Homeobox Research Articles

Mutations in the WUSCHEL-related homeobox1 gene cause an increased leaflet number in soybean

WUSCHEL-related homeobox (WOX) transcription factors play a crucial role in lateral organ development in several plant species; however, their precise functions in soybean (Glycine max [L.] Merr.) were unclear. Here, we identified two independent multi-leaflet soybean mutants, mlw48-8 and mlw48-161, from a CRISPR/Cas9-engineered mutant library in the Williams 82 background. Both mutants exhibited irregular leaf margins, and the upper leaves were narrow and almost lanceolate at maturity. Molecular analysis revealed that these are allelic mutants with independent mutations in the WUSCHEL-related homeobox1 (GmWOX1A) gene. A transcriptome analysis demonstrated that GmWOX1A modulates the expression of auxin- and leaf development–related genes. Yeast two-hybrid and split-luciferase complementation imaging assays revealed that GmWOX1A interacts with the YABBY family protein GmYAB5, providing further evidence of its potential involvement in leaf development. Notably, the mlw48-161 mutant showed an increased seed number per plant. Consequently, our study not only provides valuable insights into the role of GmWOX1A in soybean leaf development but also offers a potential strategy for high-yield breeding.

The Hox-based positional memory in muscle stem cells.

The skeletal muscle is a contractile tissue distributed throughout the body with various anatomical sizes, shapes and functions. In pathological conditions, such as muscular dystrophy, age-related sarcopenia and cancer cachexia, skeletal muscles are not uniformly affected throughout the body. This region-specific vulnerability cannot be fully explained by known physiological classifications, including muscle fiber types. Accumulating evidence indicates that the expression patterns of topographic homeobox (Hox) genes provide a molecular signature of positional memory, reflecting the anatomical locations and embryonic history of muscles and their associated muscle stem cells in adult mice and humans. Hox-based positional memory is not merely a remnant of embryonic development but is expected to be an intrinsic determinant controlling muscle function because recent studies have shown that aberrant Hox genes affect muscle stem cells. In this review, we discuss the concept of Hox-based positional memory, which may offer a new perspective on the region-specific pathophysiology of muscle disorders.

Members of an array of zinc-finger proteins specify distinct Hox chromatin boundaries

Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.

Mutant NPM1 marginally impacts ribosome footprint in acute myeloid leukemia cells.

NPM1-mutated acute myeloid leukemia (AML) is the most frequent AML subtype. As wild-type NPM1 is known to orchestrate ribosome biogenesis, it has been hypothesized that altered translation may contribute to leukemogenesis and leukemia maintenance in NPM1-mutated AML. However, this hypothesis has never been investigated. We reasoned that if mutant NPM1 (NPM1c) directly impacts translation in leukemic cells, loss of NPM1c would result in acute changes in the ribosome footprint. Here, we performed ribosome footprint profiling (Ribo-seq) and bulk messenger RNA (mRNA) sequencing in two NPM1-mutated cell lines engineered to express endogenous NPM1c fused to the FKBP (F36V) degron tag (degron cells). Incubation of degron cells with the small compound dTAG-13 enables highly specific degradation of NPM1c within 4hours. As expected, RNA-sequencing data showed early loss of homeobox gene expression following NPM1c degradation, confirming the reliability of our model. In contrast, Ribo-seq data showed negligible changes in the ribosome footprint in both cell lines, implying that the presence of NPM1c does not influence ribosome abundance and positioning on mRNA. While it is predictable that NPM1c exerts its leukemogenic activity at multiple levels, ribosome footprint does not seem influenced by the presence of mutant NPM1.

