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The photosynthetic phenotype of trees undergoes changes and interactions that reflect their abilities to exploit light energy. Environmental disturbances and genetic factors have been recognized as influencing these changes and interactions, yet our understanding of the underlying biological mechanisms remains limited, particularly in stochastic environments. Here, we developed a high-dimensional stochastic differential framework (HDSD) for the genome-wide mapping of quantitative trait loci (QTLs) that regulate competition or cooperation in environment-dependent phenotypes. The framework incorporates random disturbances into system mapping, a dynamic model that views multiple traits as a system. Not only does this framework describe how QTLs regulate a single phenotype, but also how they regulate multiple phenotypes and how they interact with each other to influence phenotypic variations. To validate the proposed model, we conducted mapping experiments using chlorophyll fluorescence phenotype data from Populus simonii. Through this analysis, we identified several significant QTLs that may play a crucial role in photosynthesis in stochastic environments, in which 76 significant QTLs have already been reported to encode proteins or enzymes involved in photosynthesis through functional annotation. The constructed genetic regulatory network allows for a more comprehensive analysis of the internal genetic interactions of the photosynthesis process by visualizing the relationships between SNPs. This study shows a new way to understand the genetic mechanisms that govern the photosynthetic phenotype of trees, focusing on how environmental stochasticity and genetic variation interact to shape their light energy utilization strategies., Competing Interests: Declarations. Conflict of interest: The authors have no relevant financial or nonfinancial interests to disclose., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Calcium-dependent protein kinases (CDPKs) phosphorylate downstream target proteins in response to signals transmitted by free calcium ions (Ca 2+ , one of the second messengers) and thus play important regulatory roles in many biological processes, such as plant growth, development, and stress response., Results: A bioinformatic analysis, as well as thorough evolutionary and expression investigations, were conducted to confirm previous reports of functional evidence for plant CDPKs. Using the Phytozome database's BLAST search engine and the HMM search tool in TBtools software, we discovered that CDPKs are well conserved from green algae to flowering angiosperms in various gene family sizes. Additional investigations of the obtained CDPKs revealed high conservation of domain and motif numbers, gene architectures, and patterns. However, this conservation differed among plant species. Phylogenetic analysis demonstrated that the CDPK gene family diverged from a common ancient gene. Similarly, investigations into plant interspecies evolutionary relationships revealed common ancestral plant species, suggesting speciation of plants and evolution based on plant adaptation and diversification. A search for the driving force of CDPK gene family expansion revealed that dispersed duplication events, among other duplication events, contributed largely to CDPK gene family expansion. Gene localization analysis in P. trichocarpa  demonstrated that most CDPK genes are localized within several cell organelles and bind other kinases and proteins to perform their biological functions efficiently. Using RNA-seq data and qPCR analyses, we postulated that PtCDPKs play functional roles in abiotic stress responses by regulating cold, heat, drought and salt stress to varying extents., Conclusion: The CDPK genes are well conserved in plants and are critical entities in abiotic stress regulation, and further exploration and manipulation of these genes in the future may provide solutions to some of the challenges in agriculture, forestry and food security., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

A dynamical system obtains a wide variety of kinetic realizations, which is advantageous for the analysis of biochemical systems. A reaction network, derived from a dynamical system, may or may not possess some properties needed for a thorough analysis. We improve and extend the work of Johnston and Hong et al. on network translations to network transformations, where the network is modified while preserving the dynamical system. These transformations can shrink, extend, or retain the stoichiometric subspace. Here, we show that a positive dependent network can be translated to a weakly reversible network. Using the kinetic realizations of (1) calcium signaling in the olfactory system and (2) metabolic insulin signaling, we demonstrate the benefits of transformed systems with positive deficiency for analyzing biochemical systems. Furthermore, we present an algorithm for a network transformation of a weakly reversible non-complex factorizable kinetic (NFK) system to a weakly reversible complex factorizable kinetic (CFK) system, thereby enhancing the Subspace Coincidence Theorem for NFK systems of Nazareno et al. Finally, using the transformed kinetic realization of monolignol biosynthesis in Populus xylem, we study the structural and kinetic properties of transformed systems, including the invariance of concordance and variation of injectivity and mono-/multi-stationarity under network transformation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Brassinosteroids (BRs), one of the major classes of phytohormones are essential for various processes of plant growth, development, and adaptations to biotic and abiotic stresses. In Arabidopsis, AtCYP90D1 acts as a bifunctional cytochrome P450 monooxygenase, catalyzing C-23 hydroxylation in the brassinolide biosynthetic pathway. The present study reports the functional characterizations of PtoCYP90D1, one of the AtCYP90D1 homologous genes from Populus tomentosa. The qRT-PCR analysis showed that PtoCYP90D1 was highly expressed in roots and old leaves. Overexpression of PtoCYP90D1 (PtoCYP90D1-OE) in poplar promoted growth and biomass yield, as well as increased xylem area and cell layers. Transgenic plants exhibited a significant increase in plant height and stem diameter as compared to the wild type. In contrast, the CRISPR/Cas9-generated mutation of PtoCYP90D1 (PtoCYP90D1-KO) resulted in significantly decreased biomass production in transgenic plants. Further studies revealed that cell wall components increased significantly in PtoCYP90D1-OE lines but not in PtoCYP90D1-KO lines, as compared to wild-type plants. Overall, the findings indicate a positive role of PtoCYP90D1 in improving growth rate and elevating biomass production in poplar, which will have positive implications for its versatile industrial or agricultural applications., (© 2024. The Author(s).)

The genetic control of many plant traits can be highly complex. Both allelic variation (sequence change) and dosage variation (copy number change) contribute to a plant's phenotype. While numerous studies have investigated the effect of allelic or dosage variation, very few have documented both within the same system, leaving their relative contribution to phenotypic effects unclear. The Populus genome is highly polymorphic, and poplars are fairly tolerant of gene dosage variation. Here, using a previously established Populus hybrid F1 population, we assessed and compared the effect of natural allelic variation and induced dosage variation on biomass, phenology, and leaf morphology traits. We identified QTLs for many of these traits, but our results indicate limited overlap between the QTLs associated with natural allelic variation and induced dosage variation. Additionally, the integration of data from both allelic and dosage variation identifies a larger set of QTLs that together explain a larger percentage of the phenotypic variance. Finally, our results suggest that the effect of the large indels might mask that of allelic QTLs. Our study helps clarify the relationship between allelic and dosage variation and their effects on quantitative traits., Competing Interests: Conflicts of interest The authors declare no competing interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Around 252 million years ago (Late Permian), Earth experienced one of its most significant drought periods, coinciding with a global climate crisis, resulting in a devastating loss of forest trees with no hope of recovery. In the current epoch (Anthropocene), the worsening of drought stress is expected to significantly affect forest communities. Despite extensive efforts, there is significantly less research at the molecular level on forest trees than on annual crop species. Would it not be wise to allocate equal efforts to woody species, regardless of their importance in providing essential furniture and sustaining most terrestrial ecosystems? For instance, the poplar genome is roughly quadruple the size of the Arabidopsis genome and has 1.6 times the number of genes. Thus, a massive effort in genomic studies focusing on forest trees has become inevitable to understand their adaptation to harsh conditions. Nevertheless, with the emerging role and development of high-throughput DNA sequencing systems, there is a growing body of literature about the responses of trees under drought at the molecular and eco-physiological levels. Therefore, synthesizing these findings through contextualizing drought history and concepts is essential to understanding how woody species adapt to water-limited conditions. Comprehensive genomic research on trees is critical for preserving biodiversity and ecosystem function. Integrating molecular insights with eco-physiological analysis will enhance forest management under climate change., (© 2024 Scandinavian Plant Physiology Society.)

