Cambium Reactivation Is Closely Related to the Cell-Cycle Gene Configuration in Larix kaempferi.

Dormancy release and reactivation in temperate trees are mainly controlled by temperature and are affected by age, but the underlying molecular mechanisms are still unclear. In this study, we explored the effects of low temperatures in winter and warm temperatures in spring on dormancy release and reactivation in Larix kaempferi. Further, we established the relationships between cell-cycle genes and cambium cell division. The results showed that chilling accelerated L. kaempferi bud break overall, and the longer the duration of chilling is, the shorter the bud break time is. After dormancy release, warm temperatures induced cell-cycle gene expression; when the configuration value of the cell-cycle genes reached 4.97, the cambium cells divided and L. kaempferi reactivated. This study helps to predict the impact of climate change on wood production and provides technical support for seedling cultivation in greenhouses.

Studying plant vascular development using single-cell approaches.

Vascular cells form a highly complex and heterogeneous tissue. Its composition, function, shape, and arrangement vary with the developmental stage and between organs and species. Understanding the transcriptional regulation underpinning this complexity thus requires a high-resolution technique that is capable of capturing rapid events during vascular cell formation. Single-cell and single-nucleus RNA sequencing (sc/snRNA-seq) approaches provide powerful tools to extract transcriptional information from these lowly abundant and dynamically changing cell types, which allows the reconstruction of developmental trajectories. Here, we summarize and reflect on recent studies using single-cell transcriptomics to study vascular cell types and discuss current and future implementations of sc/snRNA-seq approaches in the field of vascular development.

Network Analysis of Metabolome and Transcriptome Revealed Regulation of Different Nitrogen Concentrations on Hybrid Poplar Cambium Development.

Secondary development is a key biological characteristic of woody plants and the basis of wood formation. Exogenous nitrogen can affect the secondary growth of poplar, and some regulatory mechanisms have been found in the secondary xylem. However, the effect of nitrogen on cambium has not been reported. Herein, we investigated the effects of different nitrogen concentrations on cambium development using combined transcriptome and metabolome analysis. The results show that, compared with 1 mM NH4NO3 (M), the layers of hybrid poplar cambium cells decreased under the 0.15 mM NH4NO3 (L) and 0.3 mM NH4NO3 (LM) treatments. However, there was no difference in the layers of hybrid poplar cambium cells under the 3 mM NH4NO3 (HM) and 5 mM NH4NO3 (H) treatments. Totals of 2365, 824, 649 and 398 DEGs were identified in the M versus (vs.) L, M vs. LM, M vs. HM and M vs. H groups, respectively. Expression profile analysis of the DEGs showed that exogenous nitrogen affected the gene expression involved in plant hormone signal transduction, phenylpropanoid biosynthesis, the starch and sucrose metabolism pathway and the ubiquitin-mediated proteolysis pathway. In M vs. L, M vs. LM, M vs. HM and M vs. H, differential metabolites were enriched in flavonoids, lignans, coumarins and saccharides. The combined analysis of the transcriptome and metabolome showed that some genes and metabolites in plant hormone signal transduction, phenylpropanoid biosynthesis and starch and sucrose metabolism pathways may be involved in nitrogen regulation in cambium development, whose functions need to be verified. In this study, from the point of view that nitrogen influences cambium development to regulate wood formation, the network analysis of the transcriptome and metabolomics of cambium under different nitrogen supply levels was studied for the first time, revealing the potential regulatory and metabolic mechanisms involved in this process and providing new insights into the effects of nitrogen on wood development.

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 evolution of ontogenetic "decision-making" in the wood of a clade of tropical plants.

Greater diversity in functional morphology should be associated with the evolution of greater ontogenetic diversity, an expectation difficult to test in most long-lived wild organisms. In the cells derived from the wood meristem (vascular cambium), plants provide extraordinary systems for reconstructing ontogenies in often long-lived organisms. The vascular cambium produces files of cells from the stem center to the periphery, with each cambial derivative "deciding" which of four cell types it differentiates into. Wood cell files remain in place, allowing tracing of the ontogenetic "decisions" taken throughout the life of a stem. We compared cell files from the Pedilanthus clade (genus Euphorbia), which span a range of growth forms from small trees and shrubs of tropical habitats to desert succulents. Using language theory, we represented wood cell types as "letters" and combinations of cell types in cell files as "words," allowing us to measure the diversity of decisions based on word frequency matrices. We also used information content metrics to compare levels of predictability in "decision-making." Our analyses identified a wider array of developmental decisions in woody trees as compared to succulent shrubs, illustrating ways that woody plants provide unparalleled systems for studying the evolution of ontogeny in long-lived, non-model species.

