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Poplars play important role in global forestry. Besides being cultivated as an agro-forestry crop, they provide mankind with fuel wood, timber wood, plywood, sports goods, raw material for paper industry and recently, they are being used as a potential biofuel source. However, a number of biotic and abiotic stresses reduce poplar yield and productivity. Further, there is a strong felt need of paper and pulp industries to have reduced lignin content or modified lignin composition of poplar wood, which will assist in manufacturing better quality paper using less environmentally hazardous chemicals. Because of the non-availability of resistance donor sources and long generation period, conventional breeding could achieve success in genetic improvement of poplars. To circumvent these bottlenecks, genetic engineering interventions have been applied for poplar crop improvement. With the availability of Populus trichocarpa genome sequence in public domain, now numerous genes and transcription factors governing various metabolic pathways got identified, which can be targeted for genetic engineering for poplar improvement. Genome editing technology has really expedited the pace of poplar improvement programmes. This review provides an insight into various research efforts focused on poplar genetic engineering for improvement of silviculturally important traits.

Tension wood (TW) is a specialized xylem tissue developed under mechanical/tension stress in angiosperm trees. TW development involves transregulation of secondary cell wall genes, which leads to altered wood properties for stress adaptation. We induced TW in the stems of black cottonwood (Populus trichocarpa, Nisqually-1) and identified two significantly repressed transcription factor (TF) genes: class B3 heat-shock TF (HSFB3-1) and MYB092. Transcriptomic analysis and chromatin immunoprecipitation (ChIP) were used to identify direct TF–DNA interactions in P. trichocarpa xylem protoplasts overexpressing the TFs. This analysis established a transcriptional regulatory network in which PtrHSFB3-1 and PtrMYB092 directly activate 8 and 11 monolignol genes, respectively. The TF–DNA interactions were verified for their specificity and transactivator roles in 35 independent CRISPR-based biallelic mutants and overexpression transgenic lines of PtrHSFB3-1 and PtrMYB092 in P. trichocarpa. The gene-edited trees (mimicking the repressed PtrHSFB3-1 and PtrMYB092 under tension stress) have stem wood composition resembling that of TW during normal growth and under tension stress (i.e., low lignin and high cellulose), whereas the overexpressors showed an opposite effect (high lignin and low cellulose). Individual overexpression of the TFs impeded lignin reduction under tension stress and restored high levels of lignin biosynthesis in the TW. This study offers biological insights to further uncover how metabolism, growth, and stress adaptation are coordinately regulated in trees.

Understanding the mechanisms behind lignin formation is an important research area with significant implications for the bioenergy and biomaterial industries. Computational models are indispensable tools for understanding this complex process. Models of the monolignol pathway in Populus trichocarpa and other plants have been developed to explore how transgenic modifications affect important bioenergy traits. Many of these models, however, only capture one level of biological organization and are unable to capture regulation across multiple biological scales. This limits their ability to predict how gene modification strategies will impact lignin and other wood properties. While the first multiscale model of lignin biosynthesis in P. trichocarpa spanned the transcript, protein, metabolic, and phenotypic layers, it did not account for cross-regulatory influences that could impact abundances of untargeted monolignol transcripts and proteins. Here, we present a multiscale model incorporating these cross-regulatory influences for predicting lignin and wood traits from transgenic knockdowns of the monolignol genes. The three main components of this multiscale model are (1) a transcript-protein model capturing cross-regulatory influences, (2) a kinetic-based metabolic model, and (3) random forest models relating the steady state metabolic fluxes to 25 physical traits. We demonstrate that including the cross-regulatory behavior results in smaller predictive error for 23 of the 25 traits. We use this multiscale model to explore the predicted impact of novel combinatorial knockdowns on key bioenergy traits, and identify the perturbation of PtrC3H3 and PtrCAld5H1&2 monolignol genes as a candidate strategy for increasing saccharification efficiencies while reducing negative impacts on wood density and height.

