Secondary Xylem Differentiation Research Articles

The transcription factor PagLBD4 represses cell differentiation and secondary cell wall biosynthesis in Populus

LBD (LATERAL ORGAN BOUNDARIES DOMAIN) transcription factors are key regulators of plant growth and development. In this study, we functionally characterized the PagLBD4 gene in Populus (Populus alba × Populus glandulosa). Overexpression of PagLBD4 (PagLBD4OE) significantly repressed secondary xylem differentiation and secondary cell wall (SCW) deposition, while CRISPR/Cas9-mediated PagLBD4 knockout (PagLBD4KO) significantly increased secondary xylem differentiation and SCW deposition. Consistent with the functional analysis, gene expression analysis revealed that SCW biosynthesis pathways were significantly down-regulated in PagLBD4OE plants but up-regulated in PagLBD4KO plants. We also performed DNA affinity purification followed by sequencing (DAP-seq) to identify genes bound by PagLBD4. Integration of RNA sequencing (RNA-seq) and DAP-seq data identified 263 putative direct target genes (DTGs) of PagLBD4, including important regulatory genes for SCW biosynthesis, such as PagMYB103 and PagIRX12. Together, our results demonstrated that PagLBD4 is a repressor of secondary xylem differentiation and SCW biosynthesis in Populus, which possibly lead to the dramatic growth repression in PagLBD4OE plants.

Exogenous applications of brassinosteroids promote secondary xylem differentiation in Eucalyptus grandis.

Brassinosteroids (BRs) play many pivotal roles in plant growth and development, especially in cell elongation and vascular development. Although its biosynthetic and signal transduction pathway have been well characterized in model plants, their biological roles in Eucalyptus grandis, a major hardwood tree providing fiber and energy worldwide, remain unclear. Here, we treated E. grandis plantlets with 24-epibrassinolide (EBL), the most active BR and/or BR biosynthesis inhibitor brassinazole. We recorded the plant growth and analyzed the cell structure of the root and stem with histochemical methods; then, we performed a secondary growth, BR synthesis, and signaling-related gene expression analysis. The results showed that the BRs dramatically increased the shoot length and diameter, and the exogenous BR increased the xylem area of the stem and root. In this process, EgrBRI1, EgrBZR1, and EgrBZR2 expression were induced by the BR treatment, and the expressions of HD-ZIPIII and cellulose synthase genes were also altered. To further verify the effect of BRs in secondary xylem development in Eucalyptus, we used six-month-old plants as the material and directly applied EBL to the xylem and cambium of the vertical stems. The xylem area, fiber cell length, and cell numbers showed considerable increases. Several key BR-signaling genes, secondary xylem development-related transcription factor genes, and cellulose and lignin biosynthetic genes were also considerably altered. Thus, BR had regulatory roles in secondary xylem development and differentiation via the BR-signaling pathway in this woody plant.

"Pathomorphogenic" Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco.

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

Description of Intra-Annual Changes in Cambial Activity and Differentiation of Secondary Conductive Tissues of Aesculus hippocastanum Trees Affected by the Leaf Miner Cameraria ohridella

Aesculus hippocastanum trees are commonly infested by the leaf miner Cameraria ohridella, whose larval activity causes the destruction of the leaf parenchyma and induces defoliation. Pest attacks result in, e.g., production of smaller fruits and tree re-flowering in autumn. Concerning pest influence on stem structure only scarce information of narrower annual growth rings of wood has been published. Therefore, we determined the effect of the presence of the leaf miner infestation on intra-annual cambial activity and on differentiation of conductive tissues. These data were compared with phenological phases and pest activity. Pest feeding resulted in changes in onset, cessation and duration of cambial divisions, and differentiation of secondary xylem. The duration of cambial activity was about a month shorter in heavily infested trees and was connected with premature tree defoliation. Affected trees were characterised by a reduction in cambial divisions and earlier cessation of wood differentiation resulting in narrower wood rings. Furthermore, the infested trees exhibited altered wood structure, with more vessels of smaller diameters, however these changes did not affect its theoretical hydraulic conductivity. Interestingly, pest attack did not influence secondary phloem differentiation. The probable influence of long-term infestation on tree growth and condition was discussed.

