Vascular cambium stem cells: past, present and future.

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

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.

Screening genome-editing knockouts reveals the receptor-like kinase ASX role in regulations of secondary xylem development in Populus.

In trees, secondary xylem development is essential for the growth of perennial stem increments. Many signals regulate the process of development, but our knowledge of the molecular components involved in signal transduction is still limited. In this study, we identified Attenuation of Secondary Xylem (ASX) knockouts by screening genome-editing knockouts of xylem-expressed receptor-like kinases (RLKs) in Populus. The ASX role in secondary xylem development in Populus was discovered using biochemical, cellular, and genomic analyses. The ASX knockout plants had abnormal secondary stem growth but had little effect on shoot apical primary growth. ASX and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)2/4 were co-precipitated in developing xylem. Through their interaction, ASX is phosphorylated by SERK. Transcriptome analysis of developing xylem revealed that ASX deficiency inhibited the transcriptional activity of genes involved in xylem differentiation and secondary cell wall formation. By forming a complex, ASX and SERK may function as a signaling module for signal transduction required in the regulation of secondary xylem development in trees. This study shows that ASX, which encodes a RLKs, is required for secondary xylem development and sheds light on regulatory signals found in tree stem secondary growth.

RsCLE22a regulates taproot growth through an auxin signaling-related pathway in radish (Raphanus sativus L.).

CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides are a class of small molecules involved in plant growth and development. Although radish (Raphanus sativus) is an important root vegetable crop worldwide, the functions of CLE peptides in its taproot formation remain elusive. Here, a total of 48 RsCLE genes were identified from the radish genome. RNA in situ hybridization showed that RsCLE22a gene was highly expressed in the vascular cambium. Overexpression of RsCLE22a inhibited root growth by impairing stem cell proliferation in Arabidopsis, and radish plants with exogenous supplementation of RsCLE22 peptide (CLE22p) showed a similar phenotype. The vascular cambial activity was increased in RsCLE22a-silenced plants. Transcriptome analysis revealed that CLE22p altered the expression of several genes involved in meristem development and hormone signal transduction in radish. Immunolocalization results showed that CLE22p increased auxin accumulation in vascular cambium. Yeast one-hybrid and dual-luciferase assays showed that the WUSCHEL-RELATED HOMEOBOX 4 (RsWOX4) binds to RsCLE22a promoter and activates its transcription. The expression level of RsWOX4 was related to vascular cambial activity and was regulated by auxin. Furthermore, a RsCLE22a-RsWOX4 module is proposed to regulate taproot vascular cambium activity through an auxin signaling-related pathway in radish. These findings provide novel insights into the regulation of root growth in a horticultural crop.

Water lily (Nymphaea thermarum) genome reveals variable genomic signatures of ancient vascular cambium losses.

For more than 225 million y, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of angiosperms ∼140 million y ago (MYA), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium, the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem, which are produced by apical meristems and retained in nearly all seed plants. Here, we sequenced and assembled a draft genome of the water lily Nymphaea thermarum, an emerging system for the study of early flowering plant evolution, and compared it to genomes from other cambium-bearing and cambium-less lineages (e.g., monocots and Nelumbo). This revealed lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA, which influences cambial activity in other angiosperms. We also found the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent loss of vascular cambium reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent events of trait loss. Our results shed light on the evolution of herbaceousness-one of the key biological innovations associated with the earliest phases of angiosperm evolution.

The BOP-type co-transcriptional regulator NODULE ROOT1 promotes stem secondary growth of the tropical Cannabaceae tree Parasponia andersonii.