Changes in poplar (Populus trichocarpa) wood porous structure after liquid hot water (LHW) pretreatment. The aim of this research was to investigate the effect of applying different hydrothermal pretreatment conditions on the porous structure of poplar wood. Porosity is recognised as an important factor considering efficiency of an enzymatic hydrolysis as a step of bioethanol production. Native poplar wood as well as solid fractions after pretreatment performed at different temperatures (160 °C, 175 °C and 190 °C) were analysed. Porous structure was examined with an inverse size-exclusion chromatography (ISEC) method. Results indicated a significant development of the porous structure of the biomass with increasing porosity along with the growing temperature of the LHW process. The temperature of 190 °C was chosen as the most promising condition of poplar wood LHW pretreatment in terms of the efficiency of the subsequent steps of bioethanol production. The obtained results were consistent with the previous experimental data procured during analysis of the LHW pretreated poplar wood and its subsequent enzymatic hydrolysis yield.

In trees, wood formation needs carbon import from the photosynthetic source tissues. Sugar transporters play important roles in carbohydrate transport into wood-forming cells. Sugars will eventually be exported transporters (SWEETs) play essential roles in many physiological processes. However, the roles of this family in the growth and development of woody plants have not been systematically investigated. In this study, 27 SWEET genes were identified in the Populus trichocarpa genome. These SWEET genes were classified into four clades based on their phylogenetic relationships, gene structures, conserved motifs and chromosomal locations. Representative SWEET members from each clade were selected for further studies. The PagSWEETs of Populus alba × Populus glandulosa were localized to plasma membrane, vacuolar, endoplasmic reticulum or Golgi. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that PagSWEETs have distinct expression patterns in various tissues, and PagSWEET5, 7, 10b, 10c, 15b, 17a and 17c exhibited high expression levels in stems. PagSWEET7 is localized to the cytoplasmic membrane and specifically expressed in the phloem as detected by histochemical GUS ($\beta $ - glucuronidase) assays. Xylem production and xylem sugar content were greater in developing wood of PagSWEET7 overexpression than wild-type lines. Collectively, these results provide valuable information for further investigating functions of PagSWEET genes, and identify PagSWEET7 as a candidate gene for using biotechnology to modify the wood formation in poplar.

Despite the pivotal role of jasmonic acid in the outcome of plant-microorganism interactions, JA-signaling components in roots of perennial trees like western balsam poplar (Populus trichocarpa) are poorly characterized. Here we decipher the poplar-root JA-perception complex centered on PtJAZ6, a co-repressor of JA-signaling targeted by the effector protein MiSSP7 from the ectomycorrhizal basidiomycete Laccaria bicolor during symbiotic development. Through protein–protein interaction studies in yeast we determined the poplar root proteins interacting with PtJAZ6. Moreover, we assessed via yeast triple-hybrid how the mutualistic effector MiSSP7 reshapes the association between PtJAZ6 and its partner proteins. In the absence of the symbiotic effector, PtJAZ6 interacts with the transcription factors PtMYC2s and PtJAM1.1. In addition, PtJAZ6 interacts with it-self and with other Populus JAZ proteins. Finally, MiSSP7 strengthens the binding of PtJAZ6 to PtMYC2.1 and antagonizes PtJAZ6 homo-/heterodimerization. We conclude that a symbiotic effector secreted by a mutualistic fungus may promote the symbiotic interaction through altered dynamics of a JA-signaling-associated protein–protein interaction network, maintaining the repression of PtMYC2.1-regulated genes.

Growth of biomass for lignocellulosic biofuels and biomaterials may take place on land unsuitable for foods, meaning the biomass plants are exposed to increased abiotic stresses. Thus, the understanding how this affects biomass composition and quality is important for downstream bioprocessing. Here, we analyzed the effect of drought and salt stress on cell wall biosynthesis in young shoots and xylem tissues of Populus trichocarpa using transcriptomic and biochemical methods. Following exposure to abiotic stress, stem tissues reduced vessel sizes, and young shoots increased xylem formation. Compositional analyses revealed a reduction in the total amount of cell wall polysaccharides. In contrast, the total lignin amount was unchanged, while the ratio of S/G lignin was significantly decreased in young shoots. Consistent with these observations, transcriptome analyses show that the expression of a subset of cell wall-related genes is tightly regulated by drought and salt stresses. In particular, the expression of a part of genes encoding key enzymes for S-lignin biosynthesis, caffeic acid O-methyltransferase and ferulate 5-hydroxylase, was decreased, suggesting the lower S/G ratio could be partly attributed to the down-regulation of these genes. Together, our data identifies a transcriptional abiotic stress response strategy in poplar, which results in adaptive changes to the plant cell wall.

