Identification of key genes involved in lignin and flavonoid accumulation during Tilia tuan seed maturation.

KEY MESSAGE: Transcriptomics and phenotypic data analysis identified 24 transcription factors (TFs) that play key roles in regulating the competitive accumulation of lignin and flavonoids. Tilia tuan Szyszyl. (T. tuan) is a timber tree species with important ecological and commercial value. However, its highly lignified pericarp results in a low seed germination rate and a long dormancy period. In addition, it is unknown whether there is an interaction between the biosynthesis of flavonoids and lignin as products of the phenylpropanoid pathway during seed development. To explore the molecular regulatory mechanism of lignin and flavonoid biosynthesis, T. tuan seeds were harvested at five stages (30, 60, 90, 120, and 150 days after pollination) for lignin and flavonoid analyses. The results showed that lignin accumulated rapidly in the early and middle stages (S1, S3, and S4), and rapid accumulation of flavonoids during the early and late stages (S1 and S5). High-throughput RNA sequencing analysis of developing seeds identified 50,553 transcripts, including 223 phenylpropanoid biosynthetic pathway genes involved in lignin accumulation grouped into 3 clusters, and 106 flavonoid biosynthetic pathway genes (FBPGs) grouped into 2 clusters. Subsequent WGCNA and time-ordered gene co-expression network (TO-GCN) analysis revealed that 24 TFs (e.g., TtARF2 and TtWRKY15) were involved in flavonoids and lignin biosynthesis regulation. The transcriptome data were validated by qRT-PCR to analyze the expression profiles of key enzyme-coding genes. This study revealed that there existed a competitive relationship between flavonoid and lignin biosynthesis pathway during the development of T. tuan seeds, that provide a foundation for the further exploration of molecular mechanisms underlying lignin and flavonoid accumulation in T. tuan seeds.

Computational dissection of genetic variation modulating the response of multiple photosynthetic phenotypes to the light environment.

BACKGROUND: The expression of biological traits is modulated by genetics as well as the environment, and the level of influence exerted by the latter may vary across characteristics. Photosynthetic traits in plants are complex quantitative traits that are regulated by both endogenous genetic factors and external environmental factors such as light intensity and CO2 concentration. The specific processes impacted occur dynamically and continuously as the growth of plants changes. Although studies have been conducted to explore the genetic regulatory mechanisms of individual photosynthetic traits or to evaluate the effects of certain environmental variables on photosynthetic traits, the systematic impact of environmental variables on the dynamic process of integrated plant growth and development has not been fully elucidated. RESULTS: In this paper, we proposed a research framework to investigate the genetic mechanism of high-dimensional complex photosynthetic traits in response to the light environment at the genome level. We established a set of high-dimensional equations incorporating environmental regulators to integrate functional mapping and dynamic screening of gene‒environment complex systems to elucidate the process and pattern of intrinsic genetic regulatory mechanisms of three types of photosynthetic phenotypes of Populus simonii that varied with light intensity. Furthermore, a network structure was established to elucidate the crosstalk among significant QTLs that regulate photosynthetic phenotypic systems. Additionally, the detection of key QTLs governing the response of multiple phenotypes to the light environment, coupled with the intrinsic differences in genotype expression, provides valuable insights into the regulatory mechanisms that drive the transition of photosynthetic activity and photoprotection in the face of varying light intensity gradients. CONCLUSIONS: This paper offers a comprehensive approach to unraveling the genetic architecture of multidimensional variations in photosynthetic phenotypes, considering the combined impact of integrated environmental factors from multiple perspectives.

Genomic insights into selection for heterozygous alleles and woody traits in Populus tomentosa.

