Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development.

Leaves of flowering plants are characterized by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyze the spatiotemporal localization of a subset of auxin-related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification and how major and minor vein ranks develop with distinct identities. The localization of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is prepatterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

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.

C2H2 zinc finger protein PagIDD15A regulates secondary wall thickening and lignin biosynthesis in poplar.

Wood production is largely determined by the activity of cambial cell proliferation, and the secondary cell wall (SCW) thickening of xylem cells determines the wood property. In this study, we identified an INDETERMINATE DOMAIN (IDD) type C2H2 zinc finger transcription factor PagIDD15A as a regulator of wood formation in Populus alba × Populus glandulosa. Downregulation of PagIDD15A expression by RNA interference (RNAi) inhibited xylem development and xylem cell secondary wall thickening. RNA-seq analysis showed that PagPAL1, PagCCR2 and PagCCoAOMT1 were downregulated in the differentiating xylem of the PagIDD15A-RNAi transgenic plants, showing that PagIDD15A may regulate SCW biosynthesis through inhibiting lignin biosynthesis. The downregulation of PagVND6-B2, PagMYB10 and PagMYC4 and upregulation of PagWRKY12 in the differentiating xylem of RNAi transgenic plants suggest that PagIDD15A may also regulate these transcription factor (TF) genes to affect SCW thickening. RT-qPCR analysis in the phloem-cambium of RNAi transgenic demonstrates that PagIDD15A may regulate the expression of the genes associated with cell proliferation, including, PagSHR (SHORTROOT), PagSCR (SCARECROW), PagCYCD3;1 (CYCLIN D3;1) and PagSMR4 (SIAMESE-RELATED4), to affect the cambial activity. This study provides the knowledge of the IDD-type C2H2 zinc finger protein in regulating wood formation.

MicroRNA257 promotes secondary growth in hybrid poplar.

Populus, a significant fast-growing tree species with global afforestation and energy potential, holds considerable economic value. The abundant production of secondary xylem by trees, which serves as a vital resource for industrial purposes and human sustenance, necessitates the orchestration of various regulatory mechanisms, encompassing transcriptional regulators and microRNAs (miRNAs). Nevertheless, the investigation of microRNA-mediated regulation of poplar secondary growth remains limited. In this study, we successfully isolated a novel microRNA (Pag-miR257) from 84 K poplar and subsequently integrated it into the 35 S overexpression vector. The overexpression of Pag-miR257 resulted in notable increases in plant height, stem diameter, and fresh weight. Additionally, the overexpression of Pag-miR257 demonstrated a significant enhancement in net photosynthetic rate. The findings from the examination of cell wall autofluorescence indicated a substantial increase in both xylem area and the number of vessels in poplar plants overexpressing Pag-miR257. Furthermore, the cell wall of the Pag-miR257 overexpressing plants exhibited thickening as observed through transmission electron microscopy. Moreover, the Fourier Transforms Infrared (FTIR) analysis and phloroglucinol-HCl staining revealed an elevation in lignin content in Pag-miR257 overexpressing poplar plants. The findings of this study suggest that microRNA257 may play a role in the control of secondary growth in poplar stems, thereby potentially enhancing the development of wood engineering techniques for improved material and energy production.

A Solanum lycopersicum polyamine oxidase contributes to the control of plant growth, xylem differentiation, and drought stress tolerance.

Polyamines are involved in several plant physiological processes. In Arabidopsis thaliana, five FAD-dependent polyamine oxidases (AtPAO1 to AtPAO5) contribute to polyamine homeostasis. AtPAO5 catalyzes the back-conversion of thermospermine (T-Spm) to spermidine and plays a role in plant development, xylem differentiation, and abiotic stress tolerance. In the present study, to verify whether T-Spm metabolism can be exploited as a new route to improve stress tolerance in crops and to investigate the underlying mechanisms, tomato (Solanum lycopersicum) AtPAO5 homologs were identified (SlPAO2, SlPAO3, and SlPAO4) and CRISPR/Cas9-mediated loss-of-function slpao3 mutants were obtained. Morphological, molecular, and physiological analyses showed that slpao3 mutants display increased T-Spm levels and exhibit changes in growth parameters, number and size of xylem elements, and expression levels of auxin- and gibberellin-related genes compared to wild-type plants. The slpao3 mutants are also characterized by improved tolerance to drought stress, which can be attributed to a diminished xylem hydraulic conductivity that limits water loss, as well as to a reduced vulnerability to embolism. Altogether, this study evidences conservation, though with some significant variations, of the T-Spm-mediated regulatory mechanisms controlling plant growth and differentiation across different plant species and highlights the T-Spm role in improving stress tolerance while not constraining growth.