In a warming climate, the ability to accurately predict and track shifting environmental conditions will be fundamental for plant survival. Environmental cues define the transitions between growth and dormancy as plants synchronise development with favourable environmental conditions, however these cues are predicted to change under future climate projections which may have profound impacts on tree survival and growth. Here, we use a quantitative genetic approach to estimate the genetic basis of spring and autumn phenology in Populus trichocarpa to determine this species capacity for climate adaptation. We measured bud burst, leaf coloration, and leaf senescence traits across two years (2017–2018) and combine these observations with measures of lifetime growth to determine how genetic correlations between phenology and growth may facilitate or constrain adaptation. Timing of transitions differed between years, although we found strong cross year genetic correlations in all traits, suggesting that genotypes respond in consistent ways to seasonal cues. Spring and autumn phenology were correlated with lifetime growth, where genotypes that burst leaves early and shed them late had the highest lifetime growth. We also identified substantial heritable variation in the timing of all phenological transitions (h2 = 0.5–0.8) and in lifetime growth (h2 = 0.8). The combination of additive variation and favourable genetic correlations in phenology traits suggests that populations of cultivated varieties of P. Trichocarpa may have the capability to adapt their phenology to climatic changes without negative impacts on growth.

We used small-angle neutron scattering (SANS) and wide-angle X-ray scattering (WAXS) to study two Populus trichocarpa variants, GW-11012 and BESC-316, with lignin contents of 17.8 and 23.2%, respec...

The multisubunit Elongator complex plays key roles in transcription by interacting with RNA polymerase II and chromatin modeling. Kti proteins have been identified as the auxiliary protein for the Elongator complex. However, our knowledge of Kti proteins in woody plants remains limited. In this study, in total 16 KTI gene homologs were identified in Populus trichocarpa. Among them, the two KTI12 candidates were named PtKTI12A and PtKTI12B. Although PtKTI12A and PtKTI12B were largely different in gene expression level and tissue specificity, both genes were induced by heat and drought stresses. PtKTI12A and PtKTI12B RNAi transgenic poplar plants showed reduced levels of modified nucleosides, in particular 5-carbamoylmethyluridine and 5-methoxycarbonylmethyl-2-thiouridine. Meanwhile, their tolerance to drought was improved when subjected to withdrawal of watering. Also, the protein products of PtKTI12A and PtKTI12B had similar subcellular localization and predicted tertiary structure. The results suggest that Kti12 proteins are involved in tRNA wobble uridine modification, stress response and drought stress tolerance in hybrid poplar.

Fungal pretreatment of Populus trichocarpa wood genetically modified to reduce lignin and alter lignin chemistry is investigated for its effectiveness as an alternative to common pretreatment methods. The goal of this work is to improve biomass utilization for biofuel and biochemical applications by increasing sugar release. Sugar release after enzymatic hydrolysis was measured after various biomass pretreatments (including wood-rot fungus, hot water, and dilute acid). In the wildtype, and in constructs downregulated in PAL, 4CL, and C3H, the fungal pretreatment resulted in substantial improvements in sugar yields, up to 2.4-fold increase in glucose yield and 6-fold increase in xylose yield after enzymatic hydrolysis compared to the unpretreated control. However, the effects of fungal pretreatment were inconsistent, and in genetic lines down-regulated in 4CL, CCoAOMT, CAld5H, and C3H, fungal pretreatment yielded similar or decreased sugar release after enzymatic hydrolysis.

We identified 15 genes encoding enzymes for phenylpropanoid biosynthesis in the genome of birch by combining phylogenetic analysis and tissue-specific expression data. Lignin is one of the most abundant terrestrial biopolymers and is essential for plant structure and defense. An essential step in lignin formation is phenylpropanoid synthesis, we identified 120 gene models in 10 protein families encoding enzymes for phenylpropanoid biosynthesis in the genome of Betula pendula. The transcript abundance was determined for all 120 genes in xylem, root, leaf, and flower tissues using RNA-seq technology. We identified 15 genes that likely encode phenylpropanoid biosynthesis enzymes during wood formation. Ten of these genes are evolutionarily conserved compared to the lignin genes in Populus trichocarpa.

