Vascular Cambium Research Articles

Identification of cambium stem cell factors and their positioning mechanism.

Wood constitutes the largest reservoir of terrestrial biomass. Composed of xylem, it arises from one side of the vascular cambium, a bifacial stem cell niche that also produces phloem on the opposing side. It is currently unknown which molecular factors endow cambium stem cell identity. Here we show that TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) ligand-activated PHLOEM INTERCALATED WITH XYLEM (PXY) receptors promote the expression of CAMBIUM-EXPRESSED AINTEGUMENTA-LIKE (CAIL) transcription factors to define cambium stem cell identity in the Arabidopsis root. By sequestrating the phloem-originated TDIF, xylem-expressed PXY confines the TDIF signaling front, resulting in the activation of CAIL expression and stem cell identity in only a narrow domain. Our findings show how signals emanating from cells on opposing sides ensure robust yet dynamically adjustable positioning of a bifacial stem cell layer.

A molecular module connects abscisic acid with auxin signals to facilitate seasonal wood formation in Populus.

Perennial trees have a recurring annual cycle of wood formation in response to environmental fluctuations. However, the precise molecular mechanisms that regulate the seasonal formation of wood remain poorly understood. Our prior study indicates that VCM1 and VCM2 play a vital role in regulating the activity of the vascular cambium by controlling the auxin homoeostasis of the cambium zone in Populus. This study indicates that abscisic acid (ABA) affects the expression of VCM1 and VCM2, which display seasonal fluctuations in relation to photoperiod changes. ABA-responsive transcription factors AREB4 and AREB13, which are predominantly expressed in stem secondary vascular tissue, bind to VCM1 and VCM2 promoters to induce their expression. Seasonal changes in the photoperiod affect the ABA amount, which is linked to auxin-regulated cambium activity via the functions of VCM1 and VCM2. Thus, the study reveals that AREB4/AREB13-VCM1/VCM2-PIN5b acts as a molecular module connecting ABA and auxin signals to control vascular cambium activity in seasonal wood formation.

Transcriptomic Remodeling Occurs During Cambium Activation and Xylem Cell Development in Taxodium ascendens

Taxodium ascendens has been extensively cultivated in the wetlands of the Yangtze River in south China and has significantly contributed to ecology and timber production. Until now, research on T. ascendens genomics has yet to be conducted due to its large and complex genome, which hinders the development of T. ascendens genomic resources. Combined with the microstructural changes during cambium cell differentiation across various growth periods, we investigate the transcriptome expression and regulatory mechanisms governing cambium activity in T. ascendens. Using RNA sequencing (RNA-Seq) technology, we identified the genes involved in the cambium development of cells at three stages (dormancy, reactivation, and activity). These genes encode the regulatory and control factors associated with the cambial activity, cell division, cell expansion, and biosynthesis of cell wall components. Blast comparison revealed that three genes (TR_DN69961_c0_g1, TRINITY_DN17100_c1_g1, TRINITY_DN111727_c0_g1) from the MYB and NAC families might regulate transcription during lignin formation in wood thickening. These results illustrate the dynamic changes in the transcriptional network during vascular cambium development. Additionally, they shed light on the genetic regulation mechanism of secondary growth in T. ascendens and guide further elucidation of the candidate genes involved in regulating cambium differentiation and wood formation.

ModelBark: a toy model to study bark formation in woody species

Abstract The study of growth of woody species is a challenging issue, primarily due to the complexity of the involved processes, which span broad spatial and temporal scales. Very often, this latter aspect almost precludes complete experimentation, thereby hindering the comparison between theoretical predictions and real-world observations. Computer simulations offer an alternative approach, allowing for data collection based on theoretical assumptions, and has been applied to the analysis of different features in plant development, as the production of secondary vascular tissues, xylem and phloem, in woody plants. However, the simulation of bark development faces added difficulties, due to the scarcity of experimental observations to base the model on. In this paper, we introduce a computer model designed to simulate bark formation based on mechanical stimuli acting on the various types of cells comprising this tissue. Our model can be conceptualized as a cellular automaton of variable size with non-local updating rules. By adjusting the parameters defining the model, we investigate the most influential factors in bark development, obtaining the most common bark types observed in trees. Furthermore, we provide an intuitive interface, making it suitable for educational purposes as well.