The pollution of soil with heavy metals (HMs) has become an environmental problem of global concern. Phytoremediation, whereby plants extract HMs from soil, can efficiently and substantially reduce HM pollution in soil in an environmentally friendly manner. Cadmium-induced protein AS8 (CIPAS8) is present in many plants and its expression is induced by HMs. In this study, PeCIPAS8 and SlCIPAS8 were transformed into 84K poplar to study their effects on tolerance to, and translocation of, cadmium (Cd) in woody plants. Localization analyses showed that two CIPAS8 proteins were localized at the plasma membrane when transiently expressed in tobacco leaf epidermal cells. Compared with wild-type 84K poplar seedlings, transgenic poplar lines overexpressing PeCIPAS8 or SlCIPAS8 showed increased Cd contents and decreased Cd tolerance. Transgenic poplar lines overexpressing PeCIPAS8 or SlCIPAS8 accumulated more Cd in the roots, stems, and leaves, but the plant height did not differ significantly, compared with wild-type 84K poplar under Cd stress during the vegetative stage. CIPAS8 increased the Cd influx rate of transgenic poplar roots compared with that of the wild type, and affected the transcription levels of other metal transporters. These findings show that CIPAS8 increases Cd flux into plant tissues and demonstrate moderate Cd sensitivity of the plant. Therefore, CIPAS8 is an influx transporter with the potential to increase the uptake of toxic HMs by woody plants growing in HM-contaminated soils., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Canker disease caused by the bacterium Lonsdalea populi is one of the most destructive diseases affecting poplar stems. However, the detailed stress response mechanisms of poplar have not been widely characterized. To explore the diverse regulatory RNA landscape and the function of key regulators in poplar subjected to L. populi stress, we integrated time-course experiment with mock-inoculation (CK) and inoculation (IN) with L. populi at the first, third, and sixth day (IN1, IN3, IN6) on Populus × euramericana cv. '74/76' (107), small RNA-seq, whole transcriptome-wide analysis, degradome analysis and transgenic experiments. A total of 98 differentially expressed (DE) miRNA, 17 974 DEmRNA, and 807 DElncRNA were identified in poplar infected by L. populi, presenting dynamic changes over the infection course. Regulatory networks among RNAs were further constructed. Notably, a network centered on ptc-miR482a in CK-vs-IN3 contained most DEGs. We show that miR482a and miR1448 are located in one transcript as a polycistron. Overexpression of pre-miR482a-miR1448 (OX482-1448) and pre-miR482a (OX482) increased poplar susceptibility to canker pathogen with reduced accumulation of reactive oxygen species, while the suppression of miR482a (STTM482) conferred poplar disease resistance. PHA7 was validated as the target of miR482a with degradome sequencing and tobacco transient co-transformation, its expression being downregulated in OX482-1448 and OX482 lines. Additionally, a series of phasiRNAs were triggered by miR482a targeting PHA7, forming regulatory cascades with more RLP, NBS-LRR, and PK genes, further verifying the defense function of miR482a. These findings provide insights for understanding the roles of ncRNAs and regulatory networks involved in poplar immunity., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant homeodomain (PHD) proteins are a family of zinc finger transcription factors that play roles in abiotic stress tolerance. However, their mechanisms in conferring salt tolerance are largely unknown. In this study, we characterized a PHD gene, PdbPHD3, from Populus davidiana × P. bolleana (Shanxin poplar) in response to salt stress. PdbPHD3 is a nuclear protein that is strongly induced by salt and abscisic acid (ABA) treatments. Overexpression of PdbPHD3 conferred salt tolerance, while silencing of PdbPHD3 increased sensitivity to salt. PdbPHD3 could enhance the activities of superoxide dismutase and peroxidase to reduce the abundance of reactive oxygen species, and enhance the osmotic potential by increasing the proline content. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed that PdbPHD3 could bind to various DNA motifs, including the G-box ("CACGTG"), PALBOXAPC ("GGACGG"), and POLLEN1LELAT52 ("TTTCTT"). ChIP-seq combined with RNA sequencing identified a transcription factor gene, squamosa promoter binding protein 3 (PdbSBP3), which is directly regulated by PdbPHD3. Overexpression and silencing of PdbSBP3 improved and decreased salt tolerance, respectively. PdbSBP3 could also regulate all the physiological changes associated with salt tolerance, similar to PdbPHD3. These results suggest that PdbPHD3 confers salt tolerance by regulating PdbSBP3 to reduce ROS accumulation and increase proline content. Therefore, the regulatory axis of PdbPHD3 and PdbSBP3 confers salt tolerance in Shanxin poplar., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The phenylpropanoid pathway is vital for plant growth and development, producing lignin and flavonoids. This study investigates PtrMYB203, a homolog of MYB repressors of proanthocyanidin (PA) biosynthesis in Populus trichocarpa, as a transcriptional repressor in the phenylpropanoid pathway of hybrid poplar (Populus alba x P. glandulosa). Overexpression of PtrMYB203 (35S::PtrMYB203) in hybrid poplar detrimentally impacted plant growth and development. Histological analysis revealed irregular xylem vessel formation and decreased lignin content, corroborated by Klason lignin assays. Moreover, 35S::PtrMYB203 transgenic poplars exhibited significant decreases in anthocyanin and PA accumulations in callus tissues, even under high light conditions. Quantitative RT-PCR analysis and protoplast-based transcriptional activation assay confirmed the downregulation of lignin and flavonoid biosynthesis genes. This genetic modification also alters the expression of several MYB transcription factors, essential for phenylpropanoid pathway regulation. Remarkably, saccharification efficiency in the 35S::PtrMYB203 poplar was improved by over 34% following hot water treatment alone. These findings suggest PtrMYB203 as a potential genetic target for enhancing wood properties for bioenergy production, providing valuable insights into the manipulation of metabolite pathways in woody perennials to advance wood biotechnology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Trehalose-6-phosphate synthase (TPS) genes play an active role in the trehalose metabolism pathway that regulates the responses of plants to diverse stresses. However, the functional identification, comparison, and conservatism of TPS genes in the responses of woody plants, especially poplars, to drought stress remain unclear. Here, the trehalose content of 84K (Populus alba × P. glandulosa) poplars was down-regulated and PagTPS and PagTPP genes had diverse response patterns under drought stress. Physicochemical properties, expression patterns, and functions of PagTPS1 and PagTPS10, two class I members of TPS gene family, were identified and compared. Transgenic 84K poplars overexpressing PagTPS1 and PagTPS10 had significantly higher trehalose content with approximately 138% and 123%, respectively, and stronger drought tolerance compared to WT. PagTPS1 and PagTPS10 promoted the expression of TPPA genes and drought-responsive genes. Accordingly, poplars inhibiting PagTPS1 and PagTPS10 expression via RNA interference had lower trehalose content and drought tolerance. Simultaneously, overexpressing PagTPS1 and PagTPS10 improved the trehalose content and drought tolerance of Arabidopsis. Overall, we proposed a model of the effects of PagTPS1 and PagTPS10 as conservative regulators on the responses of plants to drought, which would provide new insights into the functional explorations of TPS genes in plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Perennial trees have a recurring annual cycle of wood formation in response to environmental fluctuations. However, the precise molecular mechanisms that regulate the seasonal formation of wood remain poorly understood. Our prior study indicates that VCM1 and VCM2 play a vital role in regulating the activity of the vascular cambium by controlling the auxin homoeostasis of the cambium zone in Populus. This study indicates that abscisic acid (ABA) affects the expression of VCM1 and VCM2, which display seasonal fluctuations in relation to photoperiod changes. ABA-responsive transcription factors AREB4 and AREB13, which are predominantly expressed in stem secondary vascular tissue, bind to VCM1 and VCM2 promoters to induce their expression. Seasonal changes in the photoperiod affect the ABA amount, which is linked to auxin-regulated cambium activity via the functions of VCM1 and VCM2. Thus, the study reveals that AREB4/AREB13-VCM1/VCM2-PIN5b acts as a molecular module connecting ABA and auxin signals to control vascular cambium activity in seasonal wood formation., (© 2024 John Wiley & Sons Ltd.)

Populus deltoides is one of the primary tree species for bioenergy production in temperate regions. In arid/semi-arid northern China, the scarcity of water and nitrogen significantly limits the productivity of poplar plantations. The identification of relevant molecular markers can promote the breeding of resource-efficient varieties. In this study, 188 genotypes of P. deltoides from six provenances served as experimental material. Genetic differentiation analysis, analysis of molecular variance (AMOVA), principal coordinate analysis (PCoA), unweighted pair group method with arithmetic mean (UPGMA) clustering, and genetic structure analysis were performed using selected simple sequence repeat (SSR) markers. Based on these analyses, the association analysis of water-use efficiency (WUE) and nitrogen-use efficiency (NUE) were conducted using general linear model (GLM) and mixed linear model (MLM) approaches. The results showed that 15 pairs of SSR primers successfully amplified across all 188 individuals, with an average of 7.33 alleles (Na) observed per primer pair. The polymorphism information content (PIC) ranged from 0.060 to 0.897, with an average of 0.544, indicating high genetic diversity in the selected markers. The average inbreeding coefficient intra-population (Fis), inbreeding coefficient inter-population (Fit), and inter-population genetic fraction coefficient (Fst) values were 0.005, 0.135, and 0.132, respectively, indicating high heterozygosity, substantial inbreeding within populations, and moderate genetic differentiation, with an average gene flow (Nm) of 1.964, suggesting substantial gene flow between populations. Additionally, molecular variance was primarily within individuals (84.12%). Genetic structure analysis revealed four subgroups, with some degree of genetic admixture among the provenances. In the GLM model, 11 markers were significantly associated with five traits ( p < 0.05), with an average contribution rate of 15.82%. Notably, SSR132 and SSR143 were significantly associated with multiple traits ( p < 0.05). The MLM model identified two markers (SSR47 and SSR85) significantly associated with ground diameter ( p < 0.05) and one marker (SSR80) significantly associated with NUE ( p < 0.05). This study identifies loci associated with WUE and NUE, laying a foundation for future genetic improvement and marker-assisted breeding strategies in poplar.