Genetic sources of phenotypic variation have been a focus of plant studies aimed at improving agricultural yield and understanding adaptive processes. Genome-wide association studies identify the genetic background behind a trait by examining associations between phenotypes and single-nucleotide polymorphisms (SNPs). Although such studies are common, biological interpretation of the results remains a challenge; especially due to the confounding nature of population structure and the systematic biases thus introduced. Here, we propose a complementary analysis (SNPeffect) that offers putative genotype-to-phenotype mechanistic interpretations by integrating biochemical knowledge encoded in metabolic models. SNPeffect is used to explain differential growth rate and metabolite accumulation in A. thaliana and P. trichocarpa accessions as the outcome of SNPs in enzyme-coding genes. To this end, we also constructed a genome-scale metabolic model for Populus trichocarpa, the first for a perennial woody tree. As expected, our results indicate that growth is a complex polygenic trait governed by carbon and energy partitioning. The predicted set of functional SNPs in both species are associated with experimentally characterized growth-determining genes and also suggest putative ones. Functional SNPs were found in pathways such as amino acid metabolism, nucleotide biosynthesis, and cellulose and lignin biosynthesis, in line with breeding strategies that target pathways governing carbon and energy partition.

Background Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. Here, we assay 17 lines of unpretreated transgenic black cottonwood (Populus trichocarpa) utilizing a lignocellulose-degrading, metabolically engineered bacterium, Caldicellulosiruptor bescii. The poplar lines were assessed by incubation with an engineered C. bescii strain that solubilized and converted the hexose and pentose carbohydrates to ethanol and acetate. The resulting fermentation titer and biomass solubilization were then utilized as a measure of biomass recalcitrance and compared to data previously reported on the transgenic poplar samples. Results Of the 17 transgenic poplar lines examined with C. bescii, a wide variation in solubilization and fermentation titer was observed. While the wild type poplar control demonstrated relatively high recalcitrance with a total solubilization of only 20% and a fermentation titer of 7.3 mM, the transgenic lines resulted in solubilization ranging from 15 to 79% and fermentation titers from 6.8 to 29.6 mM. Additionally, a strong inverse correlation (R2 = 0.8) between conversion efficiency and lignin content was observed with lower lignin samples more easily converted and solubilized by C. bescii. Conclusions Feedstock recalcitrance can be significantly reduced with transgenic plants, but finding the correct modification may require a large sample set to identify the most advantageous genetic modifications for the feedstock. Utilizing C. bescii as a screening assay for recalcitrance, poplar lines with down-regulation of coumarate 3-hydroxylase 3 (C3H3) resulted in the highest degrees of solubilization and conversion by C. bescii. One such line, with a growth phenotype similar to the wild-type, generated more than three times the fermentation products of the wild-type poplar control, suggesting that excellent digestibility can be achieved without compromising fitness of the tree.

Salicinoids form a specific class of phenolic glycosides characteristic of the Salicaceae. Although salicinoids accumulate in large amounts and have been shown to be involved in plant defense, their biosynthesis is unclear. We identified two sulfated salicinoids, salicin-7-sulfate and salirepin-7-sulfate, in black cottonwood (Populus trichocarpa). Both compounds accumulated in high amounts in above-ground tissues including leaves, petioles, and stems, but were also found at lower concentrations in roots. A survey of salicin-7-sulfate and salirepin-7-sulfate in a subset of poplar (Populus sp.) and willow (Salix sp.) species revealed a broader distribution within the Salicaceae. To elucidate the formation of these compounds, we studied the sulfotransferase (SOT) gene family in P. trichocarpa (PtSOT). One of the identified genes, PtSOT1, was shown to encode an enzyme able to convert salicin and salirepin into salicin-7-sulfate and salirepin-7-sulfate, respectively. The expression of PtSOT1 in different organs of P. trichocarpa matched the accumulation of sulfated salicinoids in planta. Moreover, RNA interference-mediated knockdown of SOT1 in gray poplar (Populus × canescens) resulted in decreased levels of sulfated salicinoids in comparison to wild-type plants, indicating that SOT1 is responsible for their formation in planta. The presence of a nonfunctional SOT1 allele in black poplar (Populus nigra) was shown to correlate with the absence of salicin-7-sulfate and salirepin-7-sulfate in this species. Food choice experiments with leaves from wild-type and SOT1 knockdown trees suggest that sulfated salicinoids do not affect the feeding preference of the generalist caterpillar Lymantria dispar. A potential role of the sulfated salicinoids in sulfur storage and homeostasis is discussed.