Differential regulation of xylem and phloem differentiation in grape berries by GA3 and CPPU

Fruit vascular bundles play a critical role in transporting water and nutrients within fruit, ultimately impacting fruit size and quality. Gibberellic acid (GA) and the synthetic cytokinin forchlorfenuron (CPPU) are commonly used to enhance grape berry size. Understanding their effects on the structure and function of berry vascular bundles could provide new information on the regulatory mechanism of these hormones on grapevine expansion and quality. In this study, the fruitlets of ‘Pinot Noir’ and ‘Einset Seedless’ grape varieties were treated by GA3, CPPU, and a combination of both at two weeks after full bloom. The samples were collected at 20, 40 and 70 days post-treatment. The results showed that all treatments led to an increase in berry size, xylem area, phloem area, the relative abundance of large-diameter grade vessels (>6 μm), the corresponding xylem area-specific hydraulic conductivity, and water absorption in both grape varieties. Specifically, CPPU favored an increase in phloem area, while GA3 primarily increased the xylem area. The combined application had a strong effect on both xylem and phloem area. RNA-seq analysis revealed that all treatments induced variations in the expression levels of multiple hormone signal components and genes related to vascular development. GA3 treatment mainly influenced some specific transcription factors involved in xylem differentiation, while CPPU and the combination treatment induced a wide range of genes related to vascular cambium initiation, proliferation, and differentiation of phloem and xylem vessels. Overall, our findings suggest that GA3 played a significant role in xylem differentiation, whereas CPPU had a major impact on both phloem and xylem differentiation in grape berries. These results lay the groundwork for further research the precise regulation of fruit vascular development under GA3 and CPPU treatments.

Experimental Induction of Extreme Indented Growth Rings (Hazel Wood) in Pinus halepensis Miller by Wide and Long Parallel Bark and Vascular Cambium Woundings.

Indented growth rings were found long ago to be experimentally induced in Pinus halepensis Miller by thin parallel axial scratching of the bark up to the vascular cambium with a sharp blade. Here, we show that when the bark and vascular cambium of P. halepensis are wounded by wide and long parallel axial wounds ("windows") rather than by thin scratches, the induced indented growth rings become dramatically more indented. All ten trees that were wounded by long parallel "windows" responded with very strong growth (especially in the first two years) that resulted in the formation of very conspicuous, extremely indented growth rings in the wood formed in between the long and wide woundings. This is true for both the trunks that were wounded all around their circumference and those that were wounded only in part of their circumference. We also suggest further lines of research.

PagJAZ5 regulates cambium activity through coordinately modulating cytokinin concentration and signaling in poplar.

Wood is resulted from the radial growth paced by the division and differentiation of vascular cambium cells in woody plants, and phytohormones play important roles in cambium activity. Here, we identified that PagJAZ5, a key negative regulator of jasmonate (JA) signaling, plays important roles in enhancing cambium cell division and differentiation by mediating cytokinin signaling in poplar 84K (Populus alba × Populus glandulosa). PagJAZ5 is preferentially expressed in developing phloem and cambium, weakly in developing xylem cells. Overexpression (OE) of PagJAZ5m (insensitive to JA) increased cambium activity and xylem differentiation, while jaz mutants showed opposite results. Transcriptome analyses revealed that cytokinin oxidase/dehydrogenase (CKXs) and type-A response regulators (RRs) were downregulated in PagJAZ5m OE plants. The bioactive cytokinins were significantly increased in PagJAZ5m overexpressing plants and decreased in jaz5 mutants, compared with that in 84K plants. The PagJAZ5 directly interact with PagMYC2a/b and PagWOX4b. Further, we found that the PagRR5 is regulated by PagMYC2a and PagWOX4b and involved in the regulation of xylem development. Our results showed that PagJAZ5 can increase cambium activity and promote xylem differentiation through modulating cytokinin level and type-A RR during wood formation in poplar.

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.

Restorer of fertility like 30, encoding a mitochondrion-localized pentatricopeptide repeat protein, regulates wood formation in poplar.