Myocyte enhancer factor 2 upregulates expression of myostatin promoter in Yesso scallop, Patinopecten yessoensis

Myostatin (MSTN) plays an important role in muscle development in animals, especially for mammals and fishes. However, little information has been reported on the regulation of MSTN in marine invertebrates, such as bivalves. In the present study, we cloned the MSTN promoter sequence of Yesso scallop Patinopecten yessoensis, identifying 4 transcription start sites, eleven TATA boxes and one E-box. Additionally, transcription factor binding sites, including myocyte enhancer factor 2 (MEF2) and POU homeodomain protein, were identified. The interaction between the MSTN promoter and MEF2 was analyzed to reveal the transcriptional activity of different fragment sizes of promoters through the dual-luciferase reporter assays. The highest transcriptional activity was found in recombinant plasmids with the most MEF2 binding sites, indicating that this transcription factor upregulates MSTN in Yesso scallop. This study provides new insight into the regulation of muscle growth and development in this species.

Generation of Insulin-Producing Cells from Canine Bone Marrow-Derived Mesenchymal Stem Cells: A Preliminary Study.

Cell-based therapy using insulin-producing cells (IPCs) is anticipated as an alternative treatment option to insulin injection or pancreatic islet transplantation for the treatment of diabetes mellitus in both human and veterinary medicine. Several protocols were reported for the differentiation of mesenchymal stem cells (MSCs) into IPCs; to date, glucose-responsive IPCs have only been obtained from canine adipose tissue-derived MSCs (cAD-MSCs), but not from canine bone marrow-derived MSCs (cBM-MSCs). Therefore, this study aims to generate in vitro glucose-responsive IPCs from cBM-MSCs using two differentiation protocols: a two-step protocol using trichostatin (TSA) and a three-step protocol using mercaptoethanol to induce pancreatic and duodenal homeobox gene 1 (PDX-1) expression. A single experiment was carried out for each protocol. BM-MSCs from one dog were successfully cultured and expanded. Cells exposed to the two-step protocol appeared rarely grouped to form small clusters; gene expression analysis showed a slight increase in PDX-1 and insulin expression, but no insulin protein production nor secretion in the culture medium was detected either under basal conditions or following glucose stimulation. Conversely, cells exposed to the three-step protocol under a 3D culture system formed colony-like structures; insulin gene expression was upregulated compared to undifferentiated control and IPCs colonies secreted insulin in the culture medium, although insulin secretion was not enhanced by high-glucose culture conditions. The single experiment results suggest that the three-step differentiation protocol could generate IPCs from cBM-MSCs; however, further experiments are needed to confirm these data. The ability of IPCs from cBM- MSCs to produce insulin, described here for the first time, is a preliminary interesting result. Nevertheless, the IPCs' unresponsiveness to glucose, if confirmed, would affect its clinical application. Further studies are necessary to establish a differentiation protocol in this perspective.

Genome sequencing and assembly of Indian major carp, Cirrhinus mrigala (Hamilton, 1822)

The freshwater aquaculture species, Cirrhinus mrigala, commonly known as mrigal, holds significance in the carp-dominated aquaculture system, globally. Despite constituting 1.08% of the total freshwater aquaculture production, mrigal is the third most important Indian major carp. However, its genome and associated information is not available. This study aims to address this gap by generation high quality genome assembly using PacBio long reads, Illumina short reads and Hi-C scaffolding. The characterization of assembled highly contiguous genome, 1.057 Gb in size, revealed 39,091 genes with functional annotations. The orthology analysis based on direct orthologs and single copy ortholgs places C. mrigala in a distinct position within the Otophysi clade. Additionally, the study delves into Hox gene clusters, identifying 38 Hox genes distributed in seven clusters. The present genomic information offers potential applications for sustainable aquaculture management, including selection programs for economic traits.

Functional characterization of WsPR-1 reveals its interplay with cytokinin and gibberellin signaling pathways