The remobilization and resorption of plant nutrients is considered as a crucial aspect of the seasonal senescence of plant organs. In leaves, the mechanisms responsible for the relocation of valuable compounds are well understood while the related processes in roots are still being debated. Some research indicates that remobilization in roots occurs, while other studies have not found evidence of this process. Considering that the total biomass of fine roots is equal to or greater than that of leaves, clarifying the conflicting reports and ambiguities may provide critical information on the circulation of chemical elements in forest ecosystems. This study provides new information concerning the basis for remobilization processes in roots by combining physiological data with gene expression and protein levels. We suggest that, as in leaves, molecular mechanisms involved in nitrogen (N) resorption are also activated in senescent roots. An analysis of N concentration indicated that N levels decreased during the senescence of both organs. The decrease was associated with an increase in the expression of a glutamine synthetase (GS) gene and a concomitant elevation in the amount of GS—one of the most important enzymes in N metabolism. In addition, significant accumulation of carbohydrates was observed in fine roots, which may represent an adaptation to unfavorable weather conditions that would allow remobilization to occur rather than a rapid death in response to ground frost or cold. Our results provide new insights into the senescence of plant organs and clarify contentious topics related to the remobilization process in fine roots

Lignin, an abundant renewable aromatic biopolymer, is an essential macromolecule in terrestrial vascular plants. Genetic modifications affecting monolignol biosynthesis, which produces lignin monom...

Domestication of plant species has affected the evolutionary dynamics of plant pathogens in agriculture and forestry. A model system for studying the consequences of plant domestication on the evolution of an emergent plant disease is the fungal pathogen Sphaerulina musiva. This ascomycete causes leaf spot and stem canker disease of Populus spp. and their hybrids. A population genomics approach was used to determine the degree of population structure and evidence for selection on the North American population of S. musiva. In total, 122 samples of the fungus were genotyped identifying 120,016 single-nucleotide polymorphisms after quality filtering. In North America, S. musiva has low to moderate degrees of differentiation among locations. Three main genetic clusters were detected: southeastern United States, midwestern United States and Canada, and a new British Columbia cluster (BC2). Population genomics suggest that BC2 is a novel genetic cluster from central British Columbia, clearly differentiated from previously reported S. musiva from coastal British Columbia, and the product of a single migration event. Phenotypic measurements from greenhouse experiments indicate lower aggressiveness of BC2 on Populus trichocarpa. In summary, S. musiva has geographic structure across broad regions indicative of gene flow among clusters. The interconnectedness of the North American S. musiva populations across large geographic distances further supports the hypothesis of anthropogenic-facilitated transport of the pathogen.

Funder: University of Cambridge; Id: http://dx.doi.org/10.13039/501100000735, Aim: The biogeographic history affects adaptive evolution by altering the pattern of genetic variation and provides the background upon which other evolutionary processes are operating. Assessment of the consequence of such eco‐evolutionary interactions is crucial to population's genetic diversity maintenance. Yet, considering how biogeographic and multiple evolutionary processes interact in complex environments to alter genetic diversity in conservation planning remains unresolved. Location: The Pacific Northwest. Methods: Herein I document the impact of geographic landscapes and past climatic fluctuations on the evolutionary processes that drive population divergence of Populus trichocarpa by assembling genomic, climatic and species occurrence data. Results: Based on inferences of demographic history of the species and tests for niche divergence, results show that the British Columbia (BC)‐North and BC‐South populations are ancestral and subject to more recent bottlenecks than the Oregon population. Consistently, ecological niche modelling illustrates that compared to the Last Glacial Maximum (~21 Ka), the present niche suitability of the BC‐North and ‐South populations is overall marginally inferior to that of the Oregon population. However, genomic analysis demonstrates that the Oregon harbours the lowest genetic diversity, possibly due to an extended bottleneck experienced by the population. Model prediction indicates that in the 2050s, the impact of climate change is minor compared to the niche suitability of the species as a whole, but climate change will likely result in considerable genetic shifts over two generations especially in the Oregon and BC‐South populations, which will attenuate along a northward gradient. Main conclusions: This study underpins the long‐lasting impact of demographic histories and natural selection on genetic diversity, rather than highlighting the consequence of recent demographic events (e.g. bottleneck in the Holocene epoch; ~12 Ka) potentially cascading to eroding genetic diversity and yielding conservation urgency.