Brassinosteroids promote parenchyma cell and secondary xylem development in sugar beet (Beta vulgaris L.) root.

Increasing crop yield has always been an important goal in agriculture. Brassinosteroids (BRs) are growth‐promoting steroid hormones with vital roles in many root developmental processes. Sugar beet ( Beta vulgaris L.) is a root crop with a tertiary root structure. The differentiation of vascular bundles and the division of cambial cells increase root diameter. However, little is known about how BRs regulate the transverse growth of beetroot. Therefore, sugar beet with eight leaves was grown in medium containing epibrassinolide or brassinazole, an inhibitor of BR biosynthesis. BRs increased the spacing between the cambial rings by increasing the size of parenchyma cells between the rings and ultimately increasing root diameter. BRs also promoted secondary xylem differentiation. Moreover, the gene expression analysis of BvXTH33, BvSHV3, BvCESA6, BvPARVUS, and BvCEL1, which were related to the cell wall biosynthesis, indicated that BR could promote the growth of cell wall. These findings showed that BRs function in transverse development in beetroot.

MiR319a-targeted PtoTCP20 regulates secondary growth via interactions with PtoWOX4 and PtoWND6 in Populus tomentosa.

Secondary growth is a key characteristic of trees, which requires the coordination of multiple regulatory mechanisms including transcriptional regulators and microRNAs (miRNAs). However, the roles of microRNAs in the regulation of secondary growth need to be explored in depth. Here, the role of miR319a and its target, PtoTCP20, in the secondary growth of Populus tomentosa stem was investigated using genetic and molecular analyses. The expression level of miR319a gradually decreased from primary to secondary growth in P.tomentosa, while that of PtoTCP20 gradually increased. MiR319a overexpression in seedlings resulted in delayed secondary growth and decreased xylem production, while miR319a knockdown and PtoTCP20 overexpression promoted secondary growth and increased xylem production. Further analysis showed that PtoTCP20 interacted with PtoWOX4a and activated PtoWND6 transcription in vitro and in vivo. Our data show that PtoTCP20 controls vascular cambium proliferation by binding to PtoWOX4a, and promotes secondary xylem differentiation by activating PtoWND6 transcription, thereby regulating secondary growth in P.tomentosa. Our findings provide insight into the molecular mechanisms underlying secondary growth in trees.

The GATA transcription factor GNC plays an important role in photosynthesis and growth in poplar.

GATA transcription factors are involved in the regulation of diverse growth processes and environmental responses in Arabidopsis and rice. In this study, we conducted a comprehensive bioinformatic survey of the GATA family in the woody perennial Populus trichocarpa. Thirty-nine Populus GATA genes were classified into four subfamilies based on gene structure and phylogenetic relationships. Predicted cis-elements suggested potential roles of poplar GATA genes in light, phytohormone, development, and stress responses. A poplar GATA gene, PdGATA19/PdGNC (GATA nitrate-inducible carbon-metabolism-involved), was identified from a fast growing poplar clone. PdGNC expression was significantly up-regulated in leaves under both high (50 mM) and low (0.2 mM) nitrate concentrations. The CRISPR/Cas9-mediated mutant crispr-GNC showed severely retarded growth and enhanced secondary xylem differentiation. PdGNC-overexpressing transformants exhibited 25-30% faster growth, 20-28% higher biomass accumulation, and ~25% increase in chlorophyll content, photosynthetic rate, and plant height, compared with the wild type. Transcriptomic analysis showed that PdGNC was involved in photosynthetic electron transfer and carbon assimilation in the leaf, cell division and carbohydrate utilization in the stem, and nitrogen uptake in the root. These data indicated that PdGNC plays a crucial role in plant growth and is potentially useful in tree molecular breeding.

Analysis of NAC Domain Transcription Factor Genes of Tectona grandis L.f. Involved in Secondary Cell Wall Deposition.

