Weight-induced radial growth in plant stems depends on PIN3.

How multiple growth programs coordinate during development is a fundamental question in biology. During plant stem development, radial growth is continuously adjusted in response to longitudinal-growth-derived weight increase to guarantee stability.1,2,3 Here, we demonstrate that weight-stimulated stem radial growth depends on the auxin efflux carrier PIN3, which, upon weight increase, expands its cellular localization from the lower to the lateral sides of xylem parenchyma, phloem, procambium, and starch sheath cells, imposing a radial auxin flux that results in radial growth. Using the protein synthesis inhibitor cycloheximide (CHX) or the fluorescent endocytic tracer FM4-64, we reveal that this expansion of the PIN3 cellular localization domain occurs because weight increase breaks the balance between PIN3 biosynthesis and removal, favoring PIN3 biosynthesis. Experimentation using brefeldin A (BFA) treatments or arg1 and arl2 mutants further supports this conclusion. Analyses of CRISPR-Cas9 lines for Populus PIN3 orthologs reveals that PIN3 dependence of weight-induced radial growth is conserved at least in these woody species. Altogether, our work sheds new light on how longitudinal and radial growth coordinate during stem development.

Differential warming at crown scale impacts walnut primary growth onset and secondary growth rate.

Trees are exposed to significant spatio-temporal thermal variations, which can induce intracrown discrepancies in the onset and dynamics of primary and secondary growth. In recent decades, an increase in late winter and early spring temperatures has been observed, potentially accelerating bud break, cambial activation and their potential coordination. Intracrown temperature heterogeneities could lead to asymmetric tree shapes unless there is a compensatory mechanism at the crown level. An original warming experiment was conducted on young Juglans regia trees in a greenhouse. From February to August, the average temperature difference during the day between warmed and control parts was 4°C. The warming treatment advanced the date of budbreak significantly, by up to 14 days. Warming did not alter secondary growth resumption but increased growth rates, leading to higher xylem cell production (twice as many) and to an increase in radial increment (+80% compared to control). Meristems resumptions were asynchronous without coordination in response to temperature. Buds on warmed branches began to swell two weeks prior to cambial division, which was one week earlier than on control branches. A difference in carbon and water remobilisation at the end of bud ecodormancy was noted under warming. Overall, our results argue for a lack of compensatory mechanisms at the crown scale, which may lead to significant changes in tree architecture in response to intra-crown temperature heterogeneities.

PagHAM4a-PagSCL21 and PagHAM4b-PagTCP20 modules positively regulate cambial activity and its differentiation into secondary xylem in poplar.

Stem secondary xylem produced by cambial division and differentiation is the main source of tree biomass. The secondary xylem formation involves a complex transcriptional regulatory network, however, the underlying mechanism is still being explored. Here, we report that PagHAM4a and PagHAM4b are positive regulators of cambial differentiation into secondary xylem in hybrid poplar (Populus alba × Populus glandulosa clone 84K). Overexpression of PagHAM4a and PagHAM4b enhanced cambial activity and increased the number of secondary xylem cells in the stems of poplar. By contrast, single or double mutations of PagHAM4a and PagHAM4b by CRISPR-Cas9 decreased cambial activity, leading to a significant reduction of secondary xylem. Neither overexpression nor mutation of the two genes affected the size of vessels and fibers in xylem. Both PagHAM4a- and PagHAM4b-regulated gene networks were mainly centered in the stage when cambium had just initiated secondary growth, but the molecular networks regulated by the two genes were distinct. Further analysis revealed that PagSCL21 and PagTCP20 are direct targets of PagHAM4a and PagHAM4b, respectively, and their overexpression also promoted cambial differentiation into secondary xylem. Taken together, we identified two novel key regulatory modules in poplar, PagHAM4a-PagSCL21 and PagHAM4b-PagTCP20, which provide new insights into the mechanism of secondary xylem formation in trees.

Exogenous gibberellin suppressed taproot secondary thickening by inhibiting the formation and maintenance of vascular cambium in radish (Raphanus sativus L.).