Tree stems undergo a massive secondary growth in which secondary xylem and phloem tissues arise from the vascular cambium. Vascular cambium activity is driven by endogenous developmental signalling cues and environmental stimuli. Current knowledge regarding the genetic regulation of cambium activity and secondary growth is still far from complete. The tropical Cannabaceae tree Parasponia andersonii is a non-legume research model of nitrogen-fixing root nodulation. Parasponia andersonii can be transformed efficiently, making it amenable for CRISPR-Cas9-mediated reverse genetics. We considered whether P. andersonii also could be used as a complementary research system to investigate tree-related traits, including secondary growth. We established a developmental map of stem secondary growth in P. andersonii plantlets. Subsequently, we showed that the expression of the co-transcriptional regulator PanNODULE ROOT1 (PanNOOT1) is essential for controlling this process. PanNOOT1 is orthologous to Arabidopsis thaliana BLADE-ON-PETIOLE1 (AtBOP1) and AtBOP2, which are involved in the meristem-to-organ-boundary maintenance. Moreover, in species forming nitrogen-fixing root nodules, NOOT1 is known to function as a key nodule identity gene. Parasponia andersonii CRISPR-Cas9 loss-of-function Pannoot1 mutants are altered in the development of the xylem and phloem tissues without apparent disturbance of the cambium organization and size. Transcriptomic analysis showed that the expression of key secondary growth-related genes is significantly down-regulated in Pannoot1 mutants. This allows us to conclude that PanNOOT1 positively contributes to the regulation of stem secondary growth. Our work also demonstrates that P. andersonii can serve as a tree research system.

Vascular function of the T3/modern clade WUSCHEL-Related HOMEOBOX transcription factor genes predate apical meristem-maintenance function.

BACKGROUND: Plants have the lifelong ability to generate new organs due to the persistent functioning of stem cells. In seed plants, groups of stem cells are housed in the shoot apical meristem (SAM), root apical meristem (RAM), and vascular cambium (VC). In ferns, a single shoot stem cell, the apical cell, is located in the SAM, whereas each root initiates from a single shoot-derived root initial. WUSCHEL-RELATED HOMEOBOX (WOX) family transcription factors play important roles to maintain stem-cell identity. WOX genes are grouped phylogenetically into three clades. The T3WOX/modern clade has expanded greatly in angiosperms, with members functioning in multiple meristems and complex developmental programs. The model fern Ceratopteris richardii has only one well-supported T3WOX/modern WOX gene, CrWUL. Its orthologs in Arabidopsis, AtWUS, AtWOX5, and AtWOX4, function in the SAM, RAM, and VC, respectively. Identifying the function of CrWUL will provide insights on the progenitor function and the diversification of the modern WOX genes in seed plants. RESULTS: To investigate the role of CrWUL in the fern, we examined the expression and function of CrWUL and found it expresses during early root development and in vasculature but not in the SAM. Knockdown of CrWUL by RNAi produced plants with fewer roots and fewer phloem cells. When expressed in Arabidopsis cambium, CrWUL was able to complement AtWOX4 function in an atwox4 mutant, suggesting that the WOX function in VC is conserved between ferns and angiosperms. Additionally, the proposed progenitor of T3WOX genes from Selaginella kraussiana is expressed in the vasculature but not in the shoot apical meristem. In contrast to the sporophyte, the expression of CrWUL in the gametophyte exhibits a more general expression pattern and when knocked down, offered little discernable phenotypes. CONCLUSIONS: The results presented here support the occurrence of co-option of the T3WOX/modern clade gene from the gametophyte to function in vasculature and root development in the sporophyte. The function in vasculature is likely to have existed in the progenitor of lycophyte T3WOX/modern clade genes and this function predates its SAM function found in many seed plants.

Ontogenesis of Resin Ducts and Secretory Process in Protium spruceanum (Burseraceae) Stems.

The objective of this work was to characterize the ontogenesis of Protium spruceanum secretory ducts, to evaluate the effects of seasonality on that process, and to characterize the chemical nature of the resin. Morphometric, anatomical, micromorphometric, histochemical, and ultrastructural evaluations of shoot apexes and chemical analyses of the resin were performed. The ducts of schizolysigenous origin are distributed in the primary and secondary phloem. The subsecretory tissue is meristematic and can restore the secretory epithelium. Secretory epithelial cells have wall thickening resembling that of the Casparian strip that regulates secretion reflux. The main resin compounds are pentacyclic triterpenoids, α- and ß-amyrins, and α- and ß-amyrenones, which are reported here for the first time for this species. The presence of electron-dense and electron-opaque structures, in the secretory epithelial cells, are compatible with the triterpenes and mucilage identified in the resin. Rising temperatures, rainfall, and increasing day length induce the formation of ducts in the vascular cambium throughout Spring/Summer. The abundant production of resin rich in pentacyclic triterpenes indicates the potential use of the species for medicinal and cosmetic purposes. The understanding that secretory processes are concentrated during the Spring/Summer seasons will contribute to the definition of resin extraction management strategies.