The CBL-interacting protein kinase (CIPK) gene family plays important roles in plant growth, development, and responses to abiotic stresses. To date, the CIPKs have been extensively identified and characterized in various plants. However, there have been few studies on CIPKs in Dimocarpus longan Lour (D. longan). In this study we identified eight putative CIPKs in D. longan (DlCIPKs) according to RNA-seq data. The physicochemical properties, subcellular localization, conserved motifs, phylogenetic relationships, glycosylation and phosphorylation sites, and gene ontology annotation were analyzed using bioinformatics tools. The tissue-specific and heat-stress-responsive expressions were systematically investigated as well. Ten motifs widely maintained by DlCIPKs were identified. All eight DlCIPKs identified from D. longan and the 50 CIPKs selected from Zea mays, Oryza sativa, Sorghum bicolor, Vitis vinifera, Populus trichocarpa, and Arabidopsis thaliana were categorized together into seven groups; group 7 was the largest and contained 20 CIPKs. Localizations of these eight DlCIPKs were predicted in the plasma membrane, cytoplasm, chloroplast thylakoid membrane, or nucleus and were predicted to differ. All eight DlCIPKs were predicted to be phosphorylated but with different numbers of phosphorylation sites. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that among the eight DlCIPKs, only DlCIPK1, DlCIPK5, and DlCIPK8 were differently expressed; notably, between root and leaf tissues. All eight DlCIPKs responded to heat treatments. The expressions of DlCIPK1, DlCIPK4, DlCIPK5, and DlCIPK6 in D. longan leaves were upregulated to the maximum level after 1 h of heat treatment. In contrast, the expression levels of DlCIPK2, DlCIPK3, DlCIPK7, and DlCIPK8 increased significantly after 4 h of heat treatment. The results of this study will enrich our knowledge of DlCIPKs and establish a foundation for enhancing tolerance to abiotic stresses through genetic engineering in D. longan.

Populus simonii is an important tree in the genus Populus, widely distributed in the Northern Hemisphere and having a long cultivation history. Although this species has ecologically and economically important values, its genome sequence is currently not available, hindering the development of new varieties with wider adaptive and commercial traits. Here, we report a chromosome-level genome assembly of P. simonii using PacBio long-read sequencing data aided by Illumina paired-end reads and related genetic linkage maps. The assembly is 441.38 Mb in length and contain 686 contigs with a contig N50 of 1.94 Mb. With the linkage maps, 336 contigs were successfully anchored into 19 pseudochromosomes, accounting for 90.2% of the assembled genome size. Genomic integrity assessment showed that 1,347 (97.9%) of the 1,375 genes conserved among all embryophytes can be found in the P. simonii assembly. Genomic repeat analysis revealed that 41.47% of the P. simonii genome is composed of repetitive elements, of which 40.17% contained interspersed repeats. A total of 45,459 genes were predicted from the P. simonii genome sequence and 39,833 (87.6%) of the genes were annotated with one or more related functions. Phylogenetic analysis indicated that P. simonii and Populus trichocarpa should be placed in different sections, contrary to the previous classification according to morphology. The genome assembly not only provides an important genetic resource for the comparative and functional genomics of different Populus species, but also furnishes one of the closest reference sequences for identifying genomic variants in an F1 hybrid population derived by crossing P. simonii with other Populus species.

With the booming demand of the electric vehicle industry, the concentration of manganese (Mn) and cobalt (Co) flowing into land ecosystems has also increased significantly. While these transition metals can promote the growth and development of plants, they may become toxic under high concentrations. It is thus important to understand how Mn and Co are distributed in plants to develop novel germplasms for the remediation of these heavy metals in contaminated soils. Here, an MTP gene that encodes the CDF (cation diffusion facilitator) protein in Populus trichocarpa, PtrMTP6, was screened as the key gene involved in the distribution of both Mn and Co in poplar. The PtrMTP6-GFP fusion protein was co-localized with the mRFP-VSR2, showing that PtrMTP6 proteins are present at the pre-vacuolar compartment (PVC). Yeast mutant complementation assays further identified that PtrMTP6 serves as a Mn and Co transporter, reducing yeast cell toxicity after exposure to excessive Mn or Co. Histochemical analyses showed that PtrMTP6 was mainly expressed in phloem, suggesting that PtrMTP6 probably involved in the Mn and Co transport via phloem in plants. Under excess Co, PtrMTP6 overexpressing poplar lines were more severely damaged than the control due to higher Co accumulations in young tissue. PtrMTP6 overexpressing lines showed little change in their tolerance to excess Mn, although young tissues also accumulated more Mn. PtrMTP6 play important roles in Mn and Co distribution in poplar and further research on its regulation will be important to increase bioremediation in Mn and Co polluted ecosystems. We express our great appreciation to Jiayi Sun at Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, for the assistance with confocal laser scanning mi-croscopy. This work was funded by the National Natural Science Foundation of China (31770644) , Sichuan Science and Technology Program (2021YJ0508) and Sichuan International Cooperation Project (2017HH0050) .