Nuclear-mitochondrial communication is crucial for plant growth, particularly in the context of cytoplasmic male sterility (CMS) repair mechanisms linked to mitochondrial genome mutations. The restorer of fertility-like (RFL) genes, known for their role in CMS restoration, remain largely unexplored in plant development. In this study, we focused on the evolutionary relationship of RFL family genes in poplar specifically within the dioecious Salicaceae plants. PtoRFL30 was identified to be preferentially expressed in stem vasculature, suggesting a distinct correlation with vascular cambium development. Transgenic poplar plants overexpressing PtoRFL30 exhibited a profound inhibition of vascular cambial activity and xylem development. Conversely, RNA interference-mediated knockdown of PtoRFL30 led to increased wood formation. Importantly, we revealed that PtoRFL30 plays a crucial role in maintaining mitochondrial functional homeostasis. Treatment with mitochondrial activity inhibitors delayed wood development in PtoRFL30-RNAi transgenic plants. Further investigations unveiled significant variations in auxin accumulation levels within vascular tissues of PtoRFL30-transgenic plants. Wood development anomalies resulting from PtoRFL30 overexpression and knockdown were rectified by NAA and NPA treatments, respectively. Our findings underscore the essential role of the PtoRFL30-mediated mitochondrion-auxin signaling module in wood formation, shedding light on the intricate nucleus-organelle communication during secondary vascular development.

Vascular cambium stem cells: past, present and future.

Secondary xylem and phloem originate from a lateral meristem called the vascular cambium that consists of one to several layers of meristematic cells. Recent lineage tracing studies have shown that only one of the cambial cells in each radial cell file functions as the stem cell, capable of producing both secondary xylem and phloem. Here, we first review how phytohormones and signalling peptides regulate vascular cambium formation and activity. We then propose how the stem cell concept, familiar from apical meristems, could be applied tocambium studies. Finally, we discuss how this concept could set the basis for future research.

Bridging photosynthetic worlds: Creating C4-CAM chimeras through embryo grafting in maize and orchids

Traditional grafting practices exclude monocots due to their physical incompatibilities. Their distinct anatomical features, such as scattered vascular bundles and parallel venation and lack of vascular cambium, don't allow for graft healing. The recent development of embryonic grafting has disapproved the requirement of vascular cambium for grafting, instead relying on undifferentiated tissue. We grafted the embryos of two photosynthetically different plants- Zea mays (C4) and Dendrobium ovatum (CAM). We successfully generated grafts through a ‘whole embryo-protocorm’ method. The grafts showed multiple morphological characteristics, which included curling of leaves, stunted growth, and callus-like structure. We did an anatomical analysis to confirm graft union and vascular connectivity. The grafts showed over an 80 % survival rate across all morphology 30 days after grafting. The embryonic meristematic cells proliferated and healed the graft junction, thus allowing the grafted embryo to grow as an individual plant. This technique opens avenues for trait transfer between monocot crops.

WUSCHEL-RELATED HOMEOBOX genes are crucial for normal vascular organization and wood formation in poplar

Vascular cambium in tree species is a cylindrical domain of meristematic cells that are responsible for producing secondary xylem (also called wood) inward and secondary phloem outward. The poplar (Populus trichocarpa) WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family members, PtrWUSa and PtrWOX13b, were previously shown to be expressed in vascular cambium and differentiating xylem cells in poplar stems, but their functions remain unknown. Here, we investigated roles of PtrWUSa, PtrWOX13b and their close homologs in vascular organization and wood formation. Expression analysis showed that like PtrWUSa and PtrWOX13b, their close homologs, PtrWUSb, PtrWUS4a/b and PtrWOX13a/c, were also expressed in vascular cambium and differentiating xylem cells in poplar stems. PtrWUSa also exhibited a high level of expression in developing phloem fibers. Expression of PtrWUSa fused with the dominant EAR repression domain (PtrWUSa-DR) in transgenic poplar caused retarded growth of plants with twisted stems and curled leaves and a severe disruption of vascular organization. In PtrWUSa-DR stems, a drastic proliferation of cells occurred in the phloem region between vascular cambium and phloem fibers and they formed islands of ectopic vascular tissues or phloem fiber-like sclerenchyma cells. A similar proliferation of cells was also observed in PtrWUSa-DR leaf petioles and midveins. On the other hand, overexpression of PtrWOX4a-DR caused ectopic formation of vascular bundles in the cortical region, and overexpression of PtrWOX13a-DR and PtrWOX13b-DR led to a reduction in wood formation without affecting vascular organization in transgenic poplar plants. Together, these findings indicate crucial roles of PtrWUSa and PtrWOX13a/b in regulating vascular organization and wood formation, which furthers our understanding of the functions of WOX genes in regulating vascular cambium activity in tree species.