Heterozygous alleles are widespread in outcrossing and clonally propagated woody plants. The variation in heterozygosity that underlies population adaptive evolution and phenotypic variation, however, remains largely unknown. Here, we describe a de novo chromosome-level genome assembly of Populus tomentosa, an economic and ecologically important native tree in northern China. By resequencing 302 natural accessions, we determined that the South subpopulation (Pop_S) encompasses the ancestral strains of P. tomentosa, while the Northwest subpopulation (Pop_NW) and Northeast subpopulation (Pop_NE) experienced different selection pressures during population evolution, resulting in significant population differentiation and a decrease in the extent of heterozygosity. Analysis of heterozygous selective sweep regions (HSSR) suggested that selection for lower heterozygosity contributed to the local adaptation of P. tomentosa by dwindling gene expression and genetic load in the Pop_NW and Pop_NE subpopulations. Genome-wide association studies (GWAS) revealed that 88 single nucleotide polymorphisms (SNPs) within 63 genes are associated with nine wood composition traits. Among them, the selection for the homozygous AA allele in PtoARF8 is associated with reductions in cellulose and hemicellulose contents by attenuating PtoARF8 expression, and the increase in lignin content is attributable to the selection for decreases in exon heterozygosity in PtoLOX3 during adaptive evolution of natural populations. This study provides novel insights into allelic variations in heterozygosity associated with adaptive evolution of P. tomentosa in response to the local environment and identifies a series of key genes for wood component traits, thereby facilitating genomic-based breeding of important traits in perennial woody plants.

Enhanced genome-wide association reveals the role of YABBY11-NGATHA-LIKE1 in leaf serration development of Populus.

Leaf margins are complex plant morphological features that contribute to leaf shape diversity, which affects plant structure, yield, and adaptation. Although several leaf margin regulators have been identified to date, the genetic basis of their natural variation has not been fully elucidated. In this study, we profiled two distinct leaf morphology types (serrated and smooth) using the persistent homology mathematical framework (PHMF) in two poplar species (Populus tomentosa and Populus simonii, respectively). A combined genome-wide association study (GWAS) and expression quantitative trait nucleotide (eQTN) mapping were applied to create a leaf morphology control module using data from P. tomentosa and P. simonii populations. Natural variation in leaf margins was associated with YABBY11 (YAB11) transcript abundance in poplar. In P. tomentosa, PtoYAB11 carries a premature stop codon (PtoYAB11PSC), resulting in the loss of its positive regulation of NGATHA-LIKE1 (PtoNGAL-1) and RIBULOSE BISPHOSPHATE CARBOXYLASE LARGE SUBUNIT (PtoRBCL). Overexpression of PtoYAB11PSC promoted serrated leaf margins, enlarged leaves, enhanced photosynthesis, and increased biomass. Overexpression of PsiYAB11 in P. tomentosa promoted smooth leaf margins, higher stomatal density, and greater light damage repair ability. In poplar, YAB11-NGAL1 is sensitive to environmental conditions, acts as a positive regulator of leaf margin serration, and may also link environmental signaling to leaf morphological plasticity.

Correlation Analysis of the Characteristics of Spino-Pelvic Parameters and Health-Related Quality of Life After Long-Segment Fixation for Lenke-Silva Type 5 or 6 Degenerative Scoliosis.

Purpose: To retrospectively evaluate the characteristics of spino-pelvic parameters after long-segment fixation in patients with Lenke-Silva type 5 or 6 adult degenerative scoliosis and analyze the correlation between spino-pelvic parameters and health-related quality of life (HRQL). Methods: Thirty-two patients with degenerative scoliosis underwent long-segment posterior fixation were included. The spino-pelvic parameters were evaluated after surgery, and the HRQL scores were determined using the Scoliosis Research Society-22 (SRS-22), Oswestry Disability Index (ODI), and visual analog scale (VAS). Linear regression was used to analyze the correlation between changes in spino-pelvic parameters and improvements in HRQL. Results: Except for PI and TK, the other parameters showed significant differences after surgery (P < 0.05). All the scores of HRQL showed significant differences after surgery. The coefficient of correlation between ΔSVA and ΔVAS is 0.687 (P = 0.003), the coefficient of correlation between ΔSVA and ΔODI is 5.828 (P < 0.001). The coefficient of correlation between ΔLL and ΔVAS is -0.089 (P < 0.001), the coefficient of correlation between ΔLL and ΔODI is -1.553 (P = 0.003). The VAS score between the SVA ≥ 4cm and SVA < 4cm group have no significant difference, but have a significant difference in ODI. In PI-LL ≥ 20°and PI-LL < 20°group, the VAS and ODI all have a significant difference between the two groups. Conclusion: SVA and LL have an important role in maintaining the overall balance of the spine and closely related to the postoperative HRQL, better HRQL may be achieved by reducing SVA and increasing LL. Good preoperative design will help achieve the best clinical efficacy.