Gene expression is regulated by transcription factors binding to cis-elements in promoters. However, efficient cis-elements for genetic engineering are rarely reported. In this study, we identified an 11 bp cis-element in the PtoCP1 promoter that drives strong constitutive gene expression in Populus tomentosa . A 2,270 bp promoter region upstream of the PtoCP1 gene's translation start site was cloned and named ProPtoCP1. This promoter controls GUS reporter gene expression in the roots, leaves, and stems of Arabidopsis seedlings. Based on the location and density of cis-elements , the PtoCP1 promoter was divided into four fragments by 5'-end deletions. GUS staining and RT-qPCR revealed a key cis-element at -466 to -441 bp essential for gene expression. Further analysis showed that the MYB-TGACG cis-element is a positive regulator, whereas neither MYB nor TGACG alone drove gene expression. This study enhances our understanding of gene expression regulation by cis-elements and provides a valuable tool for genetic engineering., Competing Interests: The authors declare that they have no competing interests., (© 2024 Peng et al.)

Gene duplication and divergence are essential to plant evolution. The Arabidopsis type-A response regulator (ARR) family, negative regulators in cytokinin signaling, exemplifies gene expansion and differential retention. Despite extensive research, the understanding of type-A RR homologs in woody plants remains limited. In this study, the evolution history of type-A RR gene families across four rosids and one monocot has been comprehensively investigated. Focusing on Populus deltoides, a unique pair of dispersed duplicates, PdRR8 and PdFERR, is identified, and their duplication is estimated to have occurred in the common ancestor of the four rosids. The duplication remnants corresponding to PdRR8 have been retained in all rosids but the counterpart of PdFERR has been lost. In poplar, PdRR8 shows the highest expression levels in leaves, while PdFERR is specifically expressed in female floral buds. Among various external stimuli, cold strongly represses PdRR8 promoter activity, whereas 6-BA markedly inhibits that of PdFERR. Overexpression of PdRR8 in the Arabidopsis arr16arr17 double-mutant fully complements the reduced hydrotropic response. In contrast, PdFERR fails to rescue the hydrotropic defects of the mutant. Results of evolutionary, expression and functional analyses indicate that PdRR8, rather than PdFERR, is the true ortholog of the ARR16-ARR17 paralogs. Though PdRR8 and PdFERR originate from a common ancestral gene and evolve under strong negative selection, these two dispersed duplicates have exhibited differential expression and some degree of functional divergence., (© 2024. The Author(s).)

Increasing heatwaves are threatening forest ecosystems globally. Leaf thermal regulation and tolerance are important for plant survival during heatwaves, though the interaction between these processes and water availability is unclear. Genotypes of the widely distributed foundation tree species Populus fremontii were studied in a controlled common garden during a record summer heatwave-where air temperature exceeded 48 °C. When water was not limiting, all genotypes cooled leaves 2 to 5 °C below air temperatures. Homeothermic cooling was disrupted for weeks following a 72-h reduction in soil water, resulting in leaf temperatures rising 3 °C above air temperature and 1.3 °C above leaf thresholds for physiological damage, despite the water stress having little effect on leaf water potentials. Tradeoffs between leaf thermal safety and hydraulic safety emerged but, regardless of water use strategy, all genotypes experienced significant leaf mortality following water stress. Genotypes from warmer climates showed greater leaf cooling and less leaf mortality after water stress in comparison with genotypes from cooler climates. These results illustrate how brief soil water limitation disrupts leaf thermal regulation and potentially compromises plant survival during extreme heatwaves, thus providing insight into future scenarios in which ecosystems will be challenged with extreme heat and unreliable soil water access., Competing Interests: Competing interests statement:The authors declare no competing interest.

Optimizing crops for synergistic soil carbon (C) sequestration can enhance CO2 removal in food and bioenergy production systems. Yet, in bioenergy systems, we lack an understanding of how intraspecies variation in plant traits correlates with variation in soil biogeochemistry. This knowledge gap is exacerbated by both the heterogeneity and difficulty of measuring belowground traits. Here, we provide initial observations of C and nutrients in soil and root and stem tissues from a common garden field site of diverse, natural variant, Populus trichocarpa genotypes-established for aboveground biomass-to-biofuels research. Our goal was to explore the value of such field sites for evaluating genotype-specific effects on soil C, which ultimately informs the potential for optimizing bioenergy systems for both aboveground productivity and belowground C storage. To do this, we investigated variation in chemical traits at the scale of individual trees and genotypes and we explored correlations among stem, root, and soil samples. We observed substantial variation in soil chemical properties at the scale of individual trees and specific genotypes. While correlations among elements were observed both within and among sample types (soil, stem, root), above-belowground correlations were generally poor. We did not observe genotype-specific patterns in soil C in the top 10 cm, but we did observe genotype associations with soil acid-base chemistry (soil pH and base cations) and bulk density. Finally, a specific phenotype of interest (high vs low lignin) was unrelated to soil biogeochemistry. Our pilot study supports the usefulness of decade-old, genetically-variable, Populus bioenergy field test plots for understanding plant genotype effects on soil properties. Finally, this study contributes to the advancement of sampling methods and baseline data for Populus systems in the Pacific Northwest, USA. Further species- and region-specific efforts will enhance C predictability across scales in bioenergy systems and, ultimately, accelerate the identification of genotypes that optimize yield and carbon storage., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Craig et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Renewable alternatives for nonelectrifiable fossil-derived chemicals are needed and plant matter, the most abundant biomass on Earth, provide an ideal feedstock. However, the heterogeneous polymeric composition of lignocellulose makes conversion difficult. Lignin presents a formidable barrier to fermentation of nonpretreated biomass. Extensive chemical and enzymatic treatments can liberate fermentable carbohydrates from plant biomass, but microbial routes offer many advantages, including concomitant conversion to industrial chemicals. Here, testing of lignin content of nonpretreated biomass using the cellulolytic thermophilic bacterium, Anaerocellum bescii , revealed that the primary microbial degradation barrier relates to methoxy substitutions in lignin. This contrasts with optimal lignin composition for chemical pretreatment that favors high S/G ratio and low H lignin. Genetically modified poplar trees with diverse lignin compositions confirm these findings. In addition, poplar trees with low methoxy content achieve industrially relevant levels of microbial solubilization without any pretreatments and with no impact on tree fitness in greenhouse.