Merit of integrating in situ transcriptomics and anatomical information for cell annotation and lineage construction in single-cell analyses of Populus.

Cell type annotation and lineage construction are two of the most critical tasks conducted in the analyses of single-cell RNA sequencing (scRNA-seq). Four recent scRNA-seq studies of differentiating xylem propose four models on differentiating xylem development in Populus. The differences are mostly caused by the use of different strategies for cell type annotation and subsequent lineage interpretation. Here, we emphasize the necessity of using in situ transcriptomes and anatomical information to construct the most plausible xylem development model.

Cell size and xylem differentiation regulating genes from Salicornia europaea contribute to plant salt tolerance.

Cell wall is involved in plant growth and plays pivotal roles in plant adaptation to environmental stresses. Cell wall remodelling may be crucial to salt adaptation in the euhalophyte Salicornia europaea. However, the mechanism underlying this process is still unclear. Here, full-length transcriptome indicated cell wall-related genes were comprehensively regulated under salinity. The morphology and cell wall components in S. europaea shoot were largely modified under salinity. Through the weighted gene co-expression network analysis, SeXTH2 encoding xyloglucan endotransglucosylase/hydrolases, and two SeLACs encoding laccases were focused. Meanwhile, SeEXPB was focused according to expansin activity and the expression profiling. Function analysis in Arabidopsis validated the functions of these genes in enhancing salt tolerance. SeXTH2 and SeEXPB overexpression led to larger cells and leaves with hemicellulose and pectin content alteration. SeLAC1 and SeLAC2 overexpression led to more xylem vessels, increased secondary cell wall thickness and lignin content. Notably, SeXTH2 transgenic rice exhibited enhanced salt tolerance and higher grain yield. Altogether, these genes may function in the succulence and lignification process in S. europaea. This work throws light on the regulatory mechanism of cell wall remodelling in S. europaea under salinity and provides potential strategies for improving crop salt tolerance and yields.

ARGONAUTE10 controls cell fate specification and formative cell divisions in the Arabidopsis root.

A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity.

An AGO10:miR165/6 module regulates meristem activity and xylem development in the Arabidopsis root.

The RNA-silencing effector ARGONAUTE10 influences cell fate in plant shoot and floral meristems. ARGONAUTE10 also accumulates in the root apical meristem (RAM), yet its function(s) therein remain elusive. Here, we show that ARGONAUTE10 is expressed in the root cell initials where it controls overall RAM activity and length. ARGONAUTE10 is also expressed in the stele, where post-transcriptional regulation confines it to the root tip's pro-vascular region. There, variations in ARGONAUTE10 levels modulate metaxylem-vs-protoxylem specification. Both ARGONAUTE10 functions entail its selective, high-affinity binding to mobile miR165/166 transcribed in the neighboring endodermis. ARGONAUTE10-bound miR165/166 is degraded, likely via SMALL-RNA-DEGRADING-NUCLEASES1/2, thus reducing miR165/166 ability to silence, via ARGONAUTE1, the transcripts of cell fate-influencing transcription factors. These include PHABULOSA (PHB), which controls meristem activity in the initials and xylem differentiation in the pro-vasculature. During early germination, PHB transcription increases while dynamic, spatially-restricted transcriptional and post-transcriptional mechanisms reduce and confine ARGONAUTE10 accumulation to the provascular cells surrounding the newly-forming xylem axis. Adequate miR165/166 concentrations are thereby channeled along the ARGONAUTE10-deficient yet ARGONAUTE1-proficient axis. Consequently, inversely-correlated miR165/166 and PHB gradients form preferentially along the axis despite ubiquitous PHB transcription and widespread miR165/166 delivery inside the whole vascular cylinder.

PagEXPA1 combines with PagCDKB2;1 to regulate plant growth and the elongation of fibers in Populus alba × Populus glandulosa.