LncRNA PMAT-PtoMYB46 module represses PtoMATE and PtoARF2 promoting Pb2+ uptake and plant growth in poplar.

Lead (Pb2+) is one of the most toxic heavy-metal contaminants. Fast-growing woody plants with substantial biomass are ideal for bioremediation. However, the transcriptional regulation of Pb2+ uptake in woody plants remains unclear. Here, we identified 226 Pb2+-induced, differentially expressed long non-coding RNAs (DELs) in Populus tomentosa. Functional annotation revealed that these DELs mainly regulate carbon metabolism, biosynthesis of secondary metabolites, energy metabolism, and signal transduction through their potential target genes. Association and epistasis analysis showed that the lncRNA PMAT (Pb2+-induced multidrug and toxic compound extrusion (MATE) antisense lncRNA) interacts epistatically with PtoMYB46 to regulate leaf dry weight, photosynthesis rate, and transketolase activity. Genetic transformation and molecular assays showed that PtoMYB46 reduces the expression of PtoMATE directly or indirectly through PMAT, thereby reducing the secretion of citric acid (CA) and ultimately promoting Pb2+ uptake. Meanwhile, PtoMYB46 targets auxin response factor 2 (ARF2) and reduces its expression, thus positively regulating plant growth. We concluded that the PMAT-PtoMYB46-PtoMATE-PtoARF2 regulatory module control Pb2+ tolerance, uptake, and plant growth. This study demonstrates the involvement of lncRNAs in response to Pb2+ in poplar, yielding new insight into the potential for developing genetically improved woody plant varieties for phytoremediating lead-contaminated soils.

The Genetic Basis of Phosphorus Utilization Efficiency in Plants Provide New Insight into Woody Perennial Plants Improvement.

Soil nutrient restrictions are the main environmental conditions limiting plant growth, development, yield, and quality. Phosphorus (P), an essential macronutrient, is one of the most significant factors that vastly restrains the growth and development of plants. Although the total P is rich in soil, its bio-available concentration is still unable to meet the requirements of plants. To maintain P homeostasis, plants have developed lots of intricate responsive and acclimatory mechanisms at different levels, which contribute to administering the acquisition of inorganic phosphate (Pi), translocation, remobilization, and recycling of Pi. In recent years, significant advances have been made in the exploration of the utilization of P in annual plants, while the research progress in woody perennial plants is still vague. In the meanwhile, compared to annual plants, relevant reviews about P utilization in woody perennial plants are scarce. Therefore, based on the importance of P in the growth and development of plants, we briefly reviewed the latest advances on the genetic and molecular mechanisms of plants to uphold P homeostasis, P sensing, and signaling, ion transporting and metabolic regulation, and proposed the possible sustainable management strategies to fasten the P cycle in modern agriculture and new directions for future studies.

Dynamic physiological and transcriptome changes reveal a potential relationship between the circadian clock and salt stress response in Ulmus pumila.

Despite the important role the circadian clock plays in numerous critical physiological responses in plants, such as hypocotyl elongation, leaf movement, stomatal opening, flowering, and stress responses, there have been no investigations into the effect of the circadian clock on physiological and transcriptional networks under salt stress. Ulmus pumila L. has been reported to tolerate 100-150 mM NaCl treatment. We measured the diurnal variation in photosynthesis and chlorophyll fluorescence parameters and performed a time-course transcriptome analysis of 2-years-old U. pumila seedlings under salt treatment to dissect the physiological regulation and potential relationship between the circadian network and the salt stress response. Seedlings in 150 mM NaCl treatment exhibited salt-induced physiological enhancement compared to the control group. A total of 7009 differentially expressed unigenes (DEGs) were identified under salt stress, of which 16 DEGs were identified as circadian rhythm-related DEGs (crDEGs). Further analysis of dynamic expression changes revealed that DEGs involved in four crucial pathways-photosynthesis, thiamine metabolism, abscisic acid synthesis and metabolism, and the hormone-MAPK signal crosstalk pathway-are closely related to the circadian clock. Finally, we constructed a co-expression network between the circadian clock and these four crucial pathways. Our results help shed light on the molecular link between the circadian network and salt stress tolerance in U. pumila.