Key Message: Fluorescence in situ hybridization with frozen sections of root tips showed difference of chromosome territories distribution between autosome and sex-chromosome homologous pairs in Populus trichocarpa. The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biologic processes. Three-dimensional fluorescence in situ hybridization (3D-FISH) is a powerful tool for analyzing CTs, but its application in plants is limited. In this study, we established a 3D-FISH technique using frozen sections of Populus trichocarpa root tips, which was an improvement over the use of paraffin sections and enabled us to acquire good FISH signals. Using chromosome-specific oligo probes, we were able to analyze CTs in interphase nuclei in three dimensions. The distribution of chromosome pairs 17 and 19 in the 3D-preserved nuclei of P. trichocarpa root tip cells were analyzed and showed that the autosome pair 17 associated more often than sex chromosome 19. This research lays a foundation for further study of the spatial position of chromosomes in the nucleus and the relationship between gene expression and spatial localization of chromosomes in poplar., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The Lateral Organ Boundaries Domain (LBD) proteins, an exclusive family of transcription factors (TFs) found solely in plants, play pivotal roles in lateral organogenesis, stress adaptation, secondary growth, and hormonal signaling responses. In this study, a total of 55 PtLBD TFs from Populus trichocarpa were identified and systematically classified into two subfamilies, designated as subfamily-I and subfamily-II with seven distinct groups based on phylogenetic analysis. Gene structure detection indicated that the difference of phase numbers linking adjacent exons contribute to the variations in splicing patterns among different PtLBD groups. Numerous transcription factor binding sites and cis-elements pertinent to hormone signaling pathways and stress response mechanisms were identified within the upstream promoter regions of the PtLBD genes. Thirty-five PtLBDs were found to be engaged in either tandem or segmental duplications, and genomic collinearity analysis revealed a stronger alignment between PtLBD genes and eudicots plants compared to their relationship with monocots. GO enrichment and temporal-spatio expression patterns showed that PtLBD7 from subfamily-I and PtLBD20 from subfamily-II, along with other 13 PtLBDs, were involved in plant growth and development biological processes. The multilayered hierarchical gene networks (ML-hGRN) mediated by PtLBD7 and PtLBD20 indicated that PtLBDs were mainly function in poplar growth and stress tolerance through a multifaceted and intricate regulatory machinery. This study lays a solid groundwork for delving deeper into the roles and underlying mechanisms of LBD transcription factors in poplar, specifically those related to plant hormones and stress tolerance., (© 2024. The Author(s).)

Fine roots play a crucial role in soil nutrient and water acquisition, significantly contributing to tree growth. Fine roots with a high specific root length (SRL) and small diameter are often considered to help trees grow fast. However, inconsistencies in the literature do not provide a clear basis on the effect of root functional traits, such as SRL or root mass density (RMD), on tree growth rate in phylogenetically related trees. Our aim was to examine relationships between tree growth rate and root functional traits, using clones displaying different growth rates in a hybrid poplar plantation located in New Liskeard, ON, Canada. Fine roots (diameter < 2 mm) samples were collected using soil cores at depths of 0-20, 20-40 and 40-60 cm, and analyzed for morphological, chemical and architectural traits. High SRL and thin fine roots were associated with the least productive clones, which is not consistent with the root economics spectrum (RES) theory. However, the most productive clone had larger fine root diameter and higher root lignin concentrations, probably reducing root construction and maintenance costs and carbon losses. Therefore, at the 0-20 and 20-40 cm depths, tree growth rates showed positive correlations with root diameter and root lignin concentrations, but negative correlations with SRL and root soluble compounds concentration. Increasing RMD at the 0-20 cm depth promoted tree growth rates, showing the importance of soil exploration in the topsoil for tree growth. We conclude that fine root variation does not always follow the RES hypothesis and argue that the rapid growth rate of trees may also be driven by fine root growth in diameter and mass in phylogenetically related trees., (© The Author(s) 2024. Published by Oxford University Press.)

Soil salinization has become a global problem and high salt concentration in soil negatively affects plant growth. In our previous study, we found that overexpression of PsAMT1.2 from Populus simonii could improve the salt tolerance of poplar, but the physiological and molecular mechanism was not well understood. To explore the regulation pathway of PsAMT1.2 in salt tolerance, we investigated the morphological, physiological and transcriptome differences between the PsAMT1.2 overexpression transgenic poplar and the wild type under salt stress. The PsAMT1.2 overexpression transgenic poplar showed better growth with increased net photosynthetic rate and higher chlorophyll content compared with wild type under salt stress. The overexpression of PsAMT1.2 increased the catalase, superoxide dismutase, peroxidase and ascorbate peroxidase activities, and therefore probably enhanced the reactive oxygen species clearance ability, which also reduced the degree of membrane lipid peroxidation under salt stress. Meanwhile, the PsAMT1.2 overexpression transgenic poplar maintained a relatively high K+/Na+ ratio under salt stress. RNA-seq analysis indicated that PsAMT1.2 might improve plant salt tolerance by regulating pathways related to the photosynthetic system, chloroplast structure, antioxidant activity and anion transport. Among the 1056 differentially expressed genes, genes related to photosystem I and photosystem II were up-regulated and genes related to chloride channel protein-related were down-regulated. The result of the present study would provide new insight into regulation mechanism of PsAMT1.2 in improving salt tolerance of poplar., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Populus cathayana × canadansis 'Xinlin 1' ('P.'xin lin 1') with the characteristics of rapid growth and high yield, is frequently attacked by herbivorous insects. However, little is known about how it defenses against Hyphantria cunea (H. cunea) at molecular and biochemical levels. Differences in the transcriptome and metabolome were analyzed after 'P. 'xin lin 1' leaves were fed to H. cunea for 0h, 2h, 4h, 8h, 16h and 24h. In the five comparison groups including 2h vs. CK, 4h vs. CK, 8h vs. CK, 16h vs. CK, and 24h vs. CK, a total of 8925 genes and 842 metabolites were differentially expressed. A total of 825 transcription factors (TFs) were identified, which encoded 56 TF families. The results showed that the top four families with the highest number of TFs were AP2/ERF, MYB, C2C2, bHLH. Analyses of leaves which were fed to H. cunea showed that the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were significantly enriched in plant hormone signal transduction pathway, MAPK signaling pathway, flavonoid, flavone and flavonol and anthocyanin biosynthesis pathway. Additionally, there were a number of genes significantly up-regulated in MAPK signaling pathway. Some compounds involved in plant hormone signal transduction and flavonoid/flavone and flavonol/ anthocyanin pathways such as jasmonic acid (JA), jasmonoyl-L-Isoleucine (JA-Ile), kaempferol and cyanidin-3-O-glucoside were induced in infested 'P.'xin lin 1'. This study provides a new understanding for exploring the dynamic response mechanism of poplar to the infestation of H. cunea., (© 2024. The Author(s).)

LESION SIMULATING DISEASE1 (LSD) family genes play a key role in plant response to abiotic and biotic stress. However, their functions in the resistance of tree to drought stress are still largely not clear. Here, five LSD family genes in poplar genome were identified. Phylogenetic and collinear relationship analysis showed that they belonged to LSD, LSD-one-like 1 (LOL1) and LSD-one-like 2 (LOL2) subfamilies, and experienced two segmental duplication events. PagLSDs were highly conserved in gene structure, and all PagLSDs contained at least two LSD domains. Expression pattern and cis-acting element analyses showed that PagLSDs were widely expressed in different organs, significantly induced by polyethylene glycol, and possessed a great number of plant growth, development, plant hormones, and biotic and abiotic stress elements in their promoter regions. Further physiological experiments with transgenic poplar plants revealed that overexpression of PagLOL1b significantly enhanced the drought tolerance of transgenic plants. The improved drought tolerance was closely associated with the significant increase in stomatal closure, water use efficiency, antioxidant enzyme gene expression and antioxidant enzyme activity in transgenic plants. The results in our study imply that PagLOL1b has great potential in the engineering of new tree varieties resistant to drought stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Our study utilized genome-wide association studies (GWAS) to link nucleotide variants to traits in Populus trichocarpa, a species with rapid linkage disequilibrium decay. The aim was to overcome the challenge of interpreting statistical associations at individual loci without sufficient biological context, which often leads to reliance solely on gene annotations from unrelated model organisms. We employed an integrative approach that included GWAS targeting multiple traits using three individual techniques for lignocellulose phenotyping, expression quantitative trait loci (eQTL) analysis to construct transcriptional regulatory networks around each candidate locus and co-expression analysis to provide biological context for these networks, using lignocellulose biosynthesis in Populus trichocarpa as a case study. The research identified three candidate genes potentially involved in lignocellulose formation, including one previously recognized gene (Potri.005G116800/VND1, a critical regulator of secondary cell wall formation) and two genes (Potri.012G130000/AtSAP9 and Potri.004G202900/BIC1) with newly identified putative roles in lignocellulose biosynthesis. Our integrative approach offers a framework for providing biological context to loci associated with trait variation, facilitating the discovery of new genes and regulatory networks., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Chlorophyll (Chl) is essential for plants to carry out photosynthesis, growth and development processes. Growth-regulating factors (GRFs) play a vital role in regulating Chl degradation in plants. However, the molecular mechanism by which GRF5 regulates Chl degradation in poplar remains unknown. Here we found that overexpression of PpnGRF5-1 increased Chl content in leaves and promoted chloroplast development in poplar. Overexpression of PpnGRF5-1 in poplar delayed Chl degradation induced by external factors, such as hormones, darkness and salt stress. PpnGRF5-1 responded to brassinosteroid (BR) signalling during BR-induced Chl degradation and reduced the expression levels of Chl degradation and senescence-related genes. PpnGRF5-1 inhibited the expression of Chl b reductases PagNYC1 and PagNOL. PpnGRF5-1 could interact with PagBZR1 in the nucleus. PagBZR1 also inhibited the expression of PagNYC1. In addition, we found that the protein-protein interaction between PagBZR1 and PpnGRF5-1 enhanced the inhibitory effect of PpnGRF5-1 on the Chl b reductases PagNYC1 and PagNOL. BZR1 and GRF5-1 were upregulated, and NOL and NYC1 were downregulated in triploid poplars compared to diploids. This study revealed a new mechanism by which PpnGRF5-1 regulates Chl degradation in poplars and lays the foundation for comprehensively analysing the molecular mechanism of Chl metabolism in triploid poplars., (© 2024 John Wiley & Sons Ltd.)