Pathogenesis-related protein 1 (PR-1) is an antimicrobial protein involved in systemic acquired resistance (SAR) in plants, but its regulatory role and interactions with other pathways remain unclear. In this study, we functionally characterize WsPR-1 gene of Withania somnifera in Nicotiana tabacum to elucidate its role in plant defense, growth, and development. Interestingly, transgenic tobacco plants with increased levels of cytokinin (CK) and decreased gibberellins (GAs) exhibited stunted shoot growth, an underdeveloped root system, modified leaf morphology, reduced seed pod production, and delayed leaf senescence. Transcriptional analysis revealed that WsPR-1 overexpression downregulated the GA 20-oxidase (GA20ox) gene involved in GA biosynthesis while upregulating GA 2-oxidase (GA2ox), a GA catabolic enzyme. Moreover, transcript levels of FRUITFULL (FUL) and LEAFY (NFL2) flowering genes exhibited a decrease in WsPR-1 plants, which could explain the delayed flowering and reduced seed pod development in transgenic plants. Confocal microscopy confirmed increased lignin deposition in stem cross-sections of WsPR-1 transgenic plants, supported by gene expression analysis and lignin content quantification. Additionally, our findings also suggest the involvement of Knotted1-like homeobox (KNOX) gene in enhancing cytokinin levels. This study highlights PR-1's regulatory role in plant growth and development, with potential to boost crop yields and enhance resilience.

Catalytic activity of Setd2 is essential for embryonic development in mice: establishment of a mouse model harboring patient-derived Setd2 mutation.

SETD2 is the only enzyme responsible for transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3). Mutations in SETD2 cause human diseases including cancer and developmental defects. In mice, Setd2 is essential for embryonic vascular remodeling. Given that many epigenetic modifiers have recently been found to possess noncatalytic functions, it is unknown whether the major function(s) of Setd2 is dependent on its catalytic activity or not. Here, we established a site-specific knockin mouse model harboring a cancer patient-derived catalytically dead Setd2 (Setd2-CD). We found that the essentiality of Setd2 in mouse development is dependent on its methyltransferase activity, as the Setd2CD/CD and Setd2-/- mice showed similar embryonic lethal phenotypes and largely comparable gene expression patterns. However, compared with Setd2-/-, the Setd2CD/CD mice showed less severe defects in allantois development, and single-cell RNA-seq analysis revealed differentially regulated allantois-specific 5' Hoxa cluster genes in these two models. Collectively, this study clarifies the importance of Setd2 catalytic activity in mouse development and provides a new model for comparative study of previously unrecognized Setd2 functions.

Prognostic Significance of Caudal-type Homeobox Gene 2 and Sex-determining Region Y-Box 2 Immunohistochemistry Markers in Gastric Cancer – A Clinicopathological Study

Abstract Background: Gastric cancer is one of the most commonly diagnosed cancers and the third leading cause of cancer-related deaths in the world as it has a poor prognosis due to its delayed presentation. Adenocarcinoma (intestinal or diffuse type) is the most common type histologically, and tumor, node, and metastasis staging is essential for prognosis and treatment planning. Gastric cancer patients may have different clinical presentation even within the same cancer stage group, hence it is important to evaluate additional markers in such patients which can help in prognosticating them to aid in effective management of such patients. This study evaluates two new immunohistochemistry (IHC) markers and their clinical correlation which will help in prognosticating gastric cancer patients. Materials and Methods: Biopsy tissues from 30 patients between April 2016 and April 2019, who underwent upper gastrointestinal endoscopy in a tertiary care center of Armed Forces and were found to have gastric cancer, were studied. Caudal-type homeobox gene 2 (CDX2) and sex-determining region Y-box 2 (SOX2) immunoreactivity was correlated with histomorphology, clinical presentation, and staging of tumor. Results: CDX2 positivity (P = 0.002) as well as SOX2 negativity (P = 0.058) was associated with an advanced stage of disease in our study. CDX2+/SOX2− profile was also noted to be statistically correlating (P = 0.014) with poor prognosis in gastric cancer. Conclusion: Loss of SOX2 together with positive CDX2 expression had a good correlation with advanced disease and poor outcome. Further studies should be undertaken for targeted therapy for these IHC markers.

Members of an array of zinc finger proteins specify distinct Hox chromatin boundaries.

Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CTCF, is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and MAZ, and identified a family of zinc finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.