Genes expression profiles in vascular cambium of Eucalyptus urophylla × Eucalyptus grandis at different ages.

BACKGROUND: Wood is a secondary xylem generated by vascular cambium. Vascular cambium activities mainly include cambium proliferation and vascular tissue formation through secondary growth, thereby producing new secondary phloem inward and secondary xylem outward and leading to continuous tree thickening and wood formation. Wood formation is a complex biological process, which is strictly regulated by multiple genes. Therefore, molecular level research on the vascular cambium of different tree ages can lead to the identification of both key and related genes involved in wood formation and further explain the molecular regulation mechanism of wood formation. RESULTS: In the present study, RNA-Seq and Pac-Bio Iso-Seq were used for profiling gene expression changes in Eucalyptus urophylla × Eucalyptus grandis (E. urograndis) vascular cambium at four different ages. A total of 59,770 non-redundant transcripts and 1892 differentially expressed genes (DEGs) were identified. The expression trends of the DEGs related to cell division and differentiation, cell wall biosynthesis, phytohormone, and transcription factors were analyzed. The DEGs encoding expansin, kinesin, cycline, PAL, GRP9, KNOX, C2C2-dof, REV, etc., were highly expressed in E. urograndis at three years old, leading to positive effects on growth and development. Moreover, some gene family members, such as NAC, MYB, HD-ZIP III, RPK, and RAP, play different regulatory roles in wood formation because of their sophisticated transcriptional network and function redundantly. CONCLUSIONS: These candidate genes are a potential resource to further study wood formation, especially in fast-growing and adaptable eucalyptus. The results may also serve as a basis for further research to unravel the molecular mechanism underlying wood formation.

Plasmodesmata callose binding protein 2 contributes to the regulation of cambium/phloem formation and auxin response during the tissue reunion process in incised Arabidopsis stem.

Plants are exposed to a variety of biotic and abiotic stresses, including wounding at the stem. The healing process (tissue reunion) begins immediately after stem wounding. The plant hormone auxin plays an important role during tissue reunion. In decapitated stems, auxin transport from the shoot apex is reduced and tissue reunion does not occur but is restored by application of indole-3-acetic acid (IAA). In this study, we found that plasmodesmata callose binding protein 2 (PDCB2) affects the expansion of the cambium/phloem region via changes in auxin response during the process of tissue reunion. PDCB2 was expressed in the cortex and endodermis on the incised side of stems 1-3 days after incision. PDCB2-knockout plants showed reduced callose deposition at plasmodesmata and DR5::GUS activity in the endodermis/cortex in the upper region of the incision accompanied by an increase in size of the cambium/phloem region during tissue reunion. In addition, PIN(PIN-FORMED)3, which is involved in lateral auxin transport, was induced by auxin in the cambium/phloem and endodermis/cortex in the upper part of the incision in wild type, but its expression of PIN3 was decreased in pdcb2 mutant. Our results suggest that PDCB2 contributes to the regulation of cambium/phloem development via auxin response.

Inducible, Tissue-Specific Gene Expression in Arabidopsis Using GR-LhG4-Mediated Trans-Activation.

Inducible, tissue-specific gene expression is a potent tool to study gene regulatory networks as it allows spatially and temporally controlled genetic perturbations. To this end, we generated a toolkit that covers many cell types in the three main meristems: the root apical meristem, the shoot apical meristem, and the vascular cambium. The system is based on an extensive set of driver lines expressing a synthetic transcription factor under cell type-specific promoters. Induction leads to nuclear translocation of the transcription factor and expression of response elements under control of a cognate synthetic promoter. In addition, a fluorescent reporter incorporated in driver lines allows to monitor induction. All previously generated driver lines are available from the Nottingham Arabidopsis Stock Center. This protocol describes how users can create their own constructs compatible with the existing set of lines and as well as induction and imaging procedures.

Function and Characteristic Analysis of Candidate PEAR Proteins in Populus yunnanensis.