Shoot branching from axillary bud (AB) directly determines plant architecture. However, the mechanism through which AB remains dormant or emerges to form branches as plants grow remains largely unknown. Here, the auxin-strigolactone (IAA-SL) pathway was first shown to regulate shoot branching in poplar, and we found that PagKNAT2/6b could modulate this pathway. PagKNAT2/6b was expressed mainly in the shoot apical meristem and AB and was induced by shoot apex damage. PagKNAT2/6b overexpressing poplar plants (PagKNAT2/6b OE) exhibited multiple branches that mimicked the branching phenotype of nontransgenic plants after decapitation treatment, while compared with nontransgenic controls, PagKNAT2/6b antisense transgenic poplar and Pagknat2/6b mutant lines exhibited a significantly decreased number of branches after shoot apex damage treatment. In addition, we found that PagKNAT2/6b directly inhibits the expression of the key IAA synthesis gene PagYUC6a, which is specifically expressed in the shoot apex. Moreover, overexpression of PagYUC6a in the PagKNAT2/6b OE background reduced the number of branches after shoot apex damage treatment. Overall, we conclude that PagKNAT2/6b responds to shoot apical injury and regulates shoot branching through the IAA-SL pathway. These findings may provide a theoretical basis and candidate genes for genetic engineering to create new forest tree species with different crown types., (© 2024 John Wiley & Sons Ltd.)

Poplars are economically important tree crops and biologically important model plants, which are known to be sensitive to ozone (O 3 ). Although surface O 3 is considered as a significant global environmental issue because of its phytotoxicity and greenhouse effect, the knowledge of the dose-response (DR) relationships in poplars for the assessment of O 3 risk is still limited. Hence, this study aimed at collecting data of studies with manipulative O 3 exposures of poplars within FACE (Free Air Concentration Enhancement) and OTC (Open-Top Chamber) facilities. The datasets contain studies on hybrid poplar clones and a non-hybrid native poplar (Populus nigra L.) reporting both AOT40 (Accumulated exposure Over a Threshold of 40 ppb) and POD1 (Phytotoxic Ozone Dose above a threshold of 1 nmol m -2 Projected Leaf Area [PLA] s -1 ) to compare exposure- and flux-based indices. As a result, linear regression analysis showed that the flux-based POD1 was better than the exposure-based AOT40 to explain the biomass response of poplars to O 3 . From the DR relationships, a critical level (CL) of 5.7 mmol m -2 POD1 has been derived corresponding to 4% biomass growth reduction for hybrid poplar clones, which can be considered very sensitive to O 3 , while the non-hybrid native poplar was less sensitive to O 3 (CL: 10.3 mmol m -2 POD1), although the potential risk of O 3 for this taxon is still high due to very high stomatal conductance. Moreover, the different experimental settings (OTC vs. FACE) have affected the AOT40-based DR relationships but not the POD1-based DR relationships, suggesting that poplar responses to O 3 were principally explained by stomatal O 3 uptake regardless of the different experimental settings and exposure patterns. These results highlight the importance of the flux-based approach, especially when scaling up from experimental datasets to the O 3 risk assessment for poplars at the regional or global scale., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Photosynthesis is the main source of energy for plants to sustain growth and development. Abnormalities in photosynthesis may cause defects in plant development. The elaborate regulatory mechanism underlying photosynthesis remains unclear. In this study, we identified a natural mutant from the Greater Khingan Mountains and named it as "1-T". This mutant had variegated leaf with irregular distribution of yellow and green. Chlorophyll contents and photosynthetic capacity of 1-T were significantly reduced compared to other poplar genotypes. Furthermore, a transcriptome analysis revealed 3269 differentially expressed genes (DEGs) in 1-T. The products of the DEGs were enriched in photosystem I and photosystem II. Three motifs were significantly enriched in the promoters of these DEGs. Yeast one-hybrid, Electrophoretic mobility shift assays and tobacco transient transformation experiments indicated that PdGLKs may bind to the three motifs. Further analysis indicated that these photosystem related genes were also significantly down-regulated in PdGLK-RNAi poplars. Therefore, we preliminarily concluded that the down-regulation of PdGLKs in 1-T may affect the expression of photosystem-related genes, resulting in abnormal photosystem development and thus affecting the growth and development. Our results provide new insights into the molecular mechanism of photosynthesis regulating plant growth., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Populus tomentosa, an indigenous tree species, is widely distributed and cultivated over 1,000,000 km 2 in China, contributing significantly to forest production, ecological conservation and urban-rural greening. Although a reference genome is available for P. tomentosa, the intricate interspecific hybrid origins, chromosome structural variations (SVs) and sex determination mechanisms remain confusion and unclear due to its broad and even overlapping geographical distribution, extensive morphological variations and cross infiltration among white poplar species. We conducted a haplotype-resolved de novo assembly of P. tomentosa elite individual GM107, which comprises subgenomes a and b with a total genome size of 714.9 Mb. We then analysed the formation of hybrid species and the phylogenetic evolution and sex differentiation across the entire genus. Phylogenomic analyses suggested that GM107 likely originated from a hybridisation event between P. alba (♀) and P. davidiana (♂) which diverged at approximately 3.8 Mya. A total of 1551 chromosome SVs were identified between the two subgenomes. More noteworthily, a distinctive inversion structure spanning 2.15-2.95 Mb was unveiled among Populus, Tacamahaca, Turaga, Aigeiros poplar species and Salix, highlighting a unique evolutionary feature. Intriguingly, a novel sex genotype of the ZY type, which represents a crossover between XY and ZW systems, was identified and confirmed through both natural and artificial hybrids populations. These novel insights offer significant theoretical value for the study of the species' evolutionary origins and serve as a valuable resource for ecological genetics and forest biotechnology., (© 2024 John Wiley & Sons Ltd.)

In the last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulfite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, taking into account the structural complexity of genomes, their size, and their content in unique coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by sequencing capture bisulfite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