Sound Insulation of Corrugated-Core Sandwich Panels: Modeling, Optimization and Experiment.

With the extension of the applications of sandwich panels with corrugated core, sound insulation performance has been a great concern for acoustic comfort design in many industrial fields. This paper presents a numerical and experimental study on the vibro-acoustic optimization of a finite size sandwich panel with corrugated core for maximizing the sound transmission loss. The numerical model is established by using the wave-based method, which shows a great improvement in the computational efficiency comparing to the finite element method. Constrained by the fundamental frequency and total mass, the optimization is performed by using a genetic algorithm in three different frequency bands. According to the optimization results, the frequency averaged sound transmission of the optimized models in the low, middle, and high-frequency ranges has increased, respectively, by 7.6 dB, 7.9 dB, and 11.7 dB compared to the baseline model. Benefiting from the vast number of the evolution samples, the correlation between the structural design parameters and the sound transmission characteristics is analyzed by introducing the coefficient of determination, which gives the variation of the importance of each design parameter in different frequency ranges. Finally, for validation purposes, a sound insulation test is conducted to validate the optimization results in the high-frequency range, which proves the feasibility of the optimization method in the practical engineering design of the sandwich panel.

Genetic Effects and Expression Patterns of the Nitrate Transporter (NRT) Gene Family in Populus tomentosa.

Nitrate is an important source of nitrogen for poplar trees. The nitrate transporter (NRT) gene family is generally responsible for nitrate absorption and distribution. However, few analyses of the genetic effects and expression patterns of NRT family members have been conducted in woody plants. Here, using poplar as a model, we identified and characterized 98 members of the PtoNRT gene family. We calculated the phylogenetic and evolutionary relationships of the PtoNRT family and identified poplar-specific NRT genes and their expression patterns. To construct a core triple genetic network (association - gene expression - phenotype) for leaf nitrogen content, a candidate gene family association study, weighted gene co-expression network analysis (WGCNA), and mapping of expression quantitative trait nucleotides (eQTNs) were combined, using data from 435 unrelated Populus. tomentosa individuals. PtoNRT genes exhibited distinct expression patterns between twelve tissues, circadian rhythm points, and stress responses. The association study showed that genotype combinations of allelic variations of three PtoNRT genes had a strong effect on leaf nitrogen content. WGCNA produced two co-expression modules containing PtoNRT genes. We also found that four PtoNRT genes defined thousands of eQTL signals. WGCNA and eQTL provided comprehensive analysis of poplar nitrogen-related regulatory factors, including MYB17 and WRKY21. NRT genes were found to be regulated by five plant hormones, among which abscisic acid was the main regulator. Our study provides new insights into the NRT gene family in poplar and enables the exploitation of novel genetic factors to improve the nitrate use efficiency of trees.

Integration of genome wide association studies and co-expression networks reveal roles of PtoWRKY 42-PtoUGT76C1-1 in trans-zeatin metabolism and cytokinin sensitivity in poplar.