The Vascular Cambia of Dodder and its Allies

The vascular cambia of 20 species of the dodder and its allies belonging to the CassythaceaE, Convolvulaceae and Cuscutaceae were surveyed to assess their phenetic and phylogenetic significance. In natural condition all of them contain anomalous cambia. The Cassythaceae bore transitory cambium in the normal position but atypical by asmmetric activity. (The Cambium alternatelt retards accemtuates xylem formation on several segments and there are profuse sehizongenous spaces in wood. Haustoria contains both traheids and vessels).In stems and roots of the Convolvulaceae a series of widely different anomalous cambia were found which sometimes differed in stems and roots of the same species. Stems of arborescent group had bands of interxylary phloem in concentric rings, while in the primitive twiners these are arranged in radial plates. In general, a tendency was to increase the proportion of soft tissue. In Cuscutaceae the specified anomaly was the interfascicular cambium. Ray initials were absent but fusiform initials differentiated into profuse parenchyma cells and a few vessel elements.Haustorium develops only imperforate tracheary elements. The evolutionary history vascular cambium in the Convolvulaceae was mainly one of reduction in activity and area. Thus the vesselless haustorium of Cuscutaceae. may be regarded as a reduced farm in with the vessel bearing haustorium of Cassythaceae. It 1s also concluded that the primitive condition of Cuscutaceae is possibly derived from the general condition of Convolvulaceae.

Water flow in two Cactaceae species: standardization of the method and test under different drought conditions

AbstractInvestigating water flow in the vascular system of plants is important to identify the water path and conduction capacity of different plant species, mainly in succulent species living in dry regions. This study aimed to standardize a method to track water flow in the xylem of Mammillaria bocasana and Echinocereus reichenbachii var. fitchii (Cactaceae) and test it under different drought conditions. The plants were kept under greenhouse conditions to perform the split root method, water flow tracing, and different drought treatments. The plants of each species were separated into three treatments: constant irrigation, drought with water and dye, and drought with dye. Plants were cut 24 h after staining, total and lumen diameters were taken, and the relative theoretical hydraulic conductivity was calculated. The absorption method was adequate for the tracing of water flow, and it worked in all the treatments of the drought experiment. Conducting elements, including wide-band tracheids, were stained violet and were located near the vascular cambium. In Mammillaria bocasana, half of the vascular cylinder was stained, while in Echinocereus reichenbachii var. fitchii, the entire cylinder was stained due to the cortical bundles. M. bocasana had a higher lumen diameter and relative theoretical hydraulic conductivity than E. reichenbachii var. fitchii. With the absorption staining method, the flow of water was traced, and differences in conduction under water stress conditions between the two species of cacti were detected. These results suggest that cacti species living under water stress solve the water movement in different ways.

Comparative transcriptome profiling reveals that light coordinates auxin to inhibit adventitious root formation in grapevine

Grapevine (Vitis sp.) is one of the most important economic fruit crops all over the world, and the formation of adventitious roots (ARs) is crucial for the vegetative reproduction of grapes. However, studies on the regulatory mechanisms of this process are currently lacking. In this study, we applied an efficient and convenient leave-petiole (LP) system for studying ARs, revealing a significant inhibition of root primordia formation under continuous-light treatment. The results showed that isolated ARs of grapevine were induced and originated from ray cells near the vascular cambium, with the process categorized into induction, initiation, and extension stages. LP samples under light and dark conditions were used for transcriptome sequencing and endogenous hormone measurements at three critical time points of AR formation. A total of 37 155 transcripts were obtained, and 7041 genes showed significantly different expression levels in the petiole. An integrated analysis, including Gene Ontology (GO) enrichment analysis, weighted gene co-expression network analysis (WGCNA), and hormonal content determination, showed that several genes (ARF4, LAX1, PIN1, SUS2, APX1, TPXL1, CHS3, etc.) associated with hormone signals, sugar synthesis and transport, reactive oxygen species (ROS) scavenging, cell wall biogenesis, flavonoid biosynthesis, microtubule remodeling, and some transcription factors (HY5, COP1, ERF2, MYB15, etc) played vital roles in light-induced AR formation. A hypothetical model was initially constructed, which illustrated the centrality of auxin in HY5-dependent AR formation and the complex crosstalk among various factors. The results of this study provided abundant genetic resources and a novel perspective for understanding the molecular mechanisms of AR formation in grapevine.