Introduction: The thickening of radish taproots is primarily determined by secondary growth driven by the vascular cambium and is a highly intricate process regulated by plant hormones, transcription factors, and many metabolic pathways. Gibberellin (GA), a plant hormone associated with cell elongation, is essential in secondary growth. However, the mechanism through which exogenous GA3 regulates secondary taproot growth in radishes remains unclear. Methods: Integrated morphological, anatomical, hormonal, and transcriptomic analyses of taproots in radishes treated with GA3 and its biosynthesis inhibitor paclobutrazol (PBZ) were performed to explore their effects on taproot secondary growth and key regulatory pathways. Results: GA3 significantly hindered taproot thickening by inhibiting the formation and maintenance of the vascular cambium, and PBZ promoted root development by increasing root length rather than root diameter. Transcriptome analysis revealed 2,014, 948, and 1,831 differentially expressed genes identified from the control vs. GA3, control vs. PBZ, and GA3 vs. PBZ comparisons, respectively. Kyoto Encyclopedia of Genes and Genome pathway enrichment analysis revealed that differentially expressed genes were primarily involved in the biosyntheses of secondary metabolites and metabolic pathways. GA3 significantly increased the levels of endogenous indole-acetic acid and the expression of auxin synthesis and signal transduction genes. Discussion: Exogenous GA3 significantly inhibited the expression of genes involved in the maintenance and differentiation of vascular cambium, including WOX14, ER/ERL1, and XCP2. Exogenous GA3 affects root thickening in radishes primarily by regulating hormone signal transduction pathways, vascular cambium activity, and substance and energy metabolisms. Our findings provide insights into the mechanisms underlying taproot thickening in radishes and provide a valuable gene database for future studies.

Experimental Induction of Extreme Indented Growth Rings (Hazel Wood) in Pinus halepensis Miller by Wide and Long Parallel Bark and Vascular Cambium Woundings.

Indented growth rings were found long ago to be experimentally induced in Pinus halepensis Miller by thin parallel axial scratching of the bark up to the vascular cambium with a sharp blade. Here, we show that when the bark and vascular cambium of P. halepensis are wounded by wide and long parallel axial wounds ("windows") rather than by thin scratches, the induced indented growth rings become dramatically more indented. All ten trees that were wounded by long parallel "windows" responded with very strong growth (especially in the first two years) that resulted in the formation of very conspicuous, extremely indented growth rings in the wood formed in between the long and wide woundings. This is true for both the trunks that were wounded all around their circumference and those that were wounded only in part of their circumference. We also suggest further lines of research.

A transcriptional repressor HVA regulates vascular bundle formation through auxin transport in Arabidopsis stem.

Vascular bundles transport water and photosynthate to all organs, and increased bundle number contributes to crop lodging resistance. However, the regulation of vascular bundle formation is poorly understood in the Arabidopsis stem. We report a novel semi-dominant mutant with high vascular activity, hva-d, showing increased vascular bundle number and enhanced cambium proliferation in the stem. The activation of a C2H2 zinc finger transcription factor, AT5G27880/HVA, is responsible for the hva-d phenotype. Genetic, biochemical, and fluorescent microscopic analyses were used to dissect the functions of HVA. HVA functions as a repressor and interacts with TOPLESS via the conserved Ethylene-responsive element binding factor-associated Amphiphilic Repression motif. In contrast to the HVA activation line, knockout of HVA function with a CRISPR-Cas9 approach or expression of HVA fused with an activation domain VP16 (HVA-VP16) resulted in fewer vascular bundles. Further, HVA directly regulates the expression of the auxin transport efflux facilitator PIN1, as a result affecting auxin accumulation. Genetics analysis demonstrated that PIN1 is epistatic to HVA in controlling bundle number. This research identifies HVA as a positive regulator of vascular initiation through negatively modulating auxin transport and sheds new light on the mechanism of bundle formation in the stem.

A molecular module connects abscisic acid with auxin signals to facilitate seasonal wood formation in Populus.