DNA methylation is environment-sensitive and can mediate stress responses. In trees, changes in the environment might cumulatively shape the methylome landscape over time. However, because high-resolution methylome studies usually focus on single environmental cues, the stress-specificity and long-term stability of methylation responses remain unclear. Here, we studied the methylome plasticity of a Populus nigra cv. 'Italica' clone widely distributed across Europe. Adult trees from different geographic locations were clonally propagated in a common garden experiment and exposed to cold, heat, drought, herbivory, rust infection, and salicylic acid treatments. Whole-genome bisulfite sequencing revealed stress-induced and naturally occurring DNA methylation variants. In CG/CHG contexts, the same genomic regions were often affected by multiple stresses, suggesting a generic methylome response. Moreover, these variants showed striking overlap with naturally occurring methylation variants between trees from different locations. Drought treatment triggered CHH hypermethylation of transposable elements, affecting entire superfamilies near drought-responsive genes. Thus, we revealed genomic hotspots of methylation change that are not stress-specific and that contribute to natural DNA methylation variation, and identified stress-specific hypermethylation of entire transposon superfamilies with possible functional consequences. Our results underscore the importance of studying multiple stressors in a single experiment for recognizing general versus stress-specific methylome responses., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Aquaporins (AQPs) play an essential role in membrane water transport during plant responses to water stresses centered on conventional upstream signals. Phytohormones (PHs) regulate plant growth and yield, working with transcription factors to help plants withstand environmental challenges and regulate physiological and chemical processes. The AQP gene family is important, so researchers have studied its function and regulatory system in numerous species. Yet, there is a critical gap the understanding of many of their molecular features, thus our full knowledge of AQPs is far-off. In this study, we undertook a broad examination of the AQP family gene in Populus euphratica via bioinformatics tools and analyzed the expression patterns of certain members in response to drought, salt, and hormone stress. A total of 22 AQP genes were examined in P. euphratica , and were categorized into four main groups, including TIPs, PIPs, SIPs, and NIPs based on phylogenetic analysis. Comparable exon-intron gene structures were found by gene structure examination, and similarities in motif number and pattern within the same subgroup was determined by motif analysis. The PeuAQP gene family has numerous duplications, and there is a distinct disparity in how the members of the PeuAQP family react to post-translational modifications. Abiotic stress and hormone responses may be mediated by AQPs, as indicated by the abundance of stress response elements found in 22 AQP genes, as revealed by the promoter's cis-elements prediction. Expression pattern analysis reveals that selected six AQP genes from the PIP subgroup were all expressed in the leaves, stem, and roots with varying expression levels. Moreover, qRT-PCR analysis discovered that the majority of the selected AQP members were up- or down-regulated in response to hormone treatment and abiotic stress. Remarkably, PeuAQP14 and PeuAQP15 appeared to be highly responsive to drought stress and PeuAQP15 exhibited a high response to salt stress. The foliar application of the phytohormones (SA, IAA, GA3, MeJA, and ABA) were found to either activate or inhibit PeuAQP , suggesting that they may mitigate the effects of water shortage of poplar water stress. The present work enhances our knowledge of the practical roles of AQPs in stress reactions and offers fundamental information for the AQP genes in poplar species. It also highlights a direction for producing new varieties of poplar species with drought, salt, and hormone tolerance and holds substantial scientific and ecological importance, offering a potential contribution to the conservation of poplar species in arid regions.

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. & de Not. are the two major forest diseases in the poplar plantations in China, sometimes which can destroy all the poplar seedlings or severely damage mature poplar forests. Hybrid breeding is the most direct and efficient method of controlling and managing tree diseases. However, assessing disease resistance or selecting disease-resistance clones based on In vitro stem inoculation is inefficient, time-consuming, and expensive, limiting the development of hybrid breeding of poplar stem canker disease. In this study, we proposed an alternative method to assess disease resistance to stem canker pathogens through in vivo leaf inoculation. The test materials used in this method can be on 1-year-old poplar saplings or the annual branches of perennial poplars in the greenhouse or the field. The critical step of this alternative method is the selection of inoculating leaves: the 5-7 th newly matured leaves might be the most suitable. The second critical step of the leaf inoculation method is to make wounds on plant leaves through needle pierces, providing sufficient lesions to measure disease severity. For the adequate number of leaves produced in the early stage of poplar breeding, this in vivo leaf inoculation contributes to the rapid, accurate, and large-scale screening of the disease-resistance poplar clones to stem canker pathogens. Moreover, this leaf inoculation method will also serve as an efficient method for screening pathotypes of stem canker disease pathogen C. chrysosperma, B.dothidea, or other poplar stem canker pathogens.

Drought stress seriously threatens plant growth. The improvement of plant water use efficiency (WUE) and drought tolerance through stomatal regulation is an effective strategy for coping with water shortages. Epidermal patterning factor (EPF)/EPF-like (EPFL) family proteins regulate stomatal formation and development in plants and thus contribute to plant stress adaptation. Here, our analysis revealed the presence of 14 PeEPF members in the Populus euphratica genome, which exhibited a relatively conserved gene structure with 1-3 introns. Subcellular localisation prediction revealed that 9 PeEPF members were distributed in the chloroplasts of P. euphratica , and 5 were located extracellularly. Phylogenetic analysis indicated that PeEPFs can be divided into three clades, with genes within the same clade revealing a relatively conserved structure. Furthermore, we observed the evolutionary conservation of PeEPFs and AtEPF/EPFLs in certain domains, which suggests their conserved function. The analysis of cis-acting elements suggested the possible involvement of PeEPFs in plant response to multiple hormones. Transcriptomic analysis revealed considerable changes in the expression level of PeEPFs during treatment with polyethylene glycol and abscisic acid. The overexpression of PeEPF2 resulted in low stomatal density in transgenetic lines. These findings provide a basis for gaining insights into the function of PeEPFs in response to abiotic stress.

Fifteen poplar varieties were used in a field trial to investigate the phytoremediation efficiency, stress resistance, and wood property of poplar hybrid varieties with diverse genetic backgrounds under the composite pollution of heavy metals. The coefficient of variation and clone repeatability for growth traits and Cd concentration were large. The Cd accumulation of poplar varieties 107 and QHQ reached 1.9 and 1.7 mg, respectively, followed by QHB, Ti, 69, and Pa, in which Cd accumulation reached 1.3 mg. Most of the intra-specific hybrid varieties (69, QH1, SL4, T3, and ZL46) had low Cd concentrations and small biomass, resulting in weak Cd accumulation and low phytoremediation efficiency for Cd-polluted soil. By contrast, the inter-sectional and inter-specific hybrid varieties exhibited better growth performance and accumulated higher concentrations of heavy metals than the intra-specific hybrids. The bioconcentration factor and translocation factor of Hg, As, and Pb were less than 1, indicating that poplars have low phytoremediation efficiency for these heavy metals. The hybrids between section Aigeiros and Tacamahaca (QHQ and QHB) and the inter-specific hybrid 107 within section Aigeiros were more resistant to composite heavy metal stress than the other poplar varieties were partially because of their high levels of free proline that exceeded 93 μg·g -1 FW. According to the correlation analysis of the concentrations of the different heavy metals, the poplar roots absorbed different heavy metals in a cooperative manner, indicating that elite poplar varieties with superior capacity for accumulating diverse heavy metals can be bred feasibly. Compared with the intra-specific hybrid varieties, the inter-sectional (QHQ and QHB) and inter-specific (107) hybrid varieties had higher pollution remediation efficiency, larger biomass, higher cellulose content, and lower lignin content, which is beneficial for pulpwood. Therefore, breeding and extending inter-sectional (QHQ and QHB) and inter-specific hybrid varieties can improve the phytoremediation of composite pollution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Angiosperm trees usually develop tension wood (TW) in response to gravitational stimulation. TW comprises abundant gelatinous (G-) fibers with thick G-layers primarily composed of crystalline cellulose. Understanding the pivotal factors governing G-layer formation in TW fiber remains elusive. This study elucidates the role of a Populus trichocarpa COBRA family protein, PtrCOB3, in the G-layer formation of TW fibers. PtrCOB3 expression was upregulated, and its promoter activity was enhanced during TW formation. Comparative analysis with wild-type trees revealed that ptrcob3 mutants, mediated by Cas9/gRNA gene editing, were incapable of producing G-layers within TW fibers and showed severely impaired stem lift. Fluorescence immunolabeling data revealed a dearth of crystalline cellulose in the tertiary cell wall (TCW) of ptrcob3 TW fibers. The role of PtrCOB3 in G-layer formation is contingent upon its native promoter, as evidenced by the comparative phenotypic assessments of pCOB11::PtrCOB3, pCOB3::PtrCOB3, and pCOB3::PtrCOB11 transgenic lines in the ptrcob3 background. Overexpression of PtrCOB3 under the control of its native promoter expedited G-layer formation within TW fibers. We further identified 3 transcription factors that bind to the PtrCOB3 promoter and positively regulate its transcriptional levels. Alongside the primary TCW synthesis genes, these findings enable the construction of a 2-layer transcriptional regulatory network for the G-layer formation of TW fibers. Overall, this study uncovers mechanistic insight into TW formation, whereby a specific COB protein executes the deposition of cellulose, and consequently, G-layer formation within TW fibers., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Drought is one of the major threats to forest productivity. Oxidation stress is common in drought-stressed plants, and plants need to maintain normal life activities through complex reactive oxygen scavenging mechanisms. However, the molecular links between epigenetics, oxidation stress, and drought in poplar (Populus) remain poorly understood. Here, we found that Populus plants overexpressing PtrMYB94, which encodes an R2R3-MYB transcription factor that regulates the abscisic acid signaling pathway, displayed increased tolerance to extreme drought stress via upregulation of embryogenic cell phosphoprotein 44 (PtrECPP44) expression. Further investigation revealed that PtrMYB94 could recruit the histone deacetylases PtrHDA907/908 to the promoter of PtrECPP44 and decrease acetylation at lysine residues 9, 14, and 27 of histone H3, leading to relatively low transcriptional expression levels under normal conditions. Drought induced the expression of PtrMYB94 while preventing interaction of PtrMYB94 with PtrHDA907/908, which relaxed the chromatin structure and facilitated the binding of RNA polymerase II to the PtrECPP44 promoter. The upregulation of PtrECPP44 helped poplar alleviate oxidative damage and maintain normal cell activities. This study establishes a PtrMYB94-PtrECPP44 transcriptional regulatory module modified by PtrHDA907/908 in modulating drought-induced oxidative stress recovery. Therefore, our study reveals an oxidative regulatory mechanism in response to drought stress and provides insights into molecular breeding for stress resistance in poplar., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Arbuscular mycorrhizal fungi (AMF) play a crucial role in regulating the allocation of carbon between source and sink tissues in plants and in regulating their stress responses by changing the sucrose biosynthesis, transportation, and catabolism in plants. Invertase, a key enzyme for plant development, participates in the response of plants to drought stress by regulating sucrose metabolism. However, the detailed mechanisms by which INV genes respond to drought stress in mycorrhizal plants remain unclear. This study examined the sugar content, enzyme activity, and expression profiles of INV genes of Populus simonii × P. nigra (PsnINVs) under two inoculation treatments (inoculation or non-inoculation) and two water conditions (well-watered or drought stress). Results showed that under drought stress, AMF up-regulated the expressions of PsnA/NINV1, PsnA/NINV2, PsnA/NINV3, and PsnA/NINV5 in leaves, which may be related to the enhancement of photosynthetic capacity. Additionally, AMF up-regulated the expressions of PsnA/NINV6, PsnA/NINV10, and PsnA/NINV12 in leaves, which may be related to enhancing osmotic regulation ability and drought tolerance., (© 2024 Scandinavian Plant Physiology Society. Published by John Wiley & Sons Ltd.)