NAC proteins are one of the largest families of plant-specific transcription factors (TFs). They regulate diverse complex biological processes, including secondary xylem differentiation and wood formation. Recent genomic and transcriptomic studies of Tectona grandis L.f. (teak), one of the most valuable hardwood trees in the world, have allowed identification and analysis of developmental genes. In the present work, T. grandis NAC genes were identified and analyzed regarding to their evolution and expression profile during wood formation. We analyzed the recently published T. grandis genome, and identified 130 NAC proteins that are coded by 107 gene loci. These proteins were classified into 23 clades of the NAC family, together with Populus, Eucalyptus, and Arabidopsis. Data on transcript expression revealed specific temporal and spatial expression patterns for the majority of teak NAC genes. RT-PCR indicated expression of VND genes (Tg11g04450-VND2 and Tg15g08390-VND4) related to secondary cell wall formation in xylem vessels of 16-year-old juvenile trees. Our findings open a way to further understanding of NAC transcription factor genes in T. grandis wood biosynthesis, while they are potentially useful for future studies aiming to improve biomass and wood quality using biotechnological approaches.

Regulation of secondary xylem formation in young hybrid poplars by modifying the expression levels of the PtaGLIMa gene

Due to the importance of wood in many industrial applications, a tremendous amount of research has focused on the regulation of secondary xylem formation and wood properties. In this study, we performed functional analysis of PtaGLIM1a, a LIM gene that is predominantly expressed in the differentiation of secondary xylem of the hybrid poplar (Populus tremula × P. alba). With no growth retardation, transgenic poplar plants with increased and reduced expression levels of PtaGlim1a exhibited enhanced and diminished secondary growth, respectively, accompanied by a corresponding change in their lignin abundance. This study demonstrates that the wood-associated PtaGlim1a acts as a positive regulator of secondary xylem formation in poplar trees and could potentially be utilized in modifying the synthesis of plant secondary wall lignin.

Repression of BLADE-ON-PETIOLE genes by KNOX homeodomain protein BREVIPEDICELLUS is essential for differentiation of secondary xylem in Arabidopsis root.

Repression of boundary genes by KNOTTED1-like homeodomain transcription factor BREVIPEDICELLUS promotes the differentiation of phase II secondary xylem in Arabidopsis roots. Plant growth and development relies on the activity of meristems. Boundaries are domains of restricted growth that separate forming organs and the meristem. Class I KNOX homeodomain transcription factors are important regulators of meristem maintenance. Members of this class including BREVIDICELLUS also called KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (BP/KNAT1) fulfill this function in part by spatially regulating boundary genes. The vascular cambium is a lateral meristem that allows for radial expansion of organs during secondary growth. We show here that BP/KNAT1 repression of boundary genes plays a crucial role in root secondary growth. In particular, exclusion of BLADE-ON-PETIOLE1/2 (BOP1/2) and other members of this module from xylem is required for the differentiation of lignified fibers and vessels during the xylem expansion phase of root thickening. These data reveal a previously undiscovered role for boundary genes in the root and shed light on mechanisms controlling wood development in trees.

Stem anatomy and development of inter- and intraxylary phloem in Leptadenia pyrotechnica (Forssk.) Decne. (Asclepiadaceae)

Modification of external morphology and internal structure of plants is a key feature of their successful survival in extreme habitats. They adapt to arid habitats not only by modifying their leaves, but also show several modifications in their conducting system. Therefore, the present study is aimed to investigate the pattern of secondary growth in Leptadenia pyrotechnica (Forssk.) Decne., (Asclepiadaceae), one such species growing in Kachchh district, an arid region of Gujarat State. A single ring of vascular cambium, responsible for radial growth, divided bidirectionally and formed the secondary xylem centripetally and the phloem centrifugally. After a short period of secondary xylem differentiation, small arcs of cambium began to form secondary phloem centripetally instead of secondary xylem. After a short duration of such secondary phloem formation, these segments of cambium resumed their normal function to produce secondary xylem internally. Thus, the phloem strands became embedded within the secondary xylem and formed interxylary phloem islands. Such a recurrent behavior of the vascular cambium resulted in the formation of several patches of interxylary phloem islands. In thick stems the earlier formed non-conducting interxylary phloem showed heavy accumulation of callose on the sieve plates followed by their crushing in response to the addition of new sieve elements. Development of intraxylary phloem is also observed from the cells situated on the pith margin. As secondary growth progresses further, small arcs of internal cambium get initiated between the protoxylem and intraxylary phloem. In the secondary xylem, some of the vessels are exceptionally thick-walled, which may be associated with dry habitats in order to protect the vessel from collapsing during the dryer part of the year. The inter- and intraxylary phloem may also be an adaptive feature to prevent the sieve elements to become non-conducting during summer when the temperature is much higher.