Changes in Serum HOXA9 , PCⅢ, SE-CAD Levels in AML Patients after Chemotherapy with DCAG Regimen and Their Relationship with Prognosis

To investigate the changes in serum homeobox A9 (HOXA9 ), soluble E-cadherin (SE-CAD) and type Ⅲ procollagen (PCⅢ) levels in acute myeloid leukemia (AML) patients after chemotherapy with DCAG regimen and their relationship with prognosis. The clinical data of 80 patients with relapsed/refractory AML diagnosed and treated in our hospital from March 2018 to December 2021 were retrospectively analyzed. According to different treatment regimen, the patients were divided into DCAG group (n=40) and CAG group (n=40). The clinical efficacy and changes of HOXA9 , SE-CAD and PCⅢ levels before and after treatment were compared. In addition, all patients were divided into remission group (n=58) and non-remission group (n=22) according to the clinical efficacy. Univariate and multivariate analyses were performed to analyze the risk factors affecting the prognosis of AML patients. The predictive efficacy of the three single indicators, HOXA9 , SE-CAD, and PC III, and their combination on prognosis was analyzed. Compared with before treatment, the levels of HOXA9 , SE-CAD and PCⅢ in both the DCAG and CAG groups were decreased after treatment, and the improvement of each indicator and the clinical efficacy in the DCAG group were significantly better than those in the CAG group (all P < 0.05). Multivariate analysis showed that increased bone marrow blast count, HOXA9 mRNA, SE-CAD and PCⅢ levels were independent risk factors affecting the efficacy of chemotherapy in AML patients (all P < 0.05). ROC curves showed that the combination of HOXA9 mRNA, SE-CAD and PCIII could effectively predict the prognosis of AML patients, with a sensitivity of 84.80% and a specificity of 88.20%. DCAG regimen can significantly improve the levels of HOXA9 mRNA, SE-CAD and PCⅢ in AML patients, these three indicators are all independent risk factors affecting the prognosis of AML patients, and the combination of the three indicators can effectively predict the prognosis of the patients.

PHD17 acts as a target of miR1320 to negatively control cold tolerance via JA-activated signaling in rice

Plant Homeo Domain (PHD) proteins are involved in diverse biological processes during plant growth. However, the regulation of PHD genes on rice cold stress response remains largely unknown. Here, we reported that PHD17 negatively regulated cold tolerance in rice seedlings as a cleavage target of miR1320. PHD17 expression was greatly induced by cold stress, and was down-regulated by miR1320 overexpression and up-regulated by miR1320 knockdown. Through 5′RACE and dual luciferase assays, we found that miR1320 targeted and cleaved the 3′UTR region of PHD17. PHD17 was a nuclear-localized protein and acted as a transcriptional activator in yeast. PHD17 overexpression reduced cold tolerance of rice seedlings, while knockout of PHD17 increased cold tolerance, partially via the CBF cold signaling. By combining transcriptomic and physiological analyses, we demonstrated that PHD17 modulated ROS homeostasis and flavonoid accumulation under cold stress. K-means clustering analysis revealed that differentially expressed genes in PHD17 transgenic lines were significantly enriched in the jasmonic acid (JA) biosynthesis pathway, and expression of JA biosynthesis and signaling genes was verified to be affected by PHD17. Cold stress tests applied with MeJA or IBU (JA synthesis inhibitor) further suggested the involvement of PHD17 in JA-mediated cold signaling. Taken together, our results suggest that PHD17 acts downstream of miR1320 and negatively regulates cold tolerance of rice seedlings through JA-mediated signaling pathway.

Extreme genetic signatures of local adaptation in a notorious rice pest, Chilo suppressalis

ABSTRACT Climatic variation stands as a significant driving force behind genetic differentiation and the evolution of adaptive traits. Chilo (C.) suppressalis, commonly known as the rice stem borer, is a highly destructive pest that crucially harms rice production. The lack of natural population genomics data has hindered a more thorough understanding of its climate adaptation, particularly the genetic basis underlying adaptive traits. To overcome this obstacle, our study employed completely resequenced genomes of 384 individuals to explore the population structure, demographic history, and gene flow of C. suppressalis in China. This study observed that its gene flow occurred asymmetrically, moving from central populations to peripheral populations. Using genome-wide selection scans and genotype-environment association studies, we identified potential loci that may be associated with climatic adaptation. The most robust signal was found to be associated with cold tolerance, linked to a homeobox gene, goosecoid (GSC), whose expression level was significantly different in low and high latitudes. Moreover, downregulating the expression of this gene by RNAi enhances its cold tolerance phenotypes. Our findings have uncovered and delved into the genetic foundation of the ability of C. suppressalis to adapt to its environment. This is essential in ensuring the continued effectiveness and sustainability of novel control techniques.