PEAR proteins are a type of plant-specific DNA binding with one finger (Dof) transcription factors that play a key role in the regulation of plant growth, especially during phloem cell growth and seed germination in Arabidopsis. However, the identification, characteristics and function of PEAR proteins, particularly in woody plants, need to be further studied. In the present study, 43 candidate PEAR proteins harboring the conserved Zf-Dof domain were obtained in Populus yunnanensis. Based on phylogenetic and structural analysis, 10 representative PEAR candidates were selected, belonging to different phylogenetic groups. The functions of PEAR proteins in the stress response, signal transduction, and growth regulation of stem cambium and roots undergoing vigorous cell division in Arabidopsis were revealed based on their expression patterns as characterized by qRT-PCR analysis, in accordance with the results of cis-element analysis. In vitro experiments showed that the interaction of transcription factor (E2F) and cyclin indirectly reflects the growth regulation function of PEAR through light signaling and cell-cycle regulation. Therefore, our results provide new insight into the identity of PEAR proteins and their function in stress resistance and vigorous cell division regulation of tissues in P. yunnanensis, which may serve as a basis for further investigation of the functions and characteristics of PEAR proteins in other plants.

Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem.

BACKGROUND: Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution. RESULTS: We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay. CONCLUSIONS: This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Mosaic assembly of regulatory programs for vascular cambial growth: a view from the Early Devonian.

Evidence for secondary growth extends into the Early Devonian, 407 million years ago, raising questions about tempo and mode of origination of this key developmental feature. To address such questions, we analyze anatomy in the four oldest fossil plants with well-characterized woody tissues; one of these represents a new genus, described here formally. The new fossil is documented using the cellulose acetate peel technique and associated methods. We use the paradigm of structural fingerprints to identify developmental components of cambial growth based on fossil anatomy. We integrate developmental inferences within a theoretical framework of modular regulation of secondary growth. The fossils possess structural fingerprints consistent with four different combinations of regulatory mechanisms (modules) acting in cambial growth, representing four distinct modes of secondary growth. The different modes of secondary growth demonstrate that cambial growth is an assemblage of regulatory modules whose deployment followed a mosaic pattern across woody plants, which may represent ancestors of younger lineages that exhibit woody growth. The diverse modes of wood development occupy a wide morphospace in the anatomy of wood in the Early Devonian, suggesting that the origins of secondary growth and of its modular components pre-date this interval.

Combining single-cell RNA sequencing with spatial transcriptome analysis reveals dynamic molecular maps of cambium differentiation in the primary and secondary growth of trees.

Primary and secondary growth of the tree stem are responsible for corresponding increases in trunk height and diameter. However, our molecular understanding of the biological processes that underlie these two types of growth is incomplete. In this study, we used single-cell RNA sequencing and spatial transcriptome sequencing to reveal the transcriptional landscapes of primary and secondary growth tissues in the Populus stem. Comparison between the cell atlas and differentiation trajectory of primary and secondary growth revealed different regulatory networks involved in cell differentiation from cambium to xylem precursors and phloem precursors. These regulatory networks may be controlled by auxin accumulation and distribution. Analysis of cell differentiation trajectories suggested that vessel and fiber development followed a sequential pattern of progressive transcriptional regulation. This research provides new insights into the processes of cell identity and differentiation that occur throughout primary and secondary growth of tree stems, increasing our understanding of the cellular differentiation dynamics that occur during stem growth in trees.

Ethylene controls cambium stem cell activity via promoting local auxin biosynthesis.

The vascular cambium is the main secondary meristem in plants that produces secondary phloem (outside) and xylem (inside) on opposing sides of the cambium. The phytohormone ethylene has been implicated in vascular cambium activity, but the regulatory network underlying ethylene-mediated cambial activity remains to be elucidated. Here, we found that PETAL MOVEMENT-RELATED PROTEIN1 (RhPMP1), an ethylene-inducible HOMEODOMAIN-LEUCINE ZIPPER I transcription factor in woody plant rose (Rosa hybrida), regulates local auxin biosynthesis and auxin transport to maintain cambial activity. Knockdown of RhPMP1 resulted in smaller midveins and reduced auxin content, while RhPMP1 overexpression resulted in larger midveins and increased auxin levels compared with the wild-type plants. Furthermore, we revealed that Indole-3-pyruvate monooxygenase YUCCA 10 (RhYUC10) and Auxin transporter-like protein 2 (RhAUX2), encoding an auxin biosynthetic enzyme and an auxin influx carrier, respectively, are direct downstream targets of RhPMP1. In summary, our results suggest that ethylene promotes an auxin maximum in the cambium adjacent to the xylem to maintain cambial activity.