Partial desiccation enhances induction of secondary xylem-like tracheary elements from calli of hybrid poplar (Populus sieboldii x P. grandidentata)

Calli of hybrid poplar that had been exposed to desiccation in air before transfer to the induction medium differentiated into tracheary elements at higher rates than calli without air desiccation. Cells of hybrid poplar (Populus sieboldii x P. grandidentata) in culture can be induced to differentiate into secondary xylem-like tracheary elements that form the highly developed bordered pits and broad regions of cell walls in contrast to helical or reticulate wall thickenings in primary xylem elements. We attempted to increase the rate of differentiation of tracheary elements from calli using a combination of hormonal stimulation and partial desiccation. Calli that had been exposed to desiccation in air in a clean hood for 90 min before transfer to the induction medium differentiated into tracheary elements at higher rates than calli without air desiccation. The partial desiccation treatment had no effects on the features of the induced tracheary elements and the frequencies with which they appeared. Our results show that partial desiccation can increase, approximately threefold the rate of differentiation of secondary xylem-like tracheary elements from calli of hybrid poplar. This improvement in the rate of differentiation tracheary elements in vitro should facilitate detailed future analysis of the differentiation of secondary xylem.

Fertilization Independent Endosperm genes repress NbGH3.6 and regulate the auxin level during shoot development in Nicotiana benthamiana.

The Fertilization Independent Endosperm (FIE) gene is required to restrict endosperm development without fertilization, and it represses flowering during embryo and seedling development in Arabidopsis thaliana However, the regulatory mechanism of the FIE gene in postembryonic shoot development is not well understood. Silencing of Nicotiana benthamiana homologues of the FIE gene, NbFIE1 and NbFIE2, resulted in the enhanced outgrowth of axillary buds and the impairment of secondary xylem differentiation. RNA sequencing analysis found that one of the auxin-responsive GRETCHEN HAGEN 3(GH3) family genes, NbGH3.6, was upregulated and maintained a high expression during the time course of silencing NbFIE genes. Chromatin immunoprecipiation (ChIP)-PCR results showed a lack of H3K27me3 marks on NbGH3.6 chromatin in NbFIE-silenced plants compared with negative control plants, indicating that NbGH3.6 was a direct target of NbFIE genes during postembryonic shoot development. Moreover, the free IAA content was reduced significantly in NbFIE-silenced plants, which might cause the enhanced outgrowth of axillary buds as well as impaired secondary xylem differentiation. These results clearly indicated that NbGH3.6 was a primary target of NbFIE genes during postembryonic shoot development, and NbFIE genes regulated axillary bud growth and secondary xylem formation through tuning endogenous auxin homeostasis, possibly by regulating the expression of the NbGH3.6 gene.

Effect of auxin on xylem tracheids differentiation in decapitated stems of Pinus silvestris L. and its interaction with some vitamins and growth regulators

The effects of several vitamins and substances known as important agents in regulation of cell metabolism upon secondary xylem differentiation were studied in interaction with auxin (IAA) as applied in lanoline to decapitated stems of 5-year-old &lt;i&gt;Pinus silvestris&lt;/i&gt; trees in early and late-summer. Tested substances were: gibberellic acid, kinetin, nicotinic acid, thiamine, pyridoxine, calcium panthotenate, choline chloride, riboflavin, inositol, ascorbic acid, vitamin, A (alcohol), vitamin A (ester), saponin. None of the effects of these substances appeared significant enough to indicate the involvement in the seasonal variation of the response of cambium or differentiating tracheids to auxin. However, several effects, especially those of inositol, vitamin A and pyridoxine upon cambial xylem production and further stages of tracheid differentiation were observed. Auxin (IAA) affected cambial activity and subsequent differentiation of tracheids during the earliest stages of cell ontogenesis. At these stages auxin treatment induced quantitative expression of the developmental processes involving radial growth and secondary wall formation by tracheids. In this respect, auxin did not affect cells advanced in differentiation, however, it proved to be an essential factor in the completion of the full cycle of tracheid ontogenesis.