A taxon-restricted duplicate of Iroquois3 is required for patterning the spider waist.

The chelicerate body plan is distinguished from other arthropod groups by its division of segments into 2 tagmata: the anterior prosoma ("cephalothorax") and the posterior opisthosoma ("abdomen"). Little is understood about the genetic mechanisms that establish the prosomal-opisthosomal (PO) boundary. To discover these mechanisms, we created high-quality genomic resources for the large-bodied spider Aphonopelma hentzi. We sequenced specific territories along the antero-posterior axis of developing embryos and applied differential gene expression analyses to identify putative regulators of regional identity. After bioinformatic screening for candidate genes that were consistently highly expressed in only 1 tagma (either the prosoma or the opisthosoma), we validated the function of highly ranked candidates in the tractable spider model Parasteatoda tepidariorum. Here, we show that an arthropod homolog of the Iroquois complex of homeobox genes is required for proper formation of the boundary between arachnid tagmata. The function of this homolog had not been previously characterized, because it was lost in the common ancestor of Pancrustacea, precluding its investigation in well-studied insect model organisms. Knockdown of the spider copy of this gene, which we designate as waist-less, in P. tepidariorum resulted in embryos with defects in the PO boundary, incurring discontinuous spider germ bands. We show that waist-less is required for proper specification of the segments that span the prosoma-opisthosoma boundary, which in adult spiders corresponds to the narrowed pedicel. Our results demonstrate the requirement of an ancient, taxon-restricted paralog for the establishment of the tagmatic boundary that defines Chelicerata.

Age-associated growth control modifies leaf proximodistal symmetry and enabled leaf shape diversification

Biological shape diversity is often manifested in modulation of organ symmetry and modification of the patterned elaboration of repeated shape elements.1,2,3,4,5 Whether and how these two aspects of shape determination are coordinately regulated is unclear.5,6,7 Plant leaves provide an attractive system to investigate this problem, because they often show asymmetries along the proximodistal (PD) axis of their blades, along which they can also produce repeated marginal outgrowths such as serrations or leaflets.1 One aspect of leaf shape diversity is heteroblasty, where the leaf form in a single genotype is modified with progressive plant age.8,9,10,11 In Arabidopsis thaliana, a plant with simple leaves, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) controls heteroblasty by activating CyclinD3 expression, thereby sustaining proliferative growth and retarding differentiation in adult leaves.12,13 However, the precise significance of SPL9 action for leaf symmetry and marginal patterning is unknown. By combining genetics, quantitative shape analyses, and time-lapse imaging, we show that PD symmetry of the leaf blade in A.thaliana decreases in response to an age-dependent SPL9 expression gradient, and that SPL9 action coordinately regulates the distribution and shape of marginal serrations and overall leaf form. Using comparative analyses, we demonstrate that heteroblastic growth reprogramming in Cardamine hirsuta, a complex-leafed relative of A.thaliana, also involves prolonging the duration of cell proliferation and delaying differentiation. We further provide evidence that SPL9 enables species-specific action of homeobox genes that promote leaf complexity. In conclusion, we identified an age-dependent layer of organ PD growth regulation that modulates leaf symmetry and has enabled leaf shape diversification.

Goosecoid promotes pancreatic adenocarcinoma metastasis through TGF-β signaling.