Gibberellic acid-stimulated Arabidopsis (GASA) proteins, as cysteine-rich peptides (CRPs), play roles in development and reproduction and biotic and abiotic stresses. Although the GASA gene family has been identified in plants, the knowledge about GASAs in Populus euphratica, the woody model plant for studying abiotic stress, remains limited. Here, we referenced the well-sequenced Populus trichocarpa genome, and identified the GASAs in the whole genome of P. euphratica and P. trichocarpa. 21 candidate genes in P. trichocarpa and 19 candidate genes in P. euphratica were identified and categorized into three subfamilies by phylogenetic analysis. Most GASAs with signal peptides were located extracellularly. The GASA genes in Populus have experienced multiple gene duplication events, especially in the subfamily A. The evolution of the subfamily A, with the largest number of members, can be attributed to whole-genome duplication (WGD) and tandem duplication (TD). Collinearity analysis showed that WGD genes played a leading role in the evolution of GASA genes subfamily B. The expression patterns of P. trichocarpa and P. euphratica were investigated using the PlantGenIE database and the real-time quantitative PCR (qRT-PCR), respectively. GASA genes in P. trichocarpa and P. euphratica were mainly expressed in young tissues and organs, and almost rarely expressed in mature leaves. GASA genes in P. euphratica leaves were also widely involved in hormone responses and drought stress responses. GUS activity assay showed that PeuGASA15 was widely present in various organs of the plant, especially in vascular bundles, and was induced by auxin and inhibited by mannitol dramatically. In summary, this present study provides a theoretical foundation for further research on the function of GASA genes in P. euphratica.

Populus trichocarpa has been studied as a model poplar species through biomolecular approaches and was the first tree species to be genome sequenced. In this study, we employed a high throughput RNA-sequencing (RNA-seq) mediated leaf transcriptome analysis to investigate the response of four different Populus davidiana cultivars to drought stress. Following the RNA-seq, we compared the transcriptome profiles and identified two differentially expressed genes (DEGs) with contrasting expression patterns in the drought-sensitive and tolerant groups, i.e., upregulated in the drought-tolerant P. davidiana groups but downregulated in the sensitive group. Both these genes encode a 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme required for abscisic acid (ABA) biosynthesis. The high-performance liquid chromatography (HPLC) measurements showed a significantly higher ABA accumulation in the cultivars of the drought-tolerant group following dehydration. The Arabidopsis nced3 loss-of-function mutants showed a significantly higher sensitivity to drought stress, ~90% of these plants died after 9 days of drought stress treatment. The real-time PCR analysis of several key genes indicated a strict regulation of drought stress at the transcriptional level in the P. davidiana drought-tolerant cultivars. The transgenic P. davidiana NCED3 overexpressing (OE) plants were significantly more tolerant to drought stress as compared with the NCED knock-down RNA interference (RNAi) lines. Further, the NCED OE plants accumulated a significantly higher quantity of ABA and exhibited strict regulation of drought stress at the transcriptional level. Furthermore, we identified several key differences in the amino acid sequence, predicted structure, and co-factor/ligand binding activity of NCED3 between drought-tolerant and susceptible P. davidiana cultivars. Here, we presented the first evidence of the significant role of NCED genes in regulating ABA-dependent drought stress responses in the forest tree P. davidiana and uncovered the molecular basis of NCED3 evolution associated with increased drought tolerance.