The impact of drought on phellem development: Identification of novel gene regulators and evidence of photosynthetic activity in cork oak stems

Quercus suber (cork oak) is a sustainably exploited forest resource, producing a unique renewable raw material known as cork. With drought events imposing a negative impact on tree vitality, further research is needed to enhance our understanding of the genetic and environmental factors regulating cork development, to foster the resilience of cork oak ecosystems. We focused on characterizing long-term drought-induced molecular adaptations occurring in stems, and identifying key genetic pathways regulating phellem development. One-year-old cork oak plants were grown for 6 months under well-watered, or water-deficit (WD) conditions and main stems were targeted for histological characterization and transcriptomic analysis. WD treatment reduced meristem activity at both vascular cambium and phellogen, impairing secondary growth. Transcriptional analysis revealed a global downregulation of genes related to cell division, differentiation, and cell wall biogenesis in phellem, inner bark, and xylem under WD conditions. Phellem and inner bark showed upregulation of photosynthesis-related genes, highlighting a determinant role of stem photosynthesis in the adaptation to long-term drought. We show that developing phellem cells contain chloroplasts and their abundance increases under WD. Lastly, we propose new candidate regulatory genes involved in regulating phellogen activity and demonstrate the role of phellem in drought-induced bark photosynthesis in young plants.

Physiological and developmental disturbances caused by Botryosphaeria dieback in the annual stems of grapevine.

Botryosphaeria dieback is a grapevine trunk disease caused by fungi of the Botryosphaeriaceae family, which attacks more specifically the woody tissues. The infection leads to different symptoms including a severe form with a leaf drop as well as premature plant death. Botryosphaeria dieback causes major economic losses, since no effective treatment is yet available. A better understanding is necessary to find solutions to fight this disease. In this study, our objective was to characterize the "leaf drop" form by (1) looking for the presence of pathogens in the basal internodes of stems, (2) quantifying blocked vessels by tylosis and/or gummosis, and (3) describing the impact of the disease on vine physiology (gene expression and metabolome) and development (establishment and functioning of the cambium and phellogen) at the level of xylem and phloem of basal stem internodes. Our study has shown that Botryosphaeriaceae were present in both phloem and xylem of the basal internodes of the annual stem, with xylem vessels obturated. We have also clearly demonstrated that gene expression and metabolite profiles were strongly modified in both xylem and phloem of diseased plants. Differences in stems between healthy (control, C) and diseased (D) plants were low at flowering (vines not yet symptomatic), higher at the onset of symptom expression and still present, although less marked, at full disease expression. qRT-PCR analysis showed in both phloem and xylem an overexpression of genes involved in plant defense, and a repression of genes related to meristematic activity (i.e. vascular cambium and phellogen). Metabolomic analysis showed specific fingerprints in stems of healthy and diseased plants from the onset of symptom expression, with an increase of the level of phytoalexins and mannitol, and a decrease of 1-kestose one. At the structural level, many alterations were observed in internodes, even before the onset of symptoms: a classical obstruction of xylem vessels and, for the first time, a disorganization of the secondary phloem with an obstruction of the sieve plates by callose. The disease modifies the development of both secondary phloem (liber) and phellogen. Altogether, this study combining different approaches allowed to highlight deep vine dysfunction in the internodes at the base of stems, that may explain vine decline due to Botryosphaeria dieback.

Morphological, cytological, physiological, and molecular evidence to explain how underground fleshy roots expand in herbaceous peony (Paeonia lactiflora Pall.)

