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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.)

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.)

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.)

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).)

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).)

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.)

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.)

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.

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.)

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.)

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.)

The mutualistic interaction between ectomycorrhizal fungi and trees is characterized by the coordinated exchange of soil nutrients with soluble sugars. Despite the importance of this process, the precise mechanism by which sugars are transported from host roots to colonizing hyphae remains unclear. This study aimed to identify the specific membrane transporters responsible for the unloading of sugars at the symbiotic interface, with a focus on the role of the root Sugars Will Eventually Be Exported Transporter (SWEET) uniporters. Our study used RNA sequencing and quantitative PCR to identify PtaSWEET gene expression in Populus tremula × alba-Laccaria bicolor ectomycorrhizal root tips. Our results suggest that symbiosis-induced PtaSWEET1c is primarily responsible for transporting glucