Gibberellin promotes cambium reestablishment during secondary vascular tissue regeneration after girdling in an auxin-dependent manner in Populus.

Secondary vascular tissue (SVT) development and regeneration are regulated by phytohormones. In this study, we used an in vitro SVT regeneration system to demonstrate that gibberellin (GA) treatment significantly promotes auxin-induced cambium reestablishment. Altering GA content by overexpressing or knocking down ent-kaurene synthase (KS) affected secondary growth and SVT regeneration in poplar. The poplar DELLA gene GIBBERELLIC ACID INSENSITIVE (PtoGAI) is expressed in a specific pattern during secondary growth and cambium regeneration after girdling. Overexpression of PtoGAI disrupted poplar growth and inhibited cambium regeneration, and the inhibition of cambium regeneration could be partially restored by GA application. Further analysis of the PtaDR5:GUS transgenic plants, the localization of PIN-FORMED 1 (PIN1) and the expression of auxin-related genes found that an additional GA treatment could enhance the auxin response as well as the expression of PIN1, which mediates auxin transport during SVT regeneration. Taken together, these findings suggest that GA promotes cambium regeneration by stimulating auxin signal transduction.

The PtoTCP20-miR396d-PtoGRF15 module regulates secondary vascular development in Populus.

Secondary vascular development is a key biological characteristic of woody plants and the basis of wood formation. Our understanding of gene expression regulation and dynamic changes in microRNAs (miRNAs) during secondary vascular development is still limited. Here we present an integrated analysis of the miRNA and mRNA transcriptome of six phase-specific tissues-the shoot apex, procambium, primary vascular tissue, cambium, secondary phloem, and secondary xylem-in Populus tomentosa. Several novel regulatory modules, including the PtoTCP20-miR396d-PtoGRF15 module, were identified during secondary vascular development in Populus. A series of biochemical and molecular experiments confirmed that PtoTCP20 activated transcription of the miR396d precursor gene and that miR396d targeted PtoGRF15 to downregulate its expression. Plants overexpressing miR396d (35S:miR396d) showed enhanced secondary growth and increased xylem production. Conversely, during the transition from primary to secondary vascular development, plants with downregulated PtoTCP20expression (PtoTCP20-SRDX), downregulated miR396 expression (35S:STTM396), and PtoGRF15 overexpression (35S:PtoGRF15) showed delayed secondary growth. Novel regulatory modules were identified by integrated analysis of the miRNA and mRNA transcriptome, and the regulatory role of the PtoTCP20-miR396d-PtoGRF15 signaling cascade in secondary vascular development was validated in Populus, providing information to support improvements in forest cultivation and wood properties.

MiR395c Regulates Secondary Xylem Development Through Sulfate Metabolism in Poplar.

Secondary xylem development requires the coordination of multiple regulatory factors, including plant hormones, transcription factors, and microRNAs (miRNAs). MiR395 is an important regulator involved in sulfate metabolism, but its function in plant development is unclear. This study investigated the functions of miR395c in the secondary xylem development in Populus alba × P. glandulosa. MiR395c was highly expressed in the shoot apex and secondary xylem. The overexpression of miR395c resulted in an increase in both secondary xylem width and vessel dimension, as well as a decrease in the thickness of the secondary cell wall of the xylem fiber. Further analysis showed that miR395c inhibited biosynthesis of sulfate metabolic products by targeting ATPS genes, which led to the reduction of Abscisic acid (ABA) synthesis and down-regulation of MYB46 expression. Our results indicate that miR395c regulates the secondary xylem development process via sulfate metabolism in Populus.

Non-Coding RNA Analyses of Seasonal Cambium Activity in Populus tomentosa.

