rubber industry\u0026rsquo;s sustainability.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehomeodomain\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRR2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eResponse Regulator 2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePrincipal component analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHRTS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehairy-root transformation system\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCDB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCut\u0026ndash;Dip\u0026ndash;Budding\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDifferentially expressed genes.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cb\u003eConsent for publication\u003c/b\u003e\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB1080000), the Project for Major Science and Technology Planning in Yunnan (Grant No. 202402AE09001904), Special Program for Technological Innovation of Xishuangbanna Science and Technology Bureau (2025kjcx003), Yunnan Revitalization Talent Support Program Yunling Scholar Project to Yongping Yang.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYPY, YQY and YZ conceived and designed the experiments. CLZ, KYC and MXH participated in the sample collecting and interpretation of data. CLZ, KYC and MXH performed the experiments and analysed the sequencing data. CLZ, XY and JWY wrote the manuscript. YZ, YQY and YYQ contributed with valuable discussions and revised the paper. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences for providing the plant materials.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw data of transcriptome in this study have been deposited in database Sequence Read Archive (SRA) of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/)with accession with accession NO. PRJNA1365429 (https://www.ncbi.nlm.nih.gov/sra/?term=+PRJNA1365429)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShi Lei. 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Methods Mol Biol. 2006;344:153\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1385/1-59745-131-2:153\u003c/span\u003e\u003cspan address=\"10.1385/1-59745-131-2:153\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hevea brasiliensis, HbWOX14, Hairy root transformation, Transformation efficiency","lastPublishedDoi":"10.21203/rs.3.rs-8197958/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8197958/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eWOX\u003c/em\u003e gene family, a plant-specific group of essential transcription factors, plays important regulatory roles in stem cell maintenance, organogenesis, and hormone response cascades. For rubber trees, genotype-dependent limitations in tissue culture and low genetic transformation efficiency have long posed bottlenecks to their molecular breeding progress, where optimizing root regeneration systems is a critical breakthrough point.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eTo address these challenges, we identified 17 \u003cem\u003eHbWOX\u003c/em\u003e family members from the GT1 genome of rubber trees using bioinformatic approaches, and systematically analyzed their gene structures, conserved motifs, cis-acting elements, and tissue-specific expression profiles. Our findings revealed obvious copy-number diversification within the \u003cem\u003eHbWOX\u003c/em\u003e family. Most members harbor a conserved homeodomain (HD) and share the specific motif \"PEVSSRWNPTPEQLR\", reflecting evolutionary conservation and functional specialization of this gene family in rubber trees. Notably, \u003cem\u003eHbWOX14\u003c/em\u003e exhibited root-specific and high-level expression, with its promoter region significantly enriched for auxin-responsive cis-elements. Functional verification showed that \u003cem\u003eHbWOX14\u003c/em\u003e responds rapidly to auxins such as IAA, and its expression pattern is tightly associated with root development processes. Using an \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e-mediated transformation system, overexpression of \u003cem\u003eHbWOX14\u003c/em\u003e increased the induction rate of rubber tree hairy roots from 13.3% to 66.7%, while simultaneously significantly improving root length, number, and thickness. Transcriptome and Gene Ontology (GO) enrichment analyses further demonstrated that \u003cem\u003eHbWOX14\u003c/em\u003e exerts its regulatory function by activating the auxin signaling pathway and modulating the expression of genes involved in cell division and differentiation. Additionally, it suppresses abscisic acid and jasmonic acid-mediated stress response pathways while coordinating flavonoid glycosylation-related secondary metabolic processes, collectively creating a favorable molecular environment for hairy root development.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study is the first to systematically clarify the evolutionary characteristics and functional divergence of the \u003cem\u003eHbWOX\u003c/em\u003e gene family in rubber trees, with a specific focus on verifying the key role of \u003cem\u003eHbWOX14\u003c/em\u003e in enhancing hairy root regeneration efficiency. These findings not only enrich our understanding of the plant \u003cem\u003eWOX\u003c/em\u003e gene family's functional diversity but also provide crucial theoretical basis and practical technical support for accelerating rubber tree molecular breeding and improving its genetic transformation system.\u003c/p\u003e","manuscriptTitle":"Genome-wide analysis of the HbWOX gene family and HbWOX14-mediated enhancement of hairy root transformation in Hevea brasiliensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-03 09:38:25","doi":"10.21203/rs.3.rs-8197958/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-06T07:59:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T09:54:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123074471466961205351887860516528006485","date":"2026-02-23T09:06:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167231126217232372995871745758341982391","date":"2026-02-23T05:18:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131177715786546382882216614588431588406","date":"2026-02-22T13:17:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T11:47:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-11T14:14:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231410515407150144935830506080394490651","date":"2025-12-05T06:26:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61907989332182126954763725436575404569","date":"2025-12-02T02:48:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T02:42:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-27T18:32:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-26T00:42:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-26T00:42:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-11-25T02:16:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ab4e2b3-6856-43a2-8753-1b4c1b26537c","owner":[],"postedDate":"December 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-06T08:11:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-03 09:38:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8197958","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8197958","identity":"rs-8197958","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}