Isotope Distribution Analysis in H218O Pulse-Labeled Trees Frozen with Liquid Nitrogen.

Tracer injection has long been recognized as a valuable tool for delineating tree hydraulics and assessing water transport pathways. Recently, isotope tracers have emerged as innovative instruments for investigating tree hydraulics, providing new insights into tree water dynamics. Nevertheless, there is a critical need for further research to comprehensively grasp water movement and distribution within trees. A previously introduced technique for analyzing the isotopic ratio of water in wet tissues, offering millimeter-scale resolution for visualizing tracer movement, faces challenges due to its underdeveloped sample preparation techniques. In this study, we introduced an H2 18O tracer into S. gracilistyla samples, exclusively comprising indeterminate roots, stems, and leaves, cultivated through hydroponics and grown within the current year. Our objective was to assess the axial distribution of the tracer in the xylem. Additionally, we devised a novel method for preparing frozen wet tissue samples, enhancing the repeatability and success rate of experiments. The results demonstrated that all frozen wet tissue samples exhibited an average water loss rate of less than 0.6%. Isotopic analysis of these samples unveiled a consistent decline in tracer concentration with increasing height in all Salix specimens, with three out of five samples revealing a significant isotope gradient. Our findings affirm the efficacy and practicality of combining isotopic labeling with freezing, stabilization, and preparation techniques. Looking ahead, our isotopic labeling and analysis methods are poised to transcend woody plants, finding extensive applications in plant physiology and ecohydrology.

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.

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.

Nutrient supplementation-induced metabolic profile changes and early appearance of free N-glycans in nutrient deficient tomato plants revealed by mass spectrometry.

Inorganic fertilizers are routinely used in large scale crop production for the supplementation of nitrogen, phosphorus, and potassium in nutrient poor soil. To explore metabolic changes in tomato plants grown on humic sand under different nutritional conditions, matrix-assisted laser desorption ionization (MALDI) mass spectrometry was utilized for the analysis of xylem sap. Variations in the abundances of metabolites and oligosaccharides, including free N-glycans (FNGs), were determined. Statistical analysis of the sample-related peaks revealed significant differences in the abundance ratios of multiple metabolites, including oligosaccharides, between the control plants, grown with no fertilizers, and plants raised under "ideal" and "nitrogen deficient" nutritional conditions, i.e., under the three treatment types. Among the 36 spectral features tentatively identified as oligosaccharides, the potential molecular structures for 18 species were predicted based on their accurate masses and isotope distribution patterns. To find the spectral features that account for most of the differences between the spectra corresponding to the three different treatments, multivariate statistical analysis was carried out by orthogonal partial least squares-discriminant analysis (OPLS-DA). They included both FNGs and non-FNG compounds that can be considered as early indicators of nutrient deficiency. Our results reveal that the potential nutrient deficiency indicators can be expanded to other metabolites beyond FNGs. The m/z values for 20 spectral features with the highest variable influence on projection (VIP) scores were ranked in the order of their influence on the statistical model.

LONESOME HIGHWAY-TARGET OF MONOPTEROS5 transcription factor complex promotes a predifferentiation state for xylem vessel differentiation in the root apical meristem by inducing the expression of VASCULAR-RELATED NAC-DOMAIN genes.

In Arabidopsis thaliana, heterodimers comprising two bHLH family proteins, LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS5 (TMO5) or its homolog TMO5-LIKE 1 (T5L1) control vascular development in the root apical meristem (RAM). The LHW-TMO5/T5L1 complex regulates vascular cell proliferation, vascular pattern organization, and xylem vessel differentiation; however, the mechanism of preparation for xylem vessel differentiation in the RAM remains elusive. We examined the relationship between LHW-T5L1 and VASCULAR-RELATED NAC-DOMAIN (VND) genes, which are key regulators of vessel differentiation, using reverse genetics approaches. LHW-T5L1 upregulated the expression of VND1, VND2, VND3, VND6, and VND7 but not that of other VNDs. The expression of VND1-VND3 in the RAM was decreased in lhw. In vnd1 vnd2 vnd3 triple loss-of-function mutant roots, metaxylem differentiation was delayed, and VND6 and VND7 expression was reduced. Furthermore, transcriptome analysis of VND1-overexpressing cells revealed that VND1 upregulates genes involved in the synthesis of secondary cell wall components. These results suggest that LHW-T5L1 upregulates VND1-VND3 at the early stages of vascular development in the RAM, and VNDs promote a predifferentiation state for xylem vessels by triggering low levels of VND6 and VND7 as well as genes for the synthesis of secondary cell wall materials.