Revisiting the anatomy of the monocot cambium, a novel meristem.

MAIN CONCLUSION: The monocot cambium is semi-storied, and its cells do not undergo rearrangement. The monocot cambium is a lateral meristem responsible for secondary growth in some monocotyledons of Asparagales. It is an unusual meristem, not homologous with the vascular cambia of gymnosperms and non-monocotyledonous angiosperms. Owing to the limited information available on the characteristics of this meristem, the aim of this study was to survey the structure of the monocot cambium in order to clarify the similarities and dissimilarities of this lateral meristem to the vascular cambium of trees. Using the serial sectioning analysis, we have studied the monocot cambium of three species of arborescent monocotyledons, i.e., Quiver Tree Aloe dichotoma, Dragon Tree Dracaena draco, and Joshua Tree Yucca brevifolia, native to different parts of the world. Data showed that in contrast to the vascular cambium, the monocot cambium is composed of a single type of short initials that vary in shape, and in tangential view display a semi-storied pattern. Furthermore, the cells of the monocot cambium do not undergo rearrangement. The criteria used in identifying monocot cambium initial cell are also discussed.

Brassinosteroid-BZR1/2-WAT1 module determines the high level of auxin signalling in vascular cambium during wood formation.

The tight regulation of local auxin homeostasis and signalling maxima in xylem precursor cells specifies the organising activity of the vascular cambium and consequently promotes xylem differentiation and wood formation. However, the molecular mechanisms underlying the local auxin signalling maxima in the vascular cambium are largely unknown. Here, we reveal that brassinosteroid (BR)-activated WALLS ARE THIN1 (WAT1) facilitates wood formation by enhancing local auxin signalling in the vascular cambium in Solanum lycopersicum. Growth defects and low auxin signalling readouts in the BR-deficient tomato cultivar, Micro-Tom, were associated with a novel recessive allele, Slwat1-copi, created by the insertion of a retrotransposon in the last exon of the SlWAT1 locus. Molecular and genetic studies by generating the gain-of-function and loss-of-function tomato mutants revealed that SlWAT1 is a critical regulator for fine tuning local auxin homeostasis and signalling outputs in vascular cambium to facilitate secondary growth. Finally, we discovered that BR-regulated SlBZR1/2 directly activated downstream auxin responses by SlWAT1 upregulation in xylem precursor cells to facilitate xylem differentiation and subsequent wood formation. Our data suggest that the BR-SlBZR1/2-WAT1 signalling network contributes to the high level of auxin signalling in the vascular cambium for secondary growth.

A xylem-produced peptide PtrCLE20 inhibits vascular cambium activity in Populus.

In trees, lateral growth of the stem occurs through cell divisions in the vascular cambium. Vascular cambium activity is regulated by endogenous developmental programmes and environmental cues. However, the underlying mechanisms that regulate cambium activity are largely unknown. Genomic, biochemical and genetic approaches were used here to elucidate the role of PtrCLE20, a CLAVATA3 (CLV3)/embryo surrounding region (ESR)-related peptide gene, in the regulation of lateral growth in Populus. Fifty-two peptides encoded by CLE genes were identified in the genome of Populus trichocarpa. Among them PtrCLE20 transcripts were detected in developing xylem while the PtrCLE20 peptide was mainly localized in vascular cambium cells. PtrCLE20 acted in repressing vascular cambium activity indicated by that upregulation of PtrCLE20 resulted in fewer layers of vascular cambium cells with repressed expression of the genes related to cell dividing activity. PtrCLE20 peptide also showed a repression effect on the root growth of Populus and Arabidopsis, likely through inhibiting meristematic cell dividing activity. Together, the results suggest that PtrCLE20 peptide, produced from developing xylem cells, plays a role in regulating lateral growth by repression of cambium activity in trees.

Mosaic modularity: an updated perspective and research agenda for the evolution of vascular cambial growth.