Non-coding RNA, known as long non-coding RNA (lncRNA), circular RNA (circRNA) and microRNA (miRNA), are taking part in the multiple developmental processes in plants. However, the roles of which played during the cambium activity periodicity of woody plants remain poorly understood. Here, lncRNA/circRNA-miRNA-mRNA regulatory networks of the cambium activity periodicity in Populus tomentosa was constructed, combined with morphologic observation and transcriptome profiling. Light microscopy and Periodic Acid Schiff (PAS) staining revealed that cell walls were much thicker and number of cell layers was increased during the active-dormant stage, accompanied by abundant change of polysaccharides. The novel lncRNAs and circRNAs were investigated, and we found that 2037 lncRNAs and 299 circRNAs were differentially expression during the vascular cambium period, respectively. Moreover, 1046 genes were identified as a target gene of 2037 novel lncRNAs, and 89 of which were the miRNA precursors or targets. By aligning miRNA precursors to the 7655 lncRNAs, 21 lncRNAs were identified as precursors tof 19 known miRNAs. Furthermore, the target mRNA of lncRNA/circRNA-miRNA network mainly participated in phytohormone, cell wall alteration and chlorophyll metabolism were analyzed by GO enrichment and KEGG pathway. Especially, circRNA33 and circRNA190 taking part in the phytohormone signal pathway were down-regulated during the active-dormant transition. Xyloglucan endotransglucosylase/hydrolase protein 24-like and UDP-glycosyltransferase 85A1 involved in the cell wall modification were the targets of lncRNA MSTRG.11198.1 and MSTRG.1050.1. Notably, circRNA103 and MSTRG.10851.1 regulate the cambium periodicity may interact with the miR482. These results give a new light into activity-dormancy regulation, associated with transcriptional dynamics and non-coding RNA networks of potential targets identification.

A label-free, fast and high-specificity technique for plant cell wall imaging and composition analysis.

BACKGROUND: New cell wall imaging tools permit direct visualization of the molecular architecture of cell walls and provide detailed chemical information on wall polymers, which will aid efforts to use these polymers in multiple applications; however, detailed imaging and quantification of the native composition and architecture in the cell wall remains challenging. RESULTS: Here, we describe a label-free imaging technology, coherent Raman scattering (CRS) microscopy, including coherent anti-Stokes Raman scattering (CARS) microscopy and stimulated Raman scattering (SRS) microscopy, which can be used to visualize the major structures and chemical composition of plant cell walls. We outline the major steps of the procedure, including sample preparation, setting the mapping parameters, analysis of spectral data, and image generation. Applying this rapid approach will help researchers understand the highly heterogeneous structures and organization of plant cell walls. CONCLUSIONS: This method can potentially be incorporated into label-free microanalyses of plant cell wall chemical composition based on the in situ vibrations of molecules.

The Tetracentron genome provides insight into the early evolution of eudicots and the formation of vessel elements.

BACKGROUND: Tetracentron sinense is an endemic and endangered deciduous tree. It belongs to the Trochodendrales, one of four early diverging lineages of eudicots known for having vesselless secondary wood. Sequencing and resequencing of the T. sinense genome will help us understand eudicot evolution, the genetic basis of tracheary element development, and the genetic diversity of this relict species. RESULTS: Here, we report a chromosome-scale assembly of the T. sinense genome. We assemble the 1.07 Gb genome sequence into 24 chromosomes and annotate 32,690 protein-coding genes. Phylogenomic analyses verify that the Trochodendrales and core eudicots are sister lineages and showed that two whole-genome duplications occurred in the Trochodendrales approximately 82 and 59 million years ago. Synteny analyses suggest that the γ event, resulting in paleohexaploidy, may have only happened in core eudicots. Interestingly, we find that vessel elements are present in T. sinense, which has two orthologs of AtVND7, the master regulator of vessel formation. T. sinense also has several key genes regulated by or regulating TsVND7.2 and their regulatory relationship resembles that in Arabidopsis thaliana. Resequencing and population genomics reveals high levels of genetic diversity of T. sinense and identifies four refugia in China. CONCLUSIONS: The T. sinense genome provides a unique reference for inferring the early evolution of eudicots and the mechanisms underlying vessel element formation. Population genomics analysis of T. sinense reveals its genetic diversity and geographic structure with implications for conservation.