Cytokinins are important for in vitro shoot regeneration in plants. Cytokinin N-glucosides are produced via an irreversible glycosylation pathway, which regulates the endogenous cytokinin content. Although cytokinin N-glucoside pathways have been uncovered in higher plants, no regulator has been identified to date. We performed a metabolome genome-wide association study (mGWAS), weighted gene co-expression network analysis (WGCNA), and expression quantitative trait nucleotide (eQTN) mappings to build a core triple genetic network (mGWAS-gene expression-phenotype) for the trans-zeatin N-glucoside (ZNG) metabolite using data from 435 unrelated Populus tomentosa individuals. Variation of the ZNG level in poplar is attributed to the differential transcription of PtoWRKY42, a member of WRKY multigene family group IIb. Functional analysis revealed that PtoWRKY42 negatively regulated ZNG accumulation by binding directly to the W-box of the UDP-glycosyltransferase 76C 1-1 (PtoUGT761-1) promoter. Also, PtoWRKY42 was strongly induced by leaf senescence, 6-BA, wounding, and salt stress, resulting in a reduced ZNG level. We identified PtoWRKY42, a negative regulator of cytokinin N-glucosides, which contributes to the natural variation in ZNG level and mediates ZNG accumulation by directly modulating the key glycosyltransferase gene PtoUGT76C1-1.

Gene Coexpression Network Analysis Indicates that Hub Genes Related to Photosynthesis and Starch Synthesis Modulate Salt Stress Tolerance in Ulmus pumila.

Ulmus pumila L. is an excellent afforestation and biofuel tree that produces high-quality wood, rich in starch. In addition, U. pumila is highly adaptable to adverse environmental conditions, which is conducive to its utilization for vegetating saline soils. However, little is known about the physiological responses and transcriptional regulatory network of U. pumila under salt stress. In this study, we exposed five main cultivars in saline-alkali land (Upu2, 5, 8, 11, and 12) to NaCl stress. Of the five cultivars assessed, Upu11 exhibited the highest salt resistance. Growth and biomass accumulation in Upu11 were promoted under low salt concentrations (<150 mM). However, after 3 months of continuous treatment with 150 mM NaCl, growth was inhibited, and photosynthesis declined. A transcriptome analysis conducted after 3 months of treatment detected 7009 differentially expressed unigenes (DEGs). The gene annotation indicated that these DEGs were mainly related to photosynthesis and carbon metabolism. Furthermore, PHOTOSYNTHETIC ELECTRON TRANSFERH (UpPETH), an important electron transporter in the photosynthetic electron transport chain, and UpWAXY, a key gene controlling amylose synthesis in the starch synthesis pathway, were identified as hub genes in the gene coexpression network. We identified 25 and 62 unigenes that may interact with PETH and WAXY, respectively. Overexpression of UpPETH and UpWAXY significantly increased the survival rates, net photosynthetic rates, biomass, and starch content of transgenic Arabidopsis plants under salt stress. Our findings clarify the physiological and transcriptional regulators that promote or inhibit growth under environmental stress. The identification of salt-responsive hub genes directly responsible for photosynthesis and starch synthesis or metabolism will provide targets for future genetic improvements.

Transcriptome analysis and association mapping reveal the genetic regulatory network response to cadmium stress in Populus tomentosa.

Long non-coding RNAs (lncRNAs) play essential roles in plant abiotic stress responses, but the response of lncRNA-mediated genetic networks to cadmium (Cd) treatment remain elusive in trees, the promising candidates for phytoremediation of Cd contamination. We identified 172 Cd-responsive lncRNAs and 295 differentially expressed target genes in the leaves of Cd-treated Populus tomentosa. Functional annotation revealed that these lncRNAs were involved in various processes, including photosynthesis, hormone regulation, and phenylalanine metabolism. Association studies identified 78 significant associations, representing 14 Cd-responsive lncRNAs and 28 target genes for photosynthetic and leaf physiological traits. Epistasis uncovered 83 pairwise interactions among these traits, revealing Cd-responsive lncRNA-mediated genetic networks for photosynthesis and leaf physiology in P. tomentosa. We focused on the roles of two Cd-responsive lncRNA-gene pairs, MSTRG.22608.1-PtoMYB73 and MSTRG.5634.1-PtoMYB27, in Cd tolerance of Populus, and detected insertions/deletions within lncRNAs as polymorphisms driving target gene expression. Genotype analysis of lncRNAs and heterologous overexpression of PtoMYB73 and PtoMYB27 in Arabidopsis indicated their effects on enhancing Cd tolerance, photosynthetic rate, and leaf growth, and the potential interaction mechanisms of PtoMYB73 with abiotic stresses. Our study identifies the genetic basis for the response of Populus to Cd treatment, facilitating genetic improvement of Cd tolerance in trees.