Perennial trees have a recurring annual cycle of wood formation in response to environmental fluctuations. However, the precise molecular mechanisms that regulate the seasonal formation of wood remain poorly understood. Our prior study indicates that VCM1 and VCM2 play a vital role in regulating the activity of the vascular cambium by controlling the auxin homoeostasis of the cambium zone in Populus. This study indicates that abscisic acid (ABA) affects the expression of VCM1 and VCM2, which display seasonal fluctuations in relation to photoperiod changes. ABA-responsive transcription factors AREB4 and AREB13, which are predominantly expressed in stem secondary vascular tissue, bind to VCM1 and VCM2 promoters to induce their expression. Seasonal changes in the photoperiod affect the ABA amount, which is linked to auxin-regulated cambium activity via the functions of VCM1 and VCM2. Thus, the study reveals that AREB4/AREB13-VCM1/VCM2-PIN5b acts as a molecular module connecting ABA and auxin signals to control vascular cambium activity in seasonal wood formation.

UPBEAT1-ROS-POD-PAL System under Different Xylogenesis Scenarios in Karelian Birch (Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti).

BACKGROUND: We studied UPBEAT1 (UPB1) which regulated superoxide radical / hydrogen peroxide ratio together with peroxidase (POD) activity and PAL genes expression under different ways of apical meristem development during the xylem structural elements' formation in unique woody plants B. pendula var. pendula with straight-grained wood and B. pendula var. carelica with figured wood. The differentiation process predominanced in straight-grained wood (B. pendula var. pendula) or proliferation - in the figured wood. The investigation was conducted in the radial row (cambial zone - differentiating xylem - mature xylem) during the active cambial growth period. OBJECTIVE: The study aimed to study the xylogenesis processes occurring in the 16-year-old straight-grained silver birch (Betula pendula Roth) and Karelian birch (Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti) with figured wood. METHODS: Hydrogen peroxide and superoxide radical contents and peroxidase activity were determined spectrophotometrically. Gene expression for PAL family genes and the UPBEAT1 gene was assessed using qRT-PCR. RESULTS: Principal component analysis has confirmed trees with straight-grained and figured wood to be different according to UPBEAT1-ROS-POD-PAL system functioning. CONCLUSION: The higher superoxide radical/hydrogen peroxide ratio in figured Karelian birch, along with UPBEAT1 transcription factor and PAL genes upregulation, distinguished it from straight-grained silver birch. This metabolic picture confirmed the shift of Karelian birch xylogenesis towards proliferation processes, accompanied by ROS and phenolic compounds' flow and POD activity.

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.

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The radial growth of trees plays a crucial role in determining forest carbon sequestration capacity. Understanding the growth dynamics of trees and their response to environmental factors is essential for predicting forest's carbon sink potential under future climate change. Coniferous forest trees are particularly sensitive to climate change, with growth dynamics responding rapidly to environmental shifts. We collected and analyzed data from 99 papers published between 1975 and 2023, and examined the effects of exogenous factors (such as temperature, water, and photoperiod) and endogenous factors (including tree age and species) on cambial activity and radial growth in conifers. We further explored the mechanisms underlying these effects. The results showed that climate warming had the potential to advance the onset while delayed the end of xylem differentiation stages in conifers in temperate and boreal regions. Water availability played a crucial role in regulating the timing of cambial phenology and wood formation by influencing water potential and cell turgor. Additionally, the photoperiod not only participated in regulating the start and end times of growth, but also influenced the timing of maximum growth rate occurrence. Future climate warming was expected to extend the growing season, leading to increase in growth of conifers in boreal regions and expanding forests to higher altitudes or latitudes. However, changes in precipitation patterns and increased evapotranspiration resulting from temperature increases might advance the end of growing season and reduce growth rate in arid areas. To gain a more comprehensive understanding of the relationship between radial growth and climatic factors, it is necessary to develop process-based models to elucidate the physiological mechanisms underlying wood formation and the response of trees to climatic factors.

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.

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.