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.

Active DNA demethylation regulates tracheary element differentiation in Arabidopsis.

DNA demethylation is important for the erasure of DNA methylation. The role of DNA demethylation in plant development remains poorly understood. Here, we found extensive DNA demethylation in the CHH context around pericentromeric regions and DNA demethylation in the CG, CHG, and CHH contexts at discrete genomic regions during ectopic xylem tracheary element (TE) differentiation. While loss of pericentromeric methylation occurs passively, DNA demethylation at a subset of regions relies on active DNA demethylation initiated by DNA glycosylases ROS1, DML2, and DML3. The ros1 and rdd mutations impair ectopic TE differentiation and xylem development in the young roots of Arabidopsis seedlings. Active DNA demethylation targets and regulates many genes for TE differentiation. The defect of xylem development in rdd is proposed to be caused by dysregulation of multiple genes. Our study identifies a role of active DNA demethylation in vascular development and reveals an epigenetic mechanism for TE differentiation.

Caudicles in vandoid orchids: A carotenoid-based soft material with unique properties.

130 years ago, Darwin observed that caudicles in vandoid orchids possess considerable elasticity and further hypothesized that their elasticity serves to improve pollination efficiency. However, there has been no study that seeks to either quantitatively backup Darwin's hypothesis or characterize this natural material for practical use. Here we show that vandoid caudicles are a novel kind of soft material with extremely high extensibility (1190%), low modulus (160 kPa) and density lower than that of water. Vandoid caudicles contain carotenoids that attach to basal polymers through noncovalent interactions. Inspired by the chemical structure of caudicles, we synthesize calcium-alginate/polyacrylamide hydrogels supplemented with carotenoids and demonstrate that their strength as well as stretchability are enhanced two-fold. Our findings identify a new carotenoid-based material system with unique properties that approach the current boundaries of the Ashby chart, demonstrating potential application of carotenoids as biocompatible reinforcing agent for hydrogels. STATEMENT OF SIGNIFICANCE: We have investigated the microstructure, mechanical properties and chemical components of vandoid caudicles as an elastic plant tissue and demonstrated a bio-inspired design that can enhance the elasticity of hydrogels. Existing research on vandoid caudicles are very few and mainly focus on their phylogenetics and developmental process, and the potential application of caudicles in the field of material sciences remains unexplored. Our results showed that caudicles are more stretchable than most natural and synthetic elastomers and have a modulus similar to hydrogels. Carotenoids, an important chemical component of caudicles, can be used as supplements to hydrogels to improve their strength and stretchability.

Differential regulation of auxin and cytokinin during the secondary vascular tissue regeneration in Populus trees.

Tissue regeneration upon wounding in plants highlights the developmental plasticity of plants. Previous studies have described the morphological and molecular changes of secondary vascular tissue (SVT) regeneration after large-scale bark girdling in trees. However, how phytohormones regulate SVT regeneration is still unknown. Here, we established a novel in vitro SVT regeneration system in the hybrid aspen (Populus tremula × Populus tremuloides) clone T89 to bypass the limitation of using field-grown trees. The effects of phytohormones on SVT regeneration were investigated by applying exogenous hormones and utilizing various transgenic trees. Vascular tissue-specific markers and hormonal response factors were also examined during SVT regeneration. Using this in vitro regeneration system, we demonstrated that auxin and cytokinin differentially regulate phloem and cambium regeneration. Whereas auxin is sufficient to induce regeneration of phloem prior to continuous cambium restoration, cytokinin only promotes the formation of new phloem, not cambium. The positive role of cytokinin on phloem regeneration was further confirmed in cytokinin overexpression trees. Analysis of a DR5 reporter transgenic line further suggested that cytokinin blocks the re-establishment of auxin gradients, which is required for the cambium formation. Investigation on the auxin and cytokinin signalling genes indicated these two hormones interact to regulate SVT regeneration. Taken together, the in vitro SVT regeneration system allows us to make use of various molecular and genetic tools to investigate SVT regeneration. Our results confirmed that complementary auxin and cytokinin domains are required for phloem and cambium reconstruction.