Miniature inverted repeat transposable elements cis-regulate circular RNA expression and promote ethylene biosynthesis, reducing heat tolerance in Populus tomentosa.

Transposable elements (TEs) and their reverse complementary sequence pairs (RCPs) are enriched around loci that produce circular RNAs (circRNAs) in plants. However, the function of these TE-RCP pairs in modulating circRNA expression remains elusive. Here, we identified 4609 circRNAs in poplar (Populus tomentosa) and showed that miniature inverted repeat transposable elements (MITEs)-RCPs were enriched in circRNA flanking regions. Moreover, we used expression quantitative trait nucleotide (eQTN) mapping to decipher the cis-regulatory role of MITEs. eQTN results showed that 14 single-nucleotide polymorphisms (SNPs) were significantly associated with Circ_0000408 and Circ_0003418 levels and the lead associated SNPs were located in MITE-RCP regions, indicating that MITE-RCP sequence variations affect exon circularization. Overexpression and knockdown analysis showed that Circ_0003418 positively modulated its parental gene, which encodes the RING-type E3 ligase XBAT32, and specifically increased the expression of the PtoXBAT32.5 transcript variant, which lacks the E3 ubiquitin ligase domain. Under heat stress, PtoXBAT32.5 expression was induced with up-regulation of Circ_0003418, resulting in increased production of ethylene and peroxidation of membrane lipids. Our findings thus reveal the cis-regulatory mechanism by which a MITE-RCP pair affects circRNA abundance in poplar and indicate that Circ_0003418 is a negative regulator of poplar heat tolerance via the ubiquitin-mediated protein modification pathway.

High-Temperature-Responsive Poplar lncRNAs Modulate Target Gene Expression via RNA Interference and Act as RNA Scaffolds to Enhance Heat Tolerance.

High-temperature stress is a threat to plant development and survival. Long noncoding RNAs (lncRNAs) participate in plant stress responses, but their functions in the complex stress response network remain unknown. Poplar contributes to terrestrial ecological stability. In this study, we identified 204 high-temperature-responsive lncRNAs in an abiotic stress-tolerant poplar (Populus simonii) species using strand-specific RNA sequencing (ssRNA-seq). Mimicking overexpressed and repressed candidate lncRNAs in poplar was used to illuminate their regulation pattern on targets using nano sheet mediation. These lncRNAs were predicted to target 185 genes, of which 100 were cis genes and 119 were trans genes. Gene Ontology enrichment analysis showed that anatomical structure morphogenesis and response to stress and signaling were significantly enriched. Among heat-responsive LncRNAs, TCONS_00202587 binds to upstream sequences via its secondary structure and interferes with target gene transcription. TCONS_00260893 enhances calcium influx in response to high-temperature treatment by interfering with a specific variant/isoform of the target gene. Heterogeneous expression of these two lncRNA targets promoted photosynthetic protection and recovery, inhibited membrane peroxidation, and suppressed DNA damage in Arabidopsis under heat stress. These results showed that lncRNAs can regulate their target genes by acting as potential RNA scaffolds or through the RNA interference pathway.

Effect of Preoperative Zoledronic Acid Administration on Pain Intensity after Percutaneous Vertebroplasty for Osteoporotic Vertebral Compression Fractures.