Secondary growth from a vascular cambium, present today only in seed plants and isoetalean lycophytes, has a 400-million-yr evolutionary history that involves considerably broader taxonomic diversity, most of it hidden in the fossil record. Approaching vascular cambial growth as a complex developmental process, we review data from living plants and fossils that reveal diverse modes of secondary growth. These are consistent with a modular nature of secondary growth, when considered as a tracheophyte-wide structural feature. This modular perspective identifies putative constituent developmental modules of cambial growth, for which we review developmental anatomy and regulation. Based on these data, we propose a hypothesis that explains the sources of diversity of secondary growth, considered across the entire tracheophyte clade, and opens up new avenues for exploring the origin of secondary growth. In this hypothesis, various modes of secondary growth reflect a mosaic pattern of expression of different developmental-regulatory modules among different lineages. We outline an approach that queries three information systems (living seed plants, living seed-free plants, and fossils) and integrates data on developmental regulation, anatomy, gene evolution and phylogeny to test the mosaic modularity hypothesis and its implications, and to inform efforts aimed at understanding the evolution of secondary growth.

Wood structure and function change with maturity: Age of the vascular cambium is associated with xylem changes in current-year growth.

Xylem vessel structure changes as trees grow and mature. Age- and development-related changes in xylem structure are likely related to changes in hydraulic function. We examined whether hydraulic function, including hydraulic conductivity and vulnerability to water-stress-induced xylem embolism, changed over the course of cambial development in the stems of 17 tree species. We compared current-year growth of young (1-4 years), intermediate (2-7 years), and older (3-10 years) stems occurring in series along branches. Diffuse and ring porous species were examined, but nearly all species produced only diffuse porous xylem in the distal branches that were examined irrespective of their mature xylem porosity type. Vessel diameter and length increased with cambial age. Xylem became both more conductive and more cavitation resistant with cambial age. Ring porous species had longer and wider vessels and xylem that had higher conductivity and was more vulnerable to cavitation; however, these differences between porosity types were not present in young stem samples. Understanding plant hydraulic function and architecture requires the sampling of multiple-aged tissues because plants may vary considerably in their xylem structural and functional traits throughout the plant body, even over relatively short distances and closely aged tissues.

A temporal shift in resource allocation facilitates flowering before leaf out and spring vessel maturation in precocious species.

PREMISE OF THE STUDY: New growth in the spring requires resource mobilization in the vascular system at a time when xylem and phloem function are often reduced in seasonally cold climates. As a result, the timing of leaf out and/or flowering could depend on when the vascular system resumes normal function in the spring. This study investigated whether flowering time is influenced by vascular phenology in plants that flower precociously before they have leaves. METHODS: Flower, leaf, and vascular phenology were monitored in pairs of precocious and non-precocious congeners. Differences in resource allocation were quantified by measuring bud dry mass and water content throughout the year, floral hydration was modelled, and a girdling treatment completed on branches in the field. KEY RESULTS: Precocious flowering species invested more in floral buds the year before flowering than did their non-precocious congeners, thus mobilizing less water in the spring, which allowed flowering before new vessel maturation. CONCLUSIONS: A shift in the timing of resource allocation in precocious flowering plants allowed them to flower before the production of mature vessels and minimized the significance of seasonal changes in vascular function to their flowering phenology. The low investment required to complete floral development in the spring when the plant vascular system is often compromised could explain why flowers can emerge before leaf out.

Polar auxin transport is implicated in vessel differentiation and spatial patterning during secondary growth in Populus.

PREMISE OF THE STUDY: Dimensions and spatial distribution of vessels are critically important features of woody stems, allowing for adaptation to different environments through their effects on hydraulic efficiency and vulnerability to embolism. Although our understanding of vessel development is poor, basipetal transport of auxin through the cambial zone may play an important role. METHODS: Stems of Populus tremula ×alba were treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) in a longitudinal strip along the length of the lower stem. Vessel lumen diameter, circularity, and length; xylem growth; tension wood area; and hydraulic conductivity before and after a high pressure flush were determined on both NPA-treated and control plants. KEY RESULTS: NPA-treated stems formed aberrant vessels that were short, small in diameter, highly clustered, and angular in cross section, whereas xylem formed on the untreated side of the stem contained typical vessels that were similar to those of controls. NPA-treated stems had reduced specific conductivity relative to controls, but this difference was eliminated by the high-pressure flush. The control treatment (lanolin + dimethyl sulfoxide) reduced xylem growth and increased tension wood formation, but never produced the aberrant vessel patterning seen in NPA-treated stems. CONCLUSIONS: These results are consistent with a model of vessel development in which basipetal polar auxin transport through the xylem-side cambial derivatives is required for proper expansion and patterning of vessels and demonstrate that reduced auxin transport can produce stems with altered stem hydraulic properties.