Genome-wide transcriptional profiling reveals molecular signatures of secondary xylem differentiation in Populus tomentosa.

Wood formation occurs via cell division, primary cell wall and secondary wall formation, and programmed cell death in the vascular cambium. Transcriptional profiling of secondary xylem differentiation is essential for understanding the molecular mechanisms underlying wood formation. Differential gene expression in secondary xylem differentiation of Populus has been previously investigated using cDNA microarray analysis. However, little is known about the molecular mechanisms from a genome-wide perspective. In this study, the Affymetrix poplar genome chips containing 61,413 probes were used to investigate the changes in the transcriptome during secondary xylem differentiation in Chinese white poplar (Populus tomentosa). Two xylem tissues (newly formed and lignified) were sampled for genome-wide transcriptional profiling. In total, 6843 genes (~11%) were identified with differential expression in the two xylem tissues. Many genes involved in cell division, primary wall modification, and cellulose synthesis were preferentially expressed in the newly formed xylem. In contrast, many genes, including 4-coumarate:cinnamate-4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), and caffeoyl CoA 3-O-methyltransferase (CCoAOMT), associated with lignin biosynthesis were more transcribed in the lignified xylem. The two xylem tissues also showed differential expression of genes related to various hormones; thus, the secondary xylem differentiation could be regulated by hormone signaling. Furthermore, many transcription factor genes were preferentially expressed in the lignified xylem, suggesting that wood lignification involves extensive transcription regulation. The genome-wide transcriptional profiling of secondary xylem differentiation could provide additional insights into the molecular basis of wood formation in poplar species.

Apical control of xylem formation in the pine stem. II. Responses of differentiating tracheids

The effect of auxin supplied to the main stem of 5-year-old &lt;em&gt;Pinus silvestris&lt;/em&gt; trees during various periods after decapitation upon differentiation of the secondary xylem tracheids was investigated. The results revealed the complexity of auxin involvement in the regulatory system of tracheid differentiation of secondary xylem. It is manifested both as the inductive effect to which the cells respond in the meristematic phase and in the continuous control during the consecutive stages of radial growth and maturation. A lack of auxin during the meristematic phase resulted in smaller cell diameters and reduced the daily rate of cell wall deposition even though these cells progressively grew and matured in the presence of auxin. The intensity of these two processes increased and the cells deposited thicker walls when auxin was supplied during all stages of tracheid differentiation even though the period of maturation decreased. Under these conditions tracheids of compression wood type differentiated. Continuous availability of auxin causes earlier termination of tracheid maturation while lack of auxin results in a delay of autolysis of protoplasts. In this case auxin probably functions in a system specifying information concerning the position of the cells in respect to the cambial layer.

Proteolytic activity in the stem cambial region of Pinus sylvestris L. - A contribution to the specific differentiation of secondary xylem and phloem

Proteolytic activity was studied in the differentiating xylem and phloem of Scots pine (&lt;i&gt;Pinus sylvestris&lt;/i&gt; L.) to determine the specificity of xylem and phloem differentiation. The activity of autolytic proteases was demonstrated in the differentiating xylem during spring, summer and autumn and it was not detectable during winter. It was initiated with the onset of cambial activity in spring and unchanged during subsequent stages of xylem differentiation. The same proteolytic activity was not detectable in the extract of fresh phloem tissue. It could be detected in phloem after removal of the inhibitor found in the extract. The same pH optimum was determined for proteases extracted from xylem and phloem. However, their identity remains uncertain because of different electrophoretic mobility. On the other hand the presence of protease inhibitor in phloem tissue can be an important factor im determining the specificity of xylem an phloem differentiation.