From procambium patterning to cambium activation and maintenance in the Arabidopsis root.

In addition to primary growth, which elongates the plant body, many plant species also undergo secondary growth to thicken their body. During primary vascular development, a subset of the vascular cells, called procambium and pericycle, remain undifferentiated to later gain vascular cambium and cork cambium identity, respectively. These two cambia are the lateral meristems providing secondary growth. The vascular cambium produces secondary xylem and phloem, which give plants mechanical support and transport capacity. Cork cambium produces a protective layer called cork. In this review, we focus on recent advances in understanding the formation of procambium and its gradual maturation to active cambium in the Arabidopsis thaliana root.

The LBD11-ROS feedback regulatory loop modulates vascular cambium proliferation and secondary growth in Arabidopsis.

Vascular cambium produces the phloem and xylem, vascular tissues that transport resources and provide mechanical support, making it an ideal target for crop improvement. However, much remains unknown about how vascular cambium proliferates. In this study, through pharmaceutical and genetic manipulation of reactive oxygen species (ROS) maxima, we demonstrate a direct link between levels of ROS and activity of LATERAL ORGAN BOUNDARIES DOMAIN 11 (LBD11) in maintaining vascular cambium activity. LBD11 activates the transcription of several key ROS metabolic genes, including PEROXIDASE 71 and RESPIRATORY BURST OXIDASE HOMOLOGS D and F, to generate local ROS maxima in cambium, which in turn enhance the proliferation of cambial cells. In a negative feedback mechanism, higher ROS levels then repress LBD11 expression and maintain the balance of cambial cell proliferation. Our findings thus reveal the role of a novel LBD11/ROS-dependent feedback regulatory system in maintaining vascular cambium-specific redox homeostasis and radial growth in plants.

Sucrose Signaling Contributes to the Maintenance of Vascular Cambium by Inhibiting Cell Differentiation.

Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named 'Vascular Cell Induction Culture System Using Arabidopsis Leaves' (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation, among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation in cambial cells. Physiological and genetic analyses suggested that sucrose might function through the BRI1-EMS-SUPPRESSOR1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

Complex wound response mechanisms and phellogen evolution - insights from Early Devonian euphyllophytes.

We analyze the oldest fossil occurrences of wound-response periderm to characterize the development of wound responses in early tracheophytes. The origin of periderm production by a cambium (phellogen), an innovation with key roles in protection of inner plant tissues, is poorly explored; understanding periderm development in early tracheophytes can illuminate key aspects of this process. Anatomy of wound-response tissues is characterized in serial sections in a new Early Devonian (Emsian; c. 400 Ma) euphyllophyte from Quebec (Canada) - Nebuloxyla mikmaqiana sp. nov. - and compared to previously described euphyllophyte periderm from the same fossil locality to reconstruct periderm development. Characterizing development in these oldest periderm occurrences allows us to propose a model for the development of wound-response periderm in early tracheophytes: by phellogen activity that is poorly coordinated laterally but bifacial, producing secondary tissues initially outwardly and subsequently inwardly. The earliest occurrences of wound periderm pre-date the oldest known periderm produced systemically as a regular ontogenetic stage (canonical periderm), suggesting that periderm evolved initially as a wound-response mechanism. We hypothesize that canonical periderm evolved by exaptation of this wound sealing mechanism, whose deployment was triggered by tangential tensional stresses induced in the superficial tissues by vascular cambial growth from within.

Gibberellins promote polar auxin transport to regulate stem cell fate decisions in cambium.

Vascular cambium contains bifacial stem cells, which produce secondary xylem to one side and secondary phloem to the other. However, how these fate decisions are regulated is unknown. Here we show that the positioning of an auxin signalling maximum within the cambium determines the fate of stem cell daughters. The position is modulated by gibberellin-regulated, PIN1-dependent polar auxin transport. Gibberellin treatment broadens auxin maximum from the xylem side of the cambium towards the phloem. As a result, xylem-side stem cell daughter preferentially differentiates into xylem, while phloem-side daughter retains stem cell identity. Occasionally, this broadening leads to direct specification of both daughters as xylem, and consequently, adjacent phloem-identity cell reverts to being stem cell. Conversely, reduced gibberellin levels favour specification of phloem-side stem cell daughter as phloem. Together, our data provide a mechanism by which gibberellin regulates the ratio of xylem and phloem production.