Interaction of methyl viologen-induced chloroplast and mitochondrial signalling in Arabidopsis.

Reactive oxygen species (ROS) are key signalling intermediates in plant metabolism, defence, and stress adaptation. In plants, both the chloroplast and mitochondria are centres of metabolic control and ROS production, which coordinate stress responses in other cell compartments. The herbicide and experimental tool, methyl viologen (MV) induces ROS generation in the chloroplast under illumination, but is also toxic in non-photosynthetic organisms. We used MV to probe plant ROS signalling in compartments other than the chloroplast. Taking a genetic approach in the model plant Arabidopsis (Arabidopsis thaliana), we used natural variation, QTL mapping, and mutant studies with MV in the light, but also under dark conditions, when the chloroplast electron transport is inactive. These studies revealed a light-independent MV-induced ROS-signalling pathway, suggesting mitochondrial involvement. Mitochondrial Mn SUPEROXIDE DISMUTASE was required for ROS-tolerance and the effect of MV was enhanced by exogenous sugar, providing further evidence for the role of mitochondria. Mutant and hormone feeding assays revealed roles for stress hormones in organellar ROS-responses. The radical-induced cell death1 mutant, which is tolerant to MV-induced ROS and exhibits altered mitochondrial signalling, was used to probe interactions between organelles. Our studies suggest that mitochondria are involved in the response to ROS induced by MV in plants.

Dynamic Changes of Pectin Epitopes in Cell Walls during the Development of the Procambium-Cambium Continuum in Poplar.

The change of pectin epitopes during procambium-cambium continuum development was investigated by immunolocalization in poplar. The monoclonal antibody JIM5 labels homogalacturonan (HGA) with a low degree of esterification, and the monoclonal antibody JIM7 labels HGA with a high degree of methyl-esterification. Arabinan, rather than galactan, and HGA with low degree of esterification were located in the cell walls of procambial, while HGA with a low degree of esterification was located in the tangential walls, and galactan was located in both the tangential and radial walls of procambial, yet nearly no arabinan was located in the tangential walls of the cambial cells. The changes in pectin distribution took place when periclinal divisions appeared within a procambial trace. The distribution difference of pectin epitopes was also present in procambium-cambium derivatives. The arabinan existed in all cell walls of primary xylem, but was absent from the tangential walls of secondary xylem cells. The galactan existed only in mature primary phloem. Furthermore, 19 pectin methylesterases (PMEs) genes were identified by RNA sequencing, six genes presented highly differentially and were supposed to be involved in the cell wall esterification process. The results provide direct evidence of the dynamic changes of pectin epitopes during the development of the procambium-cambium continuum in poplar.

Proteomic analysis provides insights into changes in the central metabolism of the cambium during dormancy release in poplar.

Seasonal cycling of growth and dormancy is an important feature for the woody plants growing in temperate zone, and dormancy is an effective strategy for surviving the winter stress. But the mechanisms of dormancy maintenance and its release are still not clear, especially little information is available with regard to the changes of proteome during the process. A better understanding in the function of proteins and their related metabolic pathways would expand our knowledge of the mechanisms of dormancy maintenance and its release in trees. In this study, we employed the isobaric tags for relative and absolute quantification (iTRAQ) approach with LC-MS/MS analysis to investigate the protein profile changes during dormancy release in poplar. In addition, the change of lipid, total insoluble carbohydrates and starch granules in the cambium was investigated by histochemical methods. A total of 3789 proteins were identified in poplar cambial tissues, 1996 of them were significantly altered during the dormancy release. Most of the altered proteins involved in signaling, phytohormone, energy metabolism, stress and secondary metabolism by functional analysis. Our data shows that the lipid metabolism proteins changed significantly both in the release stage of eco- and endodormancy, while the changes of carbohydrate metabolism proteins were mainly in endo-dormancy release stage. Moreover, histochemical results were consistent with the proteomic data. Our results reveal diverse stage-specific metabolism changes during the dormancy-release process induced by chilling in poplar, which provided new information regarding the regulation mechanisms of dormancy maintenance and its release in trees.