Goosecoid (GSC), translated from a homeobox gene, is a protein that participates in metastasis of various cancers. Pancreatic adenocarcinoma (PAAD) is one of the deadliest malignancies associated with a poor diagnosis and prognosis. To develop new treatment target or biomarker for PAAD, this study intended to assess the effects and the molecular mechanism of GSC on PAAD metastasis. The expressive discrepancy of GSC in PAAD and normal tissues/cells was compared by both the quantitative PCR and western blot. The effects of GSC silencing and GSC over-expression on PAAD cells and TGF-β signaling were proved by wound-healing assay, cell counting kit-8, Transwell assay and western blot. From the results, GSC mRNA and protein levels were enriched in PAAD cancer tissues and cells. GSC silencing prohibited metastasis of PAAD cells including the ability to invade, migrate and epithelial-mesenchymal transition (EMT), whereas GSC upregulation stimulated these cells behaviors above. GSC silencing reversed the effects on cellular processes induced by activation of the TGF-β pathway. Furthermore, silencing of GSC postponed tumor growth in xenograft model. In summary, GSC was abundantly expressed in PAAD, which activated the TGF-β pathway to enhance cell metastasis and tumor development.

Evolutionary patterns and functional effects of 3D chromatin structures in butterflies with extensive genome rearrangements

Chromosome rearrangements may distort 3D chromatin architectures and thus change gene regulation, yet how 3D chromatin structures evolve in insects is largely unknown. Here, we obtain chromosome-level genomes for four butterfly species, Graphium cloanthus, Graphium sarpedon, Graphium eurypylus with 2n = 30, 40, and 60, respectively, and Papilio bianor with 2n = 60. Together with large-scale Hi-C data, we find that inter-chromosome rearrangements very rarely disrupted the pre-existing 3D chromatin structure of ancestral chromosomes. However, some intra-chromosome rearrangements changed 3D chromatin structures compared to the ancestral configuration. We find that new TADs and subTADs have emerged across the rearrangement sites where their adjacent compartments exhibit uniform types. Two intra-chromosome rearrangements altered Rel and lft regulation, potentially contributing to wing patterning differentiation and host plant choice. Notably, butterflies exhibited chromatin loops between Hox gene cluster ANT-C and BX-C, unlike Drosophila. Our CRISPR-Cas9 experiments in butterflies confirm that knocking out the CTCF binding site of the loops in BX-C affected the phenotypes regulated by Antp in ANT-C, resulting in legless larva. Our results reveal evolutionary patterns of insect 3D chromatin structures and provide evidence that 3D chromatin structure changes can play important roles in the evolution of traits.

Combined detection of SHOX2 and PTGER4 methylation with serum marker CYFRA21-1 for improved diagnosis of malignant pleural mesothelioma

AimsTo investigate the performance of a combined biomarker approach using the methylation status of the short stature homeobox 2 (SHOX2) and prostaglandin E2 receptor EP4 (PTGER4) genes, along with the...

Genome mining of WOX-ARF gene linkage in Machilus pauhoi underpinned cambial activity associated with IAA induction.

As an upright tree with multifunctional economic application, Machilus pauhoi is an excellent choice in modern forestry from Lauraceae. The growth characteristics is of great significance for its molecular breeding and improvement. However, there still lack the information of WUSCHEL-related homeobox (WOX) and Auxin response factor (ARF) gene family, which were reported as specific transcription factors in plant growth as well as auxin signaling. Here, a total of sixteen MpWOX and twenty-one MpARF genes were identified from the genome of M. pauhoi. Though member of WOX conserved in the Lauraceae, MpWOX and MpARF genes were unevenly distributed on 12 chromosomes as a result of region duplication. These genes presented 45 and 142 miRNA editing sites, respectively, reflecting a potential post-transcriptional restrain. Overall, MpWOX4, MpWOX13a, MpWOX13b, MpARF6b, MpARF6c, and MpARF19a were highly co-expressed in the vascular cambium, forming a working mode as WOX-ARF complex. MpWOXs contains typical AuxRR-core and TGA-element cis-acting regulatory elements in this auxin signaling linkage. In addition, under IAA and NPA treatments, MpARF2a and MpWOX1a was highly sensitive to IAA response, showing significant changes after 6 hours of treatment. And MpWOX1a was significantly inhibited by NPA treatment. Through all these solid analysis, our findings provide a genetic foundation to growth mechanism analysis and further molecular designing breeding in Machilus pauhoi.