CsREV-CsTCP4-CsVND7 module shapes xylem patterns differentially between stem and leaf to enhance tea plant tolerance to drought.

Cultivating drought-tolerant tea varieties enhances both yield and quality of tea plants in northern China. However, the mechanisms underlying their drought tolerance remain largely unknown. Here we identified a key regulator called CsREV, which differentially regulates xylem patterns between leaves and stems, thereby conferring drought tolerance in tea plants. When drought occurs, upregulation of CsREV activates the CsVND7a-dependent xylem vessel differentiation. However, when drought persists, the vessel differentiation is hindered as CsVND7a is downregulated by CsTCP4a. This, combined with the CsREV-promoted secondary-cell-wall thickness of xylem vessel, leads to the enhanced curling of leaves, a characteristic closely associated with plant drought tolerance. Notably, this inhibitory effect of CsTCP4a on CsVND7a expression is absent in stems, allowing stem xylem vessels to continuously differentiate. Overall, the CsREV-CsTCP4-CsVND7 module is differentially utilized to shape the xylem patterns in leaves and stems, potentially balancing water transportation and utilization to improve tea plant drought tolerance.

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.

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.

Ectopic assembly of an auxin efflux control machinery shifts developmental trajectories.

Polar auxin transport in the Arabidopsis (Arabidopsis thaliana) root tip maintains high auxin levels around the stem cell niche that gradually decrease in dividing cells but increase again once they transition toward differentiation. Protophloem differentiates earlier than other proximal tissues and employs a unique auxin "canalization" machinery that is thought to balance auxin efflux with retention. It consists of a proposed activator of PIN-FORMED (PIN) auxin efflux carriers, the cAMP-, cGMP- and Calcium-dependent (AGC) kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX); its inhibitor, BREVIS RADIX (BRX); and PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K) enzymes, which promote polar PAX and BRX localization. Because of a dynamic PAX-BRX-PIP5K interplay, the net cellular output of this machinery remains unclear. In this study, we deciphered the dosage-sensitive regulatory interactions among PAX, BRX, and PIP5K by their ectopic expression in developing xylem vessels. The data suggest that the dominant collective output of the PAX-BRX-PIP5K module is a localized reduction in PIN abundance. This requires PAX-stimulated clathrin-mediated PIN endocytosis upon site-specific phosphorylation, which distinguishes PAX from other AGC kinases. An ectopic assembly of the PAX-BRX-PIP5K module is sufficient to cause cellular auxin retention and affects root growth vigor by accelerating the trajectory of xylem vessel development. Our data thus provide direct evidence that local manipulation of auxin efflux alters the timing of cellular differentiation in the root.

Microtubule-associated phase separation of MIDD1 tunes cell wall spacing in xylem vessels in Arabidopsis thaliana.

Properly patterned cell walls specify cellular functions in plants. Differentiating protoxylem and metaxylem vessel cells exhibit thick secondary cell walls in striped and pitted patterns, respectively. Cortical microtubules are arranged in distinct patterns to direct cell wall deposition. The scaffold protein MIDD1 promotes microtubule depletion by interacting with ROP GTPases and KINESIN-13A in metaxylem vessels. Here we show that the phase separation of MIDD1 fine-tunes cell wall spacing in protoxylem vessels in Arabidopsis thaliana. Compared with wild-type, midd1 mutants exhibited narrower gaps and smaller pits in the secondary cell walls of protoxylem and metaxylem vessel cells, respectively. Live imaging of ectopically induced protoxylem vessels revealed that MIDD1 forms condensations along the depolymerizing microtubules, which in turn caused massive catastrophe of microtubules. The MIDD1 condensates exhibited rapid turnover and were susceptible to 1,6-hexanediol. Loss of ROP abolished the condensation of MIDD1 and resulted in narrow cell wall gaps in protoxylem vessels. These results suggest that the microtubule-associated phase separation of MIDD1 facilitates microtubule arrangement to regulate the size of gaps in secondary cell walls. This study reveals a new biological role of phase separation in the fine-tuning of cell wall patterning.

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.

Mutation of OsNRAMP5 reduces cadmium xylem and phloem transport in rice plants and its physiological mechanism.