MiR169 and its target PagHAP2-6 regulated by ABA are involved in poplar cambium dormancy.

Dormancy is an effective strategy for perennial plants in temperate zones to survive the winter stress. MicroRNAs (miRNAs) have been well known as important regulators for various biological processes. In this study, we checked the expression of miR169 members in the cambium zone during dormancy and active growth in poplar and found that they had distinct expression patterns. We identified and characterized a dormancy-specific target gene of miR169, PagHAP2-6. 5' RACE assays confirmed the direct cleavage of PagHAP2-6 mRNA by miR169. The yeast functional complementation analysis showed that PagHAP2-6 was a homolog of Heme Activator Protein2 (HAP2)/Nuclear factor Y-A (NF-YA) transcription factor in poplar. qRT-PCR analysis indicated that PagHAP2-6 was highly expressed in the dormant stage, which was converse to the expression pattern of pag-miR169a, n, and r. In addition, the transcription of PagHAP2-6 was induced by exogenous abscisic acid (ABA), and both over-expression of PagHAP2-6 in Arabidopsis and transient co-expression assays in Nicotiana benthamiana indicated that PagHAP2-6 could increase the resistance to exogenous ABA. Taken together, the results suggested that miR169 and its target PagHAP2-6 regulated by ABA were involved in poplar cambium dormancy, which provided new insights into the regulatory mechanisms of tree dormancy-active growth transition.

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.

Deep sequencing on a genome-wide scale reveals diverse stage-specific microRNAs in cambium during dormancy-release induced by chilling in poplar.

BACKGROUND: Trees in temperate zones show periodicity by alternating active and dormant states to adapt to environmental conditions. Although phytohormones and transcriptional regulation were found to be involved in growth cessation and dormancy transition, little is known about the mechanisms of the dormancy-active growth transition, especially dormancy maintenance and release. Small RNAs are a group of short non-coding RNAs regulating gene expressions at the post-transcriptional level during plant development and the responses to environmental stress. No report on the expression profiling of small RNAs in the cambial meristem during the dormancy-active growth transition has been reported to date. RESULTS: Three small RNA libraries from the cambium of poplar, representing endodormancy induced by short day conditions, ecodormancy induced by chilling and active growth induced by long day conditions, respectively, were generated and sequenced by Illumina high-throughput sequencing technology. This yielded 123 known microRNAs (miRNAs) with significant expression changes, which included developmental-, phytohormone- and stress-related miRNAs. Interestingly, miR156 and miR172 showed opposite expression patterns in the cambial dormancy-active growth transition. Additionally, miR160, which is involved in the auxin signaling pathway, was expressed specifically during endodormancy release by chilling. Furthermore, 275 novel miRNAs expressed in the cambial zone were identified, and 34 of them had high detection frequencies and unique expression patterns. Finally, the target genes of these novel miRNAs were predicted and some were validated experimentally by 5'RACE. CONCLUSIONS: Our results provided a comprehensive analysis of small RNAs in the cambial meristem during dormancy-release at the genome-wide level and novel evidence of miRNAs involved in the regulation of this biological process.

Tissue regeneration after bark girdling: an ideal research tool to investigate plant vascular development and regeneration.