Tracheary elements that resemble secondary xylem in calli derived from the conifers, Torreya nucifera and Cryptomeria japonica

Differentiated cells were recognized in calli derived from needles of Torreya nucifera and in calli derived from immature zygotic embryos of Cryptomeria japonica. Some differentiated cells resembled tracheary elements of primary xylem with spiral or reticulate thickening of cell walls. Other cells resembled tracheary elements with thick cell walls and bordered pits, which are features of secondary xylem. These tracheary elements were formed in cell clusters. Tracheary elements in calli of T. nucifera formed more highly developed structures, such as bordered pits and spiral thickening, than those of C. japonica. Cultured cells derived from conifers might provide a good model for studies of the differentiation of secondary xylem in vitro.

Gibberellin is required for the formation of tension wood and stem gravitropism in Acacia mangium seedlings.

Angiosperm trees generally form tension wood on the upper sides of leaning stems. The formation of tension wood is an important response to gravitational stimulus. Gibberellin appears to be involved in the differentiation of secondary xylem, but it remains unclear whether gibberellin plays a key role in the formation of tension wood and plant gravitropism. Therefore, a study was designed to investigate the effects of gibberellin and of inhibitors of the synthesis of gibberellin, namely paclobutrazole and uniconazole-P, on the formation of tension wood and negative stem gravitropism in Acacia mangium seedlings. Gibberellic acid (GA(3)), paclobutrazole and uniconazole-P were applied to seedlings via the soil in which they were growing. Distilled water was applied similarly as a control. Three days after such treatment, seedlings were tilted at an angle of 45° from the vertical, and samples of stems were collected for analysis 2 weeks, 2 months and 6 months after tilting. The effects of treatments on the stem recovery degree (Rº) were analysed as an index of the negative gravitropism of seedlings, together the width of the region of tension wood in the upper part of inclined stems. It was found that GA(3) stimulated the negative gravitropism of tilted seedling stems of A. mangium, while paclobutrazole and uniconazole-P inhibited recovery to vertical growth. Moreover, GA(3) stimulated the formation of tension wood in tilted A. mangium seedlings, while paclobutrazole and uniconazole-P strongly suppressed the formation of tension wood, as assessed 2 weeks after tilting. The results suggest that gibberellin plays an important role at the initial stages of formation of tension wood and in stem gravitropism in A. mangium seedlings in response to a gravitational stimulus.

Enhancement of secondary xylem cell proliferation by Arabidopsis cyclin D overexpression in tobacco plants

Secondary xylem is composed of daughter cells produced by the vascular cambium in the stem. Cell proliferation of the secondary xylem is the result of long-range cell division in the vascular cambium. Most xylem cells have a thickened secondary cell wall, representing a large amount of biomass storage. Therefore, regulation of cell division in the vascular cambium and differentiation into secondary xylem is important for biomass production. Cell division is regulated by cell cycle regulators. In this study, we confirm that cell cycle regulators influence cell division in the vascular cambium in tobacco. We produced transgenic tobacco that expresses Arabidopsis thaliana cyclin D2;1 (AtcycD2;1) and AtE2Fa-DPa under the control of the CaMV35S promoter. Each gene is a positive regulator of the cell cycle, and is known to influence the transition from G1 phase to S phase. AtcycD2;1-overexpressing tobacco had more secondary xylem cells when compared with control plants. In order to evaluate cell division activity in the vascular cambium, we prepared a Populus trichocarpa cycB1;1 (PtcycB1;1) promoter containing a destruction box motif for ubiquitination and a β-glucuronidase-encoding gene (PtcycB1;1pro:GUS). In transgenic tobacco containing PtcycB1;1pro:GUS, GUS staining was specifically observed in meristem tissues, such as the root apical meristem and vascular cambium. In addition, mitosis-monitoring plants containing AtcycD2;1 had stronger GUS staining in the cambium when compared with control plants. Our results indicated that overexpression of AtcycD enhances cell division in the vascular cambium and increases secondary xylem differentiation in tobacco. We succeeded in inducing cell proliferation of cambium and enlargement of secondary xylem region by AtcycD overexpression. We also evaluated mitotic activity in cambium using cyclin-GUS fusion protein from poplar.