WUSCHEL-RELATED HOMEOBOX4 (WOX4)-like genes regulate cambial cell division activity and secondary growth in Populus trees.

Plant secondary growth derives from the meristematic activity of the vascular cambium. In Arabidopsis thaliana, cell divisions in the cambium are regulated by the transcription factor WOX4, a key target of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-RELATED 41 (CLE41) signaling pathway. However, function of the WOX4-like genes in plants that are dependent on a much more prolific secondary growth, such as trees, remains unclear. Here, we investigate the role of WOX4 and CLE41 homologs for stem secondary growth in Populus trees. In Populus, PttWOX4 genes are specifically expressed in the cambial region during vegetative growth, but not after growth cessation and during dormancy, possibly involving a regulation by auxin. In PttWOX4a/b RNAi trees, primary growth was not affected whereas the width of the vascular cambium was severely reduced and secondary growth was greatly diminished. Our data show that in Populus trees, PttWOX4 genes control cell division activity in the vascular cambium, and hence growth in stem girth. This activity involves the positive regulation of PttWOX4a/b through PttCLE41-related genes. Finally, expression profiling suggests that the CLE41 signaling pathway is an evolutionarily conserved program for the regulation of vascular cambium activity between angiosperm and gymnosperm tree species.

An ecophysiological and developmental perspective on variation in vessel diameter.

Variation in xylem vessel diameter is one of the most important parameters when evaluating plant water relations. This review provides a synthesis of the ecophysiological implications of variation in lumen diameter together with a summary of our current understanding of vessel development and its endogenous regulation. We analyzed inter-specific variation of the mean hydraulic vessel diameter (Dv ) across biomes, intra-specific variation of Dv under natural and controlled conditions, and intra-plant variation. We found that the Dv measured in young branches tends to stay below 30 µm in regions experiencing winter frost, whereas it is highly variable in the tropical rainforest. Within a plant, the widest vessels are often found in the trunk and in large roots; smaller diameters have been reported for leaves and small lateral roots. Dv varies in response to environmental factors and is not only a function of plant size. Despite the wealth of data on vessel diameter variation, the regulation of diameter is poorly understood. Polar auxin transport through the vascular cambium is a key regulator linking foliar and xylem development. Limited evidence suggests that auxin transport is also a determinant of vessel diameter. The role of auxin in cell expansion and in establishing longitudinal continuity during secondary growth deserve further study.

Molecular control of wood formation in trees.

Wood (also termed secondary xylem) is the most abundant biomass produced by plants, and is one of the most important sinks for atmospheric carbon dioxide. The development of wood begins with the differentiation of the lateral meristem, vascular cambium, into secondary xylem mother cells followed by cell expansion, secondary wall deposition, programmed cell death, and finally heartwood formation. Significant progress has been made in the past decade in uncovering the molecular players involved in various developmental stages of wood formation in tree species. Hormonal signalling has been shown to play critical roles in vascular cambium cell proliferation and a peptide-receptor-transcription factor regulatory mechanism similar to that controlling the activity of apical meristems is proposed to be involved in the maintenance of vascular cambium activity. It has been demonstrated that the differentiation of vascular cambium into xylem mother cells is regulated by plant hormones and HD-ZIP III transcription factors, and the coordinated activation of secondary wall biosynthesis genes during wood formation is mediated by a transcription network encompassing secondary wall NAC and MYB master switches and their downstream transcription factors. Most genes encoding the biosynthesis enzymes for wood components (cellulose, xylan, glucomannan, and lignin) have been identified in poplar and a number of them have been functionally characterized. With the availability of genome sequences of tree species from both gymnosperms and angiosperms, and the identification of a suite of wood-associated genes, it is expected that our understanding of the molecular control of wood formation in trees will be greatly accelerated.