Natural resistance associated macrophage protein 5 (NRAMP5) is a key transporter for cadmium (Cd) uptake by rice roots; however, the effect of OsNRAMP5 on Cd translocation and redistribution in rice plants remains unknown. In this study, an extremely low Cd-accumulation mutant (lcd1) and wild type (WT) plants were utilized to investigate the effect of OsNRAMP5 mutation on Cd translocation and redistribution via the xylem and phloem and its possible physiological mechanism using field, hydroponic and isotope-labelling experiments. The results showed that OsNRAMP5 mutation reduced xylem and phloem transport of Cd, due to remarkably lower Cd translocation from roots to shoots and from the leaves Ⅰ-Ⅲ to their corresponding nodes, as well as lower Cd concentrations in xylem and phloem sap of lcd1 compared to WT plants. Mutation of OsNRAMP5 reduced Cd translocation from roots to shoots in lcd1 plants by increasing Cd deposition in cellulose of root cell walls and reducing OsHMA2-and OsCCX2-mediated xylem loading of Cd, and the citric acid- and tartaric acid-mediated long-distance xylem transport of Cd. Moreover, OsNRAMP5 mutation inhibited Cd redistribution from flag leaves to nodes and panicles in lcd1 plants by increasing Cd sequestration in cellulose and vacuoles, and decreasing OsLCT1-mediated Cd phloem transport in flag leaves.

An essential role for mannan degradation in both cell growth and secondary cell wall formation.

Coordination of secondary cell wall deposition and cell expansion during plant growth is required for cell development, particularly in vascular tissues. Yet the fundamental coordination process has received little attention. We observed that the Arabidopsis endo-1,4-mannanase gene, AtMAN6, is involved in the formation of cell walls in vascular tissues. In the inflorescence stem, the man6 mutant had smaller vessel cells with thicker secondary cell walls and shorter fiber cells. Elongation growth was reduced in the root, and secondary cell wall deposition in vessel cells occurred early. Overexpression of AtMAN6 resulted in the inverse phenotypes of the man6 mutant. AtMAN6 was discovered on the plasma membrane and was specifically expressed in vessel cells during its early development. The AtMAN6 protein degraded galactoglucomannan to produce oligosaccharides, which caused secondary cell wall deposition in vessel and fiber cells to be suppressed. Transcriptome analysis revealed that the expression of genes involved in the regulation of secondary cell wall synthesis was changed in both man6 mutant and AtMAN6 overexpression plants. AtMAN6's C-terminal cysteine repeat motif (CCRM) was found to facilitate homodimerization and is required for its activity. According to the findings, the oligosaccharides produced by AtMAN6 hydrolysis may act as a signal to mediate this coordination between cell growth and secondary cell wall deposition.

Quantification of xylem connection defects during lateral root development in Arabidopsis.

The cellular and molecular mechanisms underlying the establishment of vascular connections between primary and lateral roots have recently gained significant attention. Here, I present a protocol to visualize and quantify xylem connection defects during lateral root development. I describe steps for employing stains to infer whether the defects observed in xylem bridges are associated with alterations in the xylem differentiation program, including programmed cell death and secondary cell wall deposition. For complete details on the use and execution of this protocol, please refer to Blanco-Touriñán et al. (2023) and Ursache et al. (2018).1,2.

Quantification of Xylem-Specific Thermospermine-Dependent Translation of SACL Transcripts with Dual Luciferase Reporter System.

Thermospermine (Tspm) is a polyamine found to play a crucial role in xylem development in Arabidopsis thaliana. Tspm promotes the translation of the SACL genes by counteracting the activity of a cis element in their 5'-leader region that suppresses the translation of the main ORF. Here we describe a method to test the Tspm-dependent translational regulation of the 5'-leader of the SACL mRNAs in Nicotiana benthamiana leaves and A. thaliana mesophyll protoplasts with a dual luciferase assay. The dual luciferase reporter system is used to assess gene expression and is based on the detection of the Firefly luciferase luminescence driven by a specific promoter. However, it can also be used to evaluate the cis elements found in 5'-leader that influence the translation of the main ORF in a transcript. We have used a modified version of the pGreenII 0800 LUC plasmid carrying a double 35S promoter, followed by a poly-linker sequence in phase with the Firefly luciferase gene (pGreen2x35SLUC) where the full 5'-leader sequence of SACL3 was cloned. This construct was used for Agrobacterium tumefaciens infiltration of N. benthamiana leaves and for transfection of A. thaliana mesophyll protoplasts, followed by mock or Tspm treatments. The resulting translation of the Firefly luciferase in these organisms and conditions was then tested by measuring luminescence with the dual luciferase assay and a luminometer. These experiments have allowed us to quantify the positive effect of Tspm in the translation of SACL3 transcripts.