Expansins are important plant cell wall proteins. They can loosen and soften the cell walls and lead to wall extension and cell expansion. To investigate their role in wood formation and fiber elongation, the PagEXPA1 that highly expressed in cell differentiation and expansion tissues was cloned from 84K poplar (Populus alba × P. glandulosa). The subcellular localization showed that PagEXPA1 located in the cell wall and it was highly expressed in primary stems and young leaves. Compared with non-transgenic 84K poplar, overexpression of PagEXPA1 can promote plant-growth, lignification, and fiber cell elongation, while PagEXPA1 Cas9-editing mutant lines exhibited the opposite phenotype. Transcriptome analysis revealed that DEGs were mainly enriched in some important processes, which are associated with cell wall formation and cellulose synthesis. The protein interaction prediction and expression analysis showed that PagCDKB2:1 and PagEXPA1 might have an interaction relationship. The luciferase complementary assay and bimolecular fluorescence complementary assay validated that PagEXPA1 can combined with PagCDKB2;1. So they promoted the expansion of xylem vascular tissues and the development of poplar though participating in the regulation of cell division and differentiation by programming the cell-cycle. It provides good foundation for molecular breeding of fast-growing and high-quality poplar varieties.

Evaluation of novel promoters for vascular tissue-specific gene expression in Populus.

Due to the extended generation cycle of trees, the breeding process for forest trees tends to be time-consuming. Genetic engineering has emerged as a viable approach to expedite the genetic breeding of forest trees. However, current genetic engineering techniques employed in forest trees often utilize continuous expression promoters such as CaMV 35S, which may result in unintended consequences by introducing genes into non-target tissues. Therefore, it is imperative to develop specific promoters for forest trees to facilitate targeted and precise design and breeding. In this study, we utilized single-cell RNA-Seq data and co-expression network analysis during wood formation to identify three vascular tissue-specific genes in poplar, PP2-A10, PXY, and VNS07, which are expressed in the phloem, cambium/expanding xylem, and mature xylem, respectively. Subsequently, we cloned the promoters of these three genes from '84K' poplar and constructed them into a vector containing the eyGFPuv visual selection marker, along with the 35S mini enhancer to drive GUS gene expression. Transgenic poplars expressing the ProPagPP2-A10::GUS, ProPagPXY::GUS, and ProPagVNS07::GUS constructs were obtained. To further elucidate the tissue specificity of these promoters, we employed qPCR, histochemical staining, and GUS enzyme activity. Our findings not only establish a solid foundation for the future utilization of these promoters to precisely express of specific functional genes in stems but also provide a novel perspective for the modular breeding of forest trees.

PagPXYs improve drought tolerance by regulating reactive oxygen species homeostasis in the cambium of Populus alba × P. glandulosa.

PXY (Phloem intercalated with xylem) is a receptor kinase required for directional cell division during the development of plant vascular tissue. Drought stress usually affects plant stem cell division and differentiation thereby limiting plant growth. However, the role of PXY in cambial activities of woody plants under drought stress is unclear. In this study, we analyzed the biological functions of two PXY genes (PagPXYa and PagPXYb) in poplar growth and development and in response to drought stress in a hybrid poplar (Populus alba × P. glandulosa, '84K'). Expression analysis indicated that PagPXYs, similar to their orthologs PtrPXYs in Populus trichocarpa, are mainly expressed in the stem vascular system, and related to drought. Interestingly, overexpression of PagPXYa and PagPXYb in poplar did not have a significant impact on the growth status of transgenic plants under normal condition. However, when treated with 8 % PEG6000 or 100 mM H2O2, PagPXYa and PagPXYb overexpressing lines consistently exhibited more cambium cell layers, fewer xylem cell layers, and enhanced drought tolerance compared to the non-transgenic control '84K'. In addition, PagPXYs can alleviate the damage caused by H2O2 to the cambium under drought stress, thereby maintaining the cambial division activity of poplar under drought stress, indicating that PagPXYs play an important role in plant resistance to drought stress. This study provides a new insight for further research on the balance of growth and drought tolerance in forest trees.

Methylation of microRNA genes and its effect on secondary xylem development of stem in poplar.

MicroRNAs (miRNAs) and DNA methylation are both vital regulators of gene expression. DNA methylation can affect the transcription of miRNAs, just like coding genes, through methylating the CpG islands in the gene regions of miRNAs. Although previous studies have shown that DNA methylation and miRNAs can each be involved in the process of wood formation, the relationship between the two has been relatively little studied in plant wood formation. Studies have shown that the second internode (IN2) (from top to bottom) of 3-month-old poplar trees can represent the primary stage of poplar stem development and IN8 can represent the secondary stage. There were also significant differences in DNA methylation patterns and miRNA expression patterns obtained from PS and SS. In this study, we first interactively analyzed methylation and miRNA sequencing data to identify 43 differentially expressed miRNAs regulated by differential methylation from the primary stage and secondary stage, which were found to be involved in multiple biological processes related to wood formation by enrichment analysis. In addition, six miRNA/target gene modules were finally identified as potentially involved in secondary xylem development of poplar stems through degradome sequencing and functional analysis. In conclusion, this study provides important reference information on the mechanism of interaction between different regulatory pathways of wood formation.