Growth-regulating factor 15-mediated vascular cambium differentiation positively regulates wood formation in hybrid poplar (Populus alba × P. glandulosa).

Introduction: Hybrid poplars are industrial trees in China. An understanding of the molecular mechanism underlying wood formation in hybrid poplars is necessary for molecular breeding. Although the division and differentiation of vascular cambial cells is important for secondary growth and wood formation, the regulation of this process is largely unclear. Methods: In this study, mPagGRF15 OE and PagGRF15-SRDX transgenic poplars were generated to investigate the function of PagGRF15. RNA-seq and qRT-PCR were conducted to analyze genome-wide gene expression, while ChIP‒seq and ChIP-PCR were used to identified the downstream genes regulated by PagGRF15. Results and discussion: We report that PagGRF15 from hybrid poplar (Populus alba × P. glandulosa), a growth-regulating factor, plays a critical role in the regulation of vascular cambium activity. PagGRF15 was expressed predominantly in the cambial zone of vascular tissue. Overexpression of mPagGRF15 (the mutated version of GRF15 in the miR396 target sequence) in Populus led to decreased plant height and internode number. Further stem cross sections showed that the mPagGRF15 OE plants exhibited significant changes in vascular pattern with an increase in xylem and a reduction in phloem. In addition, cambium cell files were decreased in the mPagGRF15 OE plants. However, dominant suppression of the downstream genes of PagGRF15 using PagGRF15-SRDX showed an opposite phenotype. Based on the RNA-seq and ChIP-seq results, combining qRT-PCR and ChIP-PCR analysis, candidate genes, such as WOX4b, PXY and GID1.3, were obtained and found to be mainly involved in cambial activity and xylem differentiation. Accordingly, we speculated that PagGRF15 functions as a positive regulator mediating xylem differentiation by repressing the expression of the WOX4a and PXY genes to set the pace of cambial activity. In contrast, PagGRF15 mediated the GA signaling pathway by upregulating GID1.3 expression to stimulate xylem differentiation. This study provides valuable information for further studies on vascular cambium differentiation mechanisms and genetic improvement of the specific gravity of wood in hybrid poplars.

Stem Cells and Differentiation in Vascular Tissues.

Plant vascular tissues are crucial for the long-distance transport of water, nutrients, and a multitude of signal molecules throughout the plant body and, therefore, central to plant growth and development. The intricate development of vascular tissues is orchestrated by unique populations of dedicated stem cells integrating endogenous as well as environmental cues. This review summarizes our current understanding of vascular-related stem cell biology and of vascular tissue differentiation. We present an overview of the molecular and cellular mechanisms governing the maintenance and fate determination of vascular stem cells and highlight the interplay between intrinsic and external cues. In this context, we emphasize the role of transcription factors, hormonal signaling, and epigenetic modifications. We also discuss emerging technologies and the large repertoire of cell types associated with vascular tissues, which have the potential to provide unprecedented insights into cellular specialization and anatomical adaptations to distinct ecological niches.

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.

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.

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.

Wood of trees: Cellular structure, molecular formation, and genetic engineering.

Wood is an invaluable asset to human society due to its renewable nature, making it suitable for both sustainable energy production and material manufacturing. Additionally, wood derived from forest trees plays a crucial role in sequestering a significant portion of the carbon dioxide fixed during photosynthesis by terrestrial plants. Nevertheless, with the expansion of the global population and ongoing industrialization, forest coverage has been substantially decreased, resulting in significant challenges for wood production and supply. Wood production practices have changed away from natural forests toward plantation forests. Thus, understanding the underlying genetic mechanisms of wood formation is the foundation for developing high-quality, fast-growing plantation trees. Breeding ideal forest trees for wood production using genetic technologies has attracted the interest of many. Tremendous studies have been carried out in recent years on the molecular, genetic, and cell-biological mechanisms of wood formation, and considerable progress and findings have been achieved. These studies and findings indicate enormous possibilities and prospects for tree improvement. This review will outline and assess the cellular and molecular mechanisms of wood formation, as well as studies on genetically improving forest trees, and address future development prospects.

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.