Plastid-mediated RNA interference has emerged as a promising and effective approach for pest management. By expressing high levels of double-stranded RNAs (dsRNAs) in plastid that target essential pest genes, it has been demonstrated to effectively control certain herbivorous beetles and spider mites. However, as plants are sessile organisms, they frequently experience a combination of biotic and abiotic stresses. It remains unclear whether abiotic stress, such as drought stress, influences the accumulation of dsRNAs produced in plastids and its effectiveness in controlling pests. In this study, we aimed to investigate the effects of drought stress on dsACT expression in transplastomic poplar plants and its control efficiency against the willow leaf beetle (Plagiodera versicolora). Our findings revealed that drought stress did not significantly affect the dsRNA contents in transplastomic poplar plants, but it did lead to higher mortality of insect larvae. This increased mortality may be attributed to increased levels of jasmonic acid and cysteine proteinase inhibitor induced by water deficit. These results contribute to understanding of the mechanisms linking water deficit in plants to insect performance and provide valuable insights for implementing appropriate pest control strategies under drought stress conditions., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aspen (Populus tremula L.) is a keystone species and a model system for forest tree genomics. We present an updated resource comprising a chromosome-scale assembly, population genetics and genomics data. Using the resource, we explore the genetic basis of natural variation in leaf size and shape, traits with complex genetic architecture. We generated the genome assembly using long-read sequencing, optical and high-density genetic maps. We conducted whole-genome resequencing of the Umeå Aspen (UmAsp) collection. Using the assembly and re-sequencing data from the UmAsp, Swedish Aspen (SwAsp) and Scottish Aspen (ScotAsp) collections we performed genome-wide association analyses (GWAS) using Single Nucleotide Polymorphisms (SNPs) for 26 leaf physiognomy phenotypes. We conducted Assay of Transposase Accessible Chromatin sequencing (ATAC-Seq), identified genomic regions of accessible chromatin, and subset SNPs to these regions, improving the GWAS detection rate. We identified candidate long non-coding RNAs in leaf samples, quantified their expression in an updated co-expression network, and used this to explore the functions of candidate genes identified from the GWAS. A GWAS found SNP associations for seven traits. The associated SNPs were in or near genes annotated with developmental functions, which represent candidates for further study. Of particular interest was a ~177-kbp region harbouring associations with several leaf phenotypes in ScotAsp. We have incorporated the assembly, population genetics, genomics, and GWAS data into the PlantGenIE.org web resource, including updating existing genomics data to the new genome version, to enable easy exploration and visualisation. We provide all raw and processed data to facilitate reuse in future studies., (© 2024 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Canker caused by Lonsdalea populi has seriously reduced the economic and ecological benefits of poplar. MicroRNAs play vital roles in the response of plants to biotic stress. However, there is little research about the regulatory mechanism of miRNAs among different tree varieties upon pathogen infection. To dissect miRNAs involved in L. populi resistance, three poplar varieties, 2025 (susceptible), 107 (moderately resistant) and Populus. tomentosa cv 'henan' (resistant) were selected to elucidate the expression profiles of miRNAs using small RNA-seq. A total of 227 miRNAs were identified from all varieties. Intriguingly, miR160, miR169, miR171 and miR482b-5p were only identified in the resistant variety P. tomentosa upon pathogen infection, and these miRNAs might be important candidates for future investigation to improve the tolerance of poplar to L. populi. Among all identified miRNAs, 174 were differentially expressed in all varieties. Functional annotation analysis indicated that an array of miRNAs, including miR482, miR472, miR169, miR481, and miR172, should be involved in disease resistance and phytohormone signal transduction. Furthermore, correlation analysis of small RNA-seq and RNA-seq identified a handful of L. populi-responsive miRNAs and target genes, which exhibited that miR159 and miR172 played key roles in resistant variety P. tomentosa by targeting MYB and ERF, while miR6462c-5p and miR828 were related to the susceptibility of 2025 by targeting MYB. The comprehensive integration analysis in this research provides new insights into the regulatory pathways involved in the defence response of poplar to L. populi and offers crucial candidate miRNAs-target genes modules for poplar resistance improvement., (© 2024 Scandinavian Plant Physiology Society.)