Blue light photoreceptor cryptochrome 1 promotes wood formation and anthocyanin biosynthesis in Populus.

Blue light photoreceptor cryptochrome 1 (CRY1) in herbaceous plants plays crucial roles in various developmental processes, including cotyledon expansion, hypocotyl elongation and anthocyanin biosynthesis. However, the function of CRY1 in perennial trees is unclear. In this study, we identified two ortholog genes of CRY1 (PagCRY1a and PagCRY1b) from Populus, which displayed high sequence similarity to Arabidopsis CRY1. Overexpression of PagCRY1 substantially inhibited plant growth and promoted secondary xylem development in Populus, while CRISPR/Cas9-mediated knockout of PagCRY1 enhanced plant growth and delayed secondary xylem development. Moreover, overexpression of PagCRY1 dramatically increased anthocyanin accumulation. The further analysis supported that PagCRY1 functions specifically in response to blue light. Taken together, our results demonstrated that modulating the expression of blue light photoreceptor CRY1 ortholog gene in Populus could significantly influence plant biomass production and the process of wood formation, laying a foundation for further investigating the light-regulated tree growth.

Transcription factor PagMYB31 positively regulates cambium activity and negatively regulates xylem development in poplar.

Wood formation involves consecutive developmental steps, including cell division of vascular cambium, xylem cell expansion, secondary cell wall (SCW) deposition, and programmed cell death. In this study, we identified PagMYB31 as a coordinator regulating these processes in Populus alba × Populus glandulosa and built a PagMYB31-mediated transcriptional regulatory network. PagMYB31 mutation caused fewer layers of cambial cells, larger fusiform initials, ray initials, vessels, fiber and ray cells, and enhanced xylem cell SCW thickening, showing that PagMYB31 positively regulates cambial cell proliferation and negatively regulates xylem cell expansion and SCW biosynthesis. PagMYB31 repressed xylem cell expansion and SCW thickening through directly inhibiting wall-modifying enzyme genes and the transcription factor genes that activate the whole SCW biosynthetic program, respectively. In cambium, PagMYB31 could promote cambial activity through TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/PHLOEM INTERCALATED WITH XYLEM (PXY) signaling by directly regulating CLAVATA3/ESR-RELATED (CLE) genes, and it could also directly activate WUSCHEL HOMEOBOX RELATED4 (PagWOX4), forming a feedforward regulation. We also observed that PagMYB31 could either promote cell proliferation through the MYB31-MYB72-WOX4 module or inhibit cambial activity through the MYB31-MYB72-VASCULAR CAMBIUM-RELATED MADS2 (VCM2)/PIN-FORMED5 (PIN5) modules, suggesting its role in maintaining the homeostasis of vascular cambium. PagMYB31 could be a potential target to manipulate different developmental stages of wood formation.

Analyses of high spatial resolution datasets identify genes associated with multi-layered secondary cell wall thickening in Pinus bungeana.

BACKGROUND AND AIMS: Secondary cell wall (SCW) thickening is a major cellular developmental stage determining wood structure and properties. Although the molecular regulation of cell wall deposition during tracheary element differentiation has been well established in primary growth systems, less is known about the gene regulatory processes involved in the multi-layered SCW thickening of mature trees. METHODS: Using third-generation [long-read single-molecule real-time (SMRT)] and second-generation [short-read sequencing by synthesis (SBS)] sequencing methods, we established a Pinus bungeana transcriptome resource with comprehensive functional and structural annotation for the first time. Using these approaches, we generated high spatial resolution datasets for the vascular cambium, xylem expansion regions, early SCW thickening, late SCW thickening and mature xylem tissues of 71-year-old Pinus bungeana trees. KEY RESULTS: A total of 79 390 non-redundant transcripts, 31 808 long non-coding RNAs and 5147 transcription factors were annotated and quantified in different xylem tissues at all growth and differentiation stages. Furthermore, using this high spatial resolution dataset, we established a comprehensive transcriptomic profile and found that members of the NAC, WRKY, SUS, CESA and LAC gene families are major players in early SCW formation in tracheids, whereas members of the MYB and LBD transcription factor families are highly expressed during late SCW thickening. CONCLUSIONS: Our results provide new molecular insights into the regulation of multi-layered SCW thickening in conifers. The high spatial resolution datasets provided can serve as important gene resources for improving softwoods.