Confined-microtubule assembly shapes three-dimensional cell wall structures in xylem vessels.

Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood. In metaxylem vessels, cell wall arches are formed over numerous pit membranes, forming highly organized three-dimensional cell wall structures. Here, we show that the microtubule-associated proteins, MAP70-5 and MAP70-1, regulate arch development. The map70-1 map70-5 plants formed oblique arches in an abnormal orientation in pits. Microtubules fit the aperture of developing arches in wild-type cells, whereas microtubules in map70-1 map70-5 cells extended over the boundaries of pit arches. MAP70 caused the bending and bundling of microtubules. These results suggest that MAP70 confines microtubules within the pit apertures by altering the physical properties of microtubules, thereby directing the growth of pit arches in the proper orientation. This study provides clues to understanding how plants develop three-dimensional structure of cell walls.

Changes in the physiological activity of parenchyma cells in Dalbergia odorifera xylem and its relationship with heartwood formation.

BACKGROUND: The formation of a tree's heartwood gives the wood properties such as natural decay resistance and aesthetic color, and often directly determines the value of wood products. Regulating the quantity and quality of heartwood is of great importance to the use of wood. However, the mechanism of heartwood formation has been poorly understood. RESULTS: Using Dalbergia odorifera as the study species, the number of starch grains, the morphology of the nuclei, the changes in the content of water and secondary metabolites were observed continuously in the radial direction of the xylem. The results show that from the outer toward inner sapwood, the starch grains are abundant, the length to diameter ratio of the nuclei is decreasing, and the morphology changes from elongated elliptical and then to round. In the outer transition zone, the starch grains begin to decrease abruptly and the nuclei shrink at a slower rate, with a radial width of approximately 2 mm. In the inner transition zone, the heartwood color begins to appear, the starch grains disappear and a few nuclei with reduced fluorescence are present, with a radial width of approximately 1 mm. Heartwood formation after complete disappearance of the nuclei. The moisture content of the heartwood is higher than that of the sapwood, and the inner transition zone is where the content rises. The secondary metabolites of the heartwood begin to accumulate in large quantities in the inner transition zone. CONCLUSION: Based on the physiological changes of parenchyma cells in the xylem, the radial width of the transition zone of Dalbergia odorifera is clearly defined as approximately 3 mm. Both the water and secondary metabolite abrupt changes occur at the final stage of programmed cell death, and neither is a direct cause of programmed cell death in parenchyma cells.

Transcriptome and miRNAs Profiles Reveal Regulatory Network and Key Regulators of Secondary Xylem Formation in "84K" Poplar.

Secondary xylem produced by stem secondary growth is the main source of tree biomass and possesses great economic and ecological value in papermaking, construction, biofuels, and the global carbon cycle. The secondary xylem formation is a complex developmental process, and the underlying regulatory networks and potential mechanisms are still under exploration. In this study, using hybrid poplar (Populus alba × Populus glandulosa clone 84K) as a model system, we first ascertained three representative stages of stem secondary growth and then investigated the regulatory network of secondary xylem formation by joint analysis of transcriptome and miRNAs. Notably, 7507 differentially expressed genes (DEGs) and 55 differentially expressed miRNAs (DEMs) were identified from stage 1 without initiating secondary growth to stage 2 with just initiating secondary growth, which was much more than those identified from stage 2 to stage 3 with obvious secondary growth. DEGs encoding transcription factors and lignin biosynthetic enzymes and those associated with plant hormones were found to participate in the secondary xylem formation. MiRNA-target analysis revealed that a total of 85 DEMs were predicted to have 2948 putative targets. Among them, PagmiR396d-PagGRFs, PagmiR395c-PagGA2ox1/PagLHW/PagSULTR2/PagPolyubiquitin 1, PagmiR482d-PagLAC4, PagmiR167e-PagbHLH62, and PagmiR167f/g/h-PagbHLH110 modules were involved in the regulating cambial activity and its differentiation into secondary xylem, cell expansion, secondary cell wall deposition, and programmed cell death. Our results give new insights into the regulatory network and mechanism of secondary xylem formation.