Rare variants contribute significantly to the 'missing heritability' of quantitative traits. The genome-wide characteristics of rare variants and their roles in environmental adaptation of woody plants remain unexplored. Utilizing genome-wide rare variant association study (RVAS), expression quantitative trait loci (eQTL) mapping, genetic transformation, and molecular experiments, we explored the impact of rare variants on stomatal morphology and drought adaptation in Populus. Through comparative analysis of five world-wide Populus species, we observed the influence of mutational bias and adaptive selection on the distribution of rare variants. RVAS identified 75 candidate genes correlated with stomatal size (SS)/stomatal density (SD), and a rare haplotype in the promoter of serine/arginine-rich splicing factor PtoRSZ21 emerged as the foremost association signal governing SS. As a positive regulator of drought tolerance, PtoRSZ21 can recruit the core splicing factor PtoU1-70K to regulate alternative splicing (AS) of PtoATG2b (autophagy-related 2). The rare haplotype PtoRSZ21 hap2 weakens binding affinity to PtoMYB61, consequently affecting PtoRSZ21 expression and SS, ultimately resulting in differential distribution of Populus accessions in arid and humid climates. This study enhances the understanding of regulatory mechanisms that underlie AS induced by rare variants and might provide targets for drought-tolerant varieties breeding in Populus., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Wood production is largely determined by the activity of cambial cell proliferation, and the secondary cell wall (SCW) thickening of xylem cells determines the wood property. In this study, we identified an INDETERMINATE DOMAIN (IDD) type C2H2 zinc finger transcription factor PagIDD15A as a regulator of wood formation in Populus alba × Populus glandulosa. Downregulation of PagIDD15A expression by RNA interference (RNAi) inhibited xylem development and xylem cell secondary wall thickening. RNA-seq analysis showed that PagPAL1, PagCCR2 and PagCCoAOMT1 were downregulated in the differentiating xylem of the PagIDD15A-RNAi transgenic plants, showing that PagIDD15A may regulate SCW biosynthesis through inhibiting lignin biosynthesis. The downregulation of PagVND6-B2, PagMYB10 and PagMYC4 and upregulation of PagWRKY12 in the differentiating xylem of RNAi transgenic plants suggest that PagIDD15A may also regulate these transcription factor (TF) genes to affect SCW thickening. RT-qPCR analysis in the phloem-cambium of RNAi transgenic demonstrates that PagIDD15A may regulate the expression of the genes associated with cell proliferation, including, PagSHR (SHORTROOT), PagSCR (SCARECROW), PagCYCD3;1 (CYCLIN D3;1) and PagSMR4 (SIAMESE-RELATED4), to affect the cambial activity. This study provides the knowledge of the IDD-type C2H2 zinc finger protein in regulating wood formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The formation of adventitious roots (AR) is an essential step in the vegetative propagation of economically woody species. Reactive oxygen species (ROS) function as signaling molecules in regulating root growth and development. Here, we identified an R2R3-MYB transcription factor PagMYB180 as a regulator of AR formation in hybrid poplar (Populus alba × Populus glandulosa). PagMYB180 was specifically expressed in the vascular tissues of poplar roots, stems and leaves, and its protein was localized in the nucleus and acted as a transcriptional repressor. Both dominant repression and overexpression of PagMYB180 resulted in a significant reduction of AR quantity, a substantial increase of AR length, and an elevation of both the quantity and length of lateral roots (LR) compared to the wild type (WT) plants. Furthermore, PagMYB180 regulates programmed cell death (PCD) in root cortex cells, which is associated with elevated levels of ROS. Transcriptome and reverse transcription-quantitative PCR (RT-qPCR) analyses revealed that a series of differentially expressed genes are related to ROS, PCD and ethylene synthesis. Taken together, these results suggest that PagMYB180 may regulate AR development via a ROS/PCD-dependent pathway in poplar., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Vascular cambium in tree species is a cylindrical domain of meristematic cells that are responsible for producing secondary xylem (also called wood) inward and secondary phloem outward. The poplar (Populus trichocarpa) WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family members, PtrWUSa and PtrWOX13b, were previously shown to be expressed in vascular cambium and differentiating xylem cells in poplar stems, but their functions remain unknown. Here, we investigated roles of PtrWUSa, PtrWOX13b and their close homologs in vascular organization and wood formation. Expression analysis showed that like PtrWUSa and PtrWOX13b, their close homologs, PtrWUSb, PtrWUS4a/b and PtrWOX13a/c, were also expressed in vascular cambium and differentiating xylem cells in poplar stems. PtrWUSa also exhibited a high level of expression in developing phloem fibers. Expression of PtrWUSa fused with the dominant EAR repression domain (PtrWUSa-DR) in transgenic poplar caused retarded growth of plants with twisted stems and curled leaves and a severe disruption of vascular organization. In PtrWUSa-DR stems, a drastic proliferation of cells occurred in the phloem region between vascular cambium and phloem fibers and they formed islands of ectopic vascular tissues or phloem fiber-like sclerenchyma cells. A similar proliferation of cells was also observed in PtrWUSa-DR leaf petioles and midveins. On the other hand, overexpression of PtrWOX4a-DR caused ectopic formation of vascular bundles in the cortical region, and overexpression of PtrWOX13a-DR and PtrWOX13b-DR led to a reduction in wood formation without affecting vascular organization in transgenic poplar plants. Together, these findings indicate crucial roles of PtrWUSa and PtrWOX13a/b in regulating vascular organization and wood formation, which furthers our understanding of the functions of WOX genes in regulating vascular cambium activity in tree species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Anoplophora glabripennis (ALB) is one of the most devastating wood boring insects of poplars. Populus deltoides 'Shalinyang (PdS), a new poplar variety, shows strong resistance to ALB infestation. However, the molecular mechanism of insect resistance in PdS is unclear. Here, we found that lignan content was much higher in PdS phloem after ALB infestation than in healthy trees, and that adding lignan to artificial diet significantly reduced: larval weight; digestive enzyme activity (cellulase [CL], polygalacturonase [PG]); detoxification enzyme activity (carboxylesterase [CarE], glutathione S-transferase [GSH-ST]); and defense enzyme activity (Catalase [CAT]). We further identified the lignan biosynthesis-related PdPLR1 gene (Pinoresinol-lariciresinol reductase, PLR) based on transcriptome analysis, and it was significantly up-regulated in the PdS phloem attacked by ALB. Overexpression of PdPLR1 in Arabidopsis increased th lignan content. In contrast, silencing PdPLR1 in PdS significantly decreased expression levels of PdPLR1 and lignan content by 82.45% and 56.85%. However, silencing PdPLR1 increased the number of adults ovipositions and eggs hatching. The activity of CL, PG, CarE, GSH-ST and CAT and the biomass of larvae fed on phloem of PdS with silenced PdPLR1 were significantly higher than in the control. Taken together, up regulation of PdPLR1 enhanced PdS resistance to ALB by regulating lignan synthesis. Our findings provide in-depth insights into the molecular mechanisms of PdS-ALB interactions, which lay the foundation for understanding of defense in poplars to pest infection., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jian feng Liu reports financial support was provided by National Natural Science Foundation of China (No. 32271891)., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

LBD (LATERAL ORGAN BOUNDARIES DOMAIN) transcription factors are key regulators of plant growth and development. In this study, we functionally characterized the PagLBD4 gene in Populus (Populus alba × Populus glandulosa). Overexpression of PagLBD4 (PagLBD4OE) significantly repressed secondary xylem differentiation and secondary cell wall (SCW) deposition, while CRISPR/Cas9-mediated PagLBD4 knockout (PagLBD4KO) significantly increased secondary xylem differentiation and SCW deposition. Consistent with the functional analysis, gene expression analysis revealed that SCW biosynthesis pathways were significantly down-regulated in PagLBD4OE plants but up-regulated in PagLBD4KO plants. We also performed DNA affinity purification followed by sequencing (DAP-seq) to identify genes bound by PagLBD4. Integration of RNA sequencing (RNA-seq) and DAP-seq data identified 263 putative direct target genes (DTGs) of PagLBD4, including important regulatory genes for SCW biosynthesis, such as PagMYB103 and PagIRX12. Together, our results demonstrated that PagLBD4 is a repressor of secondary xylem differentiation and SCW biosynthesis in Populus, which possibly lead to the dramatic growth repression in PagLBD4OE plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The genetic control underlying natural variation in lignin content and composition in trees is not fully understood. We performed a systems genetic analysis to uncover the genetic regulation of lignin biosynthesis in a natural 'SwAsp' population of aspen (Populus tremula) trees. We analyzed gene expression by RNA sequencing (RNA-seq) in differentiating xylem tissues, and lignin content and composition using Pyrolysis-GC-MS in mature wood of 268 trees from 99 genotypes. Abundant variation was observed for lignin content and composition, and genome-wide association study identified proteins in the pentose phosphate pathway and arabinogalactan protein glycosylation among the top-ranked genes that are associated with these traits. Variation in gene expression and the associated genetic polymorphism was revealed through the identification of 312 705 local and 292 003 distant expression quantitative trait loci (eQTL). A co-expression network analysis suggested modularization of lignin biosynthesis and novel functions for the lignin-biosynthetic CINNAMYL ALCOHOL DEHYDROGENASE 2 and CAFFEOYL-CoA O-METHYLTRANSFERASE 3. PHENYLALANINE AMMONIA LYASE 3 was co-expressed with HOMEOBOX PROTEIN 5 (HB5), and the role of HB5 in stimulating lignification was demonstrated in transgenic trees. The systems genetic approach allowed linking natural variation in lignin biosynthesis to trees´ responses to external cues such as mechanical stimulus and nutrient availability., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Plant tau glutathione S-transferase (GSTU) is a kind of multiple functions enzyme, but its specific roles in poplar disease resistance remain uncertain. In this study, 27 PdbGSTU-encoding genes from Populus davidiana × P. bollena were cloned and their protein architectures and phylogenetic relationships were subsequently analyzed. Expression analysis revealed that PdbGSTUs were differentially expressed under Alternaria alternate infection. Then, the PdbGSTU10 was further induced by phytohormones and H 2 O 2 , especially salicylic acid (SA), indicating its potential role in the pathogen defense of poplar. Subsequently, gain- and loss-of-function assays showed that overexpressed PdbGSTU10 activated antioxidant enzymes and significantly decreased reactive oxygen species (ROS) content, ultimately improving the resistance to A. alternate in poplar. Conversely, silencing PdbGSTU10 had the opposite effect. Moreover, overexpressed PdbGSTU10 also increased the content of SA and induced the expression of SA signal-related genes. These results showed that PdbGSTU10 may enhance disease resistance in poplar by scavenging ROS and affecting the SA signaling pathway. Our findings contribute to the understanding of the functions of GSTU in woody plants, particularly in disease resistance., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)