Aims This study explored the specific molecular mechanism of Laccaria bicolor to help Populus trichocarpa resist infection by Botryosphaeria dothidea. Methods and Results Transcriptome technology was used to sequence P. trichocarpa under disease stress, and a total of 6379 differentially expressed genes (DEGs) were identified. A total of 536 new DEGs were induced by L. bicolor during the infection of B. dothidea. L. bicolor helps to prevent and alleviate the infection of B. dothidea by regulating related genes in the cell wall pathway, signal transduction pathway, disease-resistant protein synthesis pathway and antioxidant enzyme synthesis pathway of P. trichocarpa. Conclusion The inoculation of L. bicolor can regulate the expression of genes in the cell wall pathway and enhance the physical defense capabilities of plants. Under disease stress conditions, L. bicolor can regulate signal transduction pathways, disease-resistant related pathways and reactive oxygen species (ROS) clearance pathways to help P. trichocarpa alleviate the disease. Significance and Impact of the Study The research reveals the mechanism of L. bicolor inducing resistance to canker of P. trichocarpa from the molecular level and provides a theoretical basis for the practical application of mycorrhizal fungi to improve plant disease resistance.

The major facilitator superfamily (MFS) is one of the largest known membrane transporter families. MFSs are involved in many essential functions, but studies on the MFS family in poplar have not yet been reported. Here, we identified 41 MFS genes from Populus trichocarpa (PtrMFSs). We built a phylogenetic tree, which clearly divided members of PtrMFS into six groups with specific gene structures and protein motifs/domains. The promoter regions contain various cis-acting elements involved in stress and hormone responsiveness. Genes derived from segmental duplication events are unevenly distributed in 17 poplar chromosomes. Collinearity analysis showed that PtrMFS genes are conserved and homologous to corresponding genes from four other species. Transcriptome data indicated that 40 poplar MFS genes were differentially expressed when treated with Fusarium oxysporum. Co-expression networks and gene function annotations of MFS genes showed that MFS genes tightly co-regulated and closely related in function of transmembrane transport. Taken together, we systematically analyzed structure and function of genes and proteins in the PtrMFS family. Evidence indicated that poplar MFS genes play key roles in plant development and response to a biological stressor.

As the most abundant tissue on Earth1, xylem is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells2. In most angiosperms, fusiform cells are a combination of vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed together by tracheids in other vascular plants2. However, little is known about the developmental programs and evolutionary relationships of these xylem cell types. Through both single-cell and laser-capture microdissection transcriptomic profiling, here we demonstrate the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Cross-species analyses of single-cell trajectories reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed character3, 4, exhibit strong transcriptomic similarity to vessel elements but not libriform fibers, suggesting that water transportation, instead of mechanical support, is the major feature. We also found that the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. This evo-developmental framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history5.

Background Trees such as Populus are planted extensively for reforestation and afforestation. However, their successful establishment greatly depends upon ambient environmental conditions and their relative resistance to abiotic and biotic stresses. Polyphenol oxidase (PPO) is a ubiquitous metalloproteinase in plants, which plays crucial roles in mediating plant resistance against biotic and abiotic stresses. Although the whole genome sequence of Populus trichocarpa has long been published, little is known about the PPO genes in Populus, especially those related to drought stress, mechanical damage, and insect feeding. Additionally, there is a paucity of information regarding hormonal responses at the whole genome level. Results A genome-wide analysis of the poplar PPO family was performed in the present study, and 18 PtrPPO genes were identified. Bioinformatics and qRT-PCR were then used to analyze the gene structure, phylogeny, chromosomal localization, gene replication, cis-elements, and expression patterns of PtrPPOs. Sequence analysis revealed that two-thirds of the PtrPPO genes lacked intronic sequences. Phylogenetic analysis showed that all PPO genes were categorized into 11 groups, and woody plants harbored many PPO genes. Eighteen PtrPPO genes were disproportionally localized on 19 chromosomes, and 3 pairs of segmented replication genes and 4 tandem repeat genomes were detected in poplars. Cis-acting element analysis identified numerous growth and developmental elements, secondary metabolism processes, and stress-related elements in the promoters of different PPO members. Furthermore, PtrPPO genes were expressed preferentially in the tissues and fruits of young plants. In addition, the expression of some PtrPPOs could be significantly induced by polyethylene glycol, abscisic acid, and methyl jasmonate, thereby revealing their potential role in regulating the stress response. Currently, we identified potential upstream TFs of PtrPPOs using bioinformatics. Conclusions Comprehensive analysis is helpful for selecting candidate PPO genes for follow-up studies on biological function, and progress in understanding the molecular genetic basis of stress resistance in forest trees might lead to the development of genetic resources.