Introduction: This study aimed to compare and analyze the effect of preoperative zoledronic acid (ZOL) administration on pain intensity after percutaneous vertebroplasty (PVP) for osteoporotic vertebral compression fracture (OVCF). Methods: The study included 242 patients with OVCFs who underwent PVP in our hospital between January 2015 and June 2018. The patients were randomly assigned to either a ZOL group (n = 121) or a control group (n = 121). The patients in the ZOL group were treated preoperatively with intravenous infusion of 5 mg ZOL. Those in the control group were treated without ZOL. All the patients were followed up for 1 year. Results: No statistically significant differences in age, sex, weight, and body mass index (BMI) were found between the two groups. During the follow-up period, the visual analog scale score and Oswestry dysfunction index score in the ZOL group were lower than those in the control group. The bone mineral density at 6 or 12 months after treatment was significantly higher and the levels of the bone metabolism markers were significantly lower in the ZOL group than in the control group (P < 0.05 for both). Two patients in the treatment group had new vertebral fractures, whereas 13 patients in the control group had new vertebral fractures, which translate to recompression vertebral fracture incidence rates of 1.7% and 10.7%, respectively. The incidence rate of mild adverse reactions was significantly higher in the ZOL group than in the control group, but all the cases were endurable. Conclusion: Intravenous infusion of ZOL before PVP can effectively reduce postoperative pain intensity, reduce bone loss, increase bone density, reduce the risk of refracture, and improve patient quality of life.

Changes in DNA Methylation in Response to 6-Benzylaminopurine Affect Allele-Specific Gene Expression in Populus Tomentosa.

Cytokinins play important roles in the growth and development of plants. Physiological and photosynthetic characteristics are common indicators to measure the growth and development in plants. However, few reports have described the molecular mechanisms of physiological and photosynthetic changes in response to cytokinin, particularly in woody plants. DNA methylation is an essential epigenetic modification that dynamically regulates gene expression in response to the external environment. In this study, we examined genome-wide DNA methylation variation and transcriptional variation in poplar (Populus tomentosa) after short-term treatment with the synthetic cytokinin 6-benzylaminopurine (6-BA). We identified 460 significantly differentially methylated regions (DMRs) in response to 6-BA treatment. Transcriptome analysis showed that 339 protein-coding genes, 262 long non-coding RNAs (lncRNAs), and 15,793 24-nt small interfering RNAs (siRNAs) were differentially expressed under 6-BA treatment. Among these, 79% were differentially expressed between alleles in P. tomentosa, and 102,819 allele-specific expression (ASE) loci in 19,200 genes were detected showing differences in ASE levels after 6-BA treatment. Combined DNA methylation and gene expression analysis demonstrated that DNA methylation plays an important role in regulating allele-specific gene expression. To further investigate the relationship between these 6-BA-responsive genes and phenotypic variation, we performed SNP analysis of 460 6-BA-responsive DMRs via re-sequencing using a natural population of P. tomentosa, and we identified 206 SNPs that were significantly associated with growth and wood properties. Association analysis indicated that 53% of loci with allele-specific expression had primarily dominant effects on poplar traits. Our comprehensive analyses of P. tomentosa DNA methylation and the regulation of allele-specific gene expression suggest that DNA methylation is an important regulator of imbalanced expression between allelic loci.

Identification of a transcriptional regulatory module that reduces leaf temperature in poplar under heat stress.

A stable leaf temperature provides plants with a suitable microenvironment for photosynthesis. With global warming, extreme temperatures have become more frequent and severe; therefore, it is increasingly important to understand the fine regulation of leaf temperature under heat stress. In this study, five poplar species (Populus tomentosa, Populus simonii, Populus euphratica, Populus deltoides and Populus trichocarpa) that live in different native environments were used to analyze leaf temperature regulation. Leaf temperatures were more stable in Populus simonii and Populus euphratica (adapted to water-deficient regions) under elevated ambient temperature. Although transpiration contributes strongly to leaf cooling in poplar, the thicker epidermis and mesophyll and lower absorbance of Populus simonii and Populus euphratica leaves also help reduce leaf temperature, since their leaves absorb less radiation. Co-expression network and association analysis of a natural population of P. simonii indicated that PsiMYB60.2, PsiMYB61.2 and PsiMYB61.1 play dominant roles in coordinating leaf temperature, stomatal conductance and transpiration rate in response to heat stress. Individuals with CT-GT-GT genotypes of these three candidate genes have significantly higher water-use efficiency, and balance leaf temperature cooling with photosynthetic efficiency. Therefore, our findings have clarified the genetic basis of leaf cooling among poplar species and laid the foundation for molecular breeding of thermostable, water-conserving poplar varieties.