Regeneration is a common strategy for plants to survive the intrinsic and extrinsic challenges they face through their life cycle, and it may occur upon wounding. Bark girdling is applied to improve fruit production or harvest bark as medicinal material. When tree bark is removed, the cambium and phloem will be peeled off. After a small strip of bark is removed from trees, newly formed periderm and wound cambium develop from the callus on the surface of the trunk, and new phloem is subsequently derived from the wound cambium. However, after large-scale girdling, the newly formed sieve elements (SEs) appear earlier than the regenerated cambium, and both of them derive from differentiating xylem cells rather than from callus. This secondary vascular tissue regeneration mainly involves three key stages: callus formation and xylem cell dedifferentiation; SEs appearance and wound cambium formation. The new bark is formed within 1 month in poplar, Eucommia; thus, it provides high temporal resolution of regenerated tissues at different stages. In this review, we will illustrate the morphology, gene expression and phytohormone regulation of vascular tissue regeneration after large-scale girdling in trees, and also discuss the potential utilization of the bark girdling system in studies of plant vascular development and tissue regeneration.

The regulation of cambial activity in Chinese fir (Cunninghamia lanceolata) involves extensive transcriptome remodeling.

Chinese fir (Cunninghamia lanceolata), a commercially important tree for the timber and pulp industry, is widely distributed in southern China and northern Vietnam, but its large and complex genome has hindered the development of genomic resources. Few efforts have focused on analysis of the modulation of transcriptional networks in vascular cambium during the transition from active growth to dormancy in conifers. Here, we used Illumina sequencing to analyze the global transcriptome alterations at the different stages of vascular cambium development in Chinese fir. By analyzing dynamic changes in the transcriptome of vascular cambium based on our RNA sequencing (RNA-Seq) data at the dormant, reactivating and active stages, many potentially interesting genes were identified that encoded putative regulators of cambial activity, cell division, cell expansion and cell wall biosynthesis and modification. In particular, the genes involved in transcriptional regulation and hormone signaling were highlighted to reveal their biological importance in the cambium development and wood formation. Our results reveal the dynamics of transcriptional networks and identify potential key components in the regulation of vascular cambium development in Chinese fir, which will contribute to the in-depth study of cambial differentiation and wood-forming candidate genes in conifers.

The plant vascular system: evolution, development and functions.

The emergence of the tracheophyte-based vascular system of land plants had major impacts on the evolution of terrestrial biology, in general, through its role in facilitating the development of plants with increased stature, photosynthetic output, and ability to colonize a greatly expanded range of environmental habitats. Recently, considerable progress has been made in terms of our understanding of the developmental and physiological programs involved in the formation and function of the plant vascular system. In this review, we first examine the evolutionary events that gave rise to the tracheophytes, followed by analysis of the genetic and hormonal networks that cooperate to orchestrate vascular development in the gymnosperms and angiosperms. The two essential functions performed by the vascular system, namely the delivery of resources (water, essential mineral nutrients, sugars and amino acids) to the various plant organs and provision of mechanical support are next discussed. Here, we focus on critical questions relating to structural and physiological properties controlling the delivery of material through the xylem and phloem. Recent discoveries into the role of the vascular system as an effective long-distance communication system are next assessed in terms of the coordination of developmental, physiological and defense-related processes, at the whole-plant level. A concerted effort has been made to integrate all these new findings into a comprehensive picture of the state-of-the-art in the area of plant vascular biology. Finally, areas important for future research are highlighted in terms of their likely contribution both to basic knowledge and applications to primary industry.

The proteasome is responsible for caspase-3-like activity during xylem development.

Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase-3, which displays Asp-Glu-Val-Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase-3 is absent in plants, caspase-3-like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase-3-like activity exists in xylem cell death. In this study, we showed that caspase-3-like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase-3-like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase-3-like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven ß subunits encoded by 12 genes. Pharmacological assays showed that Ac-DEVD-CHO, a caspase-3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto-lactacystin ß-lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6-induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase-3-like activity and is involved in xylem development.