Transcription factor PbNAC71 regulates xylem and vessel development to control plant height.

Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, "Yunnan" quince (Cydonia oblonga Mill.) had a dwarfing effect on "Zaosu" pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising "Zaosu" (scion) grafted onto "Yunnan" quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 downregulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants.

Identification of MeC3HDZ1/MeCNA as a potential regulator of cassava storage root development.

The storage root (SR) of cassava is the main staple food in sub-Saharan Africa, where it feeds over 500 million people. However, little is known about the genetic and molecular regulation underlying its development. Unraveling such regulation would pave the way for biotechnology approaches aimed at enhancing cassava productivity. Anatomical studies indicate that SR development relies on the massive accumulation of xylem parenchyma, a cell-type derived from the vascular cambium. The C3HDZ family of transcription factors regulate cambial cells proliferation and xylem differentiation in Arabidopsis and other species. We thus aimed at identifying C3HDZ proteins in cassava and determining whether any of them shows preferential activity in the SR cambium and/or xylem. Using phylogeny and synteny studies, we identified eight C3HDZ proteins in cassava, namely MeCH3DZ1-8. We observed that MeC3HDZ1 is the MeC3HDZ gene displaying the highest expression in SR and that, within that organ, the gene also shows high expression in cambium and xylem. In-silico analyses revealed the existence of a number of potential C3HDZ targets displaying significant preferential expression in the SR. Subsequent Y1H analyses proved that MeC3HDZ1 can bind canonical C3HDZ binding sites, present in the promoters of these targets. Transactivation assays demonstrated that MeC3HDZ1 can regulate the expression of genes downstream of promoters harboring such binding sites, thereby demonstrating that MeC3HDZ1 has C3HDZ transcription factor activity. We conclude that MeC3HDZ1 may be a key factor for the regulation of storage root development in cassava, holding thus great promise for future biotechnology applications.

Trees need closure too: Wound-induced secondary vascular tissue regeneration.

Trees play a pivotal role in terrestrial ecosystems as well as being an important natural resource. These attributes are primarily associated with the capacity of trees to continuously produce woody tissue from the vascular cambium, a ring of stem cells located just beneath the bark. Long-lived trees are exposed to a myriad of biological and environmental stresses that may result in wounding, leading to a loss of bark and the underlying vascular cambium. This affects both wood formation and the quality of timber arising from the tree. In addition, the exposed wound site is a potential entry point for pathogens that cause disease. In response to wounding, trees have the capacity to regenerate lost or damaged tissues at this site. Investigating gene expression changes associated with different stages of wound healing reveals complex and dynamic changes in the activity of transcription factors, signalling pathways and hormone responses. In this review we summarise these data and discuss how they relate to our current understanding of vascular cambium formation and xylem differentiation during secondary growth. Based on this analysis, a model for wound healing that provides the conceptual foundations for future studies aimed at understanding this intriguing process is proposed.

Seasonal Developing Xylem Transcriptome Analysis of Pinus densiflora Unveils Novel Insights for Compression Wood Formation.

Wood is the most important renewable resource not only for numerous practical utilizations but also for mitigating the global climate crisis by sequestering atmospheric carbon dioxide. The compressed wood (CW) of gymnosperms, such as conifers, plays a pivotal role in determining the structure of the tree through the reorientation of stems displaced by environmental forces and is characterized by a high content of lignin. Despite extensive studies on many genes involved in wood formation, the molecular mechanisms underlying seasonal and, particularly, CW formation remain unclear. This study examined the seasonal dynamics of two wood tissue types in Pinus densiflora: CW and opposite wood (OW). RNA sequencing of developing xylem for two consecutive years revealed comprehensive transcriptome changes and unique differences in CW and OW across seasons. During growth periods, such as spring and summer, we identified 2255 transcripts with differential expression in CW, with an upregulation in lignin biosynthesis genes and significant downregulation in stress response genes. Notably, among the laccases critical for monolignol polymerization, PdeLAC17 was found to be specifically expressed in CW, suggesting its vital role in CW formation. PdeERF4, an ERF transcription factor preferentially expressed in CW, seems to regulate PdeLAC17 activity. This research provides an initial insight into the transcriptional regulation of seasonal CW development in P. densiflora, forming a foundation for future studies to enhance our comprehension of wood formation in gymnosperms.