The fleshy roots of herbaceous peonies serve as vital storage organs and are the primary source material for Chinese herbal medicine. Moreover, in certain cultivars, these roots can be propagated by root cuttings. However, the specific mechanisms governing the expansion and development of fleshy roots in herbaceous peonies currently remain unclear. To elucidate patterns of changes during the enlargement and development of herbaceous peony fleshy roots, this study assessed fleshy roots in six growth and development periods (P1–P6). By integrating environmental monitoring data, morphological, cytological, physiological and biochemical, as well as molecular characteristics across these developmental periods were compared. Both precipitation and temperature influenced the development of fleshy roots. The fastest enlargement of fleshy roots occurred under the following growth conditions: daily average precipitation = 5.6 mm; monthly average air temperature = 25.6°C; monthly average soil temperature = 26.3°C. Phloem and xylem, produced by the activity of vascular cambium, are important components of fleshy roots. Secondary xylem thickness consistently increased across the six developmental periods, resulting in the radial expansion of fleshy roots. When average monthly air and soil temperatures were about 26°C, starch accumulated gradually in fleshy roots, and carbohydrates were mostly starch-based. However, when the average monthly air and soil temperatures decreased to about 11.0°C, soluble sugar content increased. Endogenous hormones such as IAA, ZR, GA3, MeJA, and ABA played regulatory roles throughout the development of fleshy roots, and their levels and dynamic equilibrium were correlated with expansion rate. Elevated IAA and ZR content, and stabilized IAA/ABA and MeJA/ABA ratios, favored fleshy root expansion. Trends in the expression of three genes that play key roles in the expansion of fleshy roots (PlEXLB3, PlAUX1 and PlWRKY13) were consistent with morphological and physiological data, but PlHXK1 and PlIPT3 also play important roles. These findings lay a foundation for studying, in greater depth, the regulatory mechanisms governing the enlargement of herbaceous peony fleshy roots, and offer practical advice pertaining to field cultivation and management practices of this ornamental plant.

Dimensional variations in fusiform and ray initials over different seasons of year in Juglans regia Linn. from temperate type of climate of Kashmir Himalaya

Dimensional variations in fusiform and ray initials over different seasons of year in Juglans regia Linn. from temperate type of climate of Kashmir Himalaya

Growth-regulating factor 15-mediated vascular cambium differentiation positively regulates wood formation in hybrid poplar (Populus alba × P. glandulosa).

Hybrid poplars are industrial trees in China. An understanding of the molecular mechanism underlying wood formation in hybrid poplars is necessary for molecular breeding. Although the division and differentiation of vascular cambial cells is important for secondary growth and wood formation, the regulation of this process is largely unclear. In this study, mPagGRF15 OE and PagGRF15-SRDX transgenic poplars were generated to investigate the function of PagGRF15. RNA-seq and qRT-PCR were conducted to analyze genome-wide gene expression, while ChIP‒seq and ChIP-PCR were used to identified the downstream genes regulated by PagGRF15. We report that PagGRF15 from hybrid poplar (Populus alba × P. glandulosa), a growth-regulating factor, plays a critical role in the regulation of vascular cambium activity. PagGRF15 was expressed predominantly in the cambial zone of vascular tissue. Overexpression of mPagGRF15 (themutated version of GRF15 in the miR396 target sequence) in Populus led to decreased plant height and internode number. Further stem cross sections showed that the mPagGRF15 OE plants exhibited significant changes in vascular pattern with an increase in xylem and a reduction in phloem. In addition, cambium cell files were decreased in the mPagGRF15 OE plants. However, dominant suppression of the downstream genes of PagGRF15 using PagGRF15-SRDX showed an opposite phenotype. Based on the RNA-seq and ChIP-seq results, combining qRT-PCR and ChIP-PCR analysis, candidate genes, such as WOX4b, PXY and GID1.3, were obtained and found to be mainly involved in cambial activity and xylem differentiation. Accordingly, we speculated that PagGRF15 functions as a positive regulator mediating xylem differentiation by repressing the expression of the WOX4a and PXY genes to set the pace of cambial activity. In contrast, PagGRF15 mediated the GA signaling pathway by upregulating GID1.3 expression to stimulate xylem differentiation. This study provides valuable information for further studies on vascular cambium differentiation mechanisms and genetic improvement of the specific gravity of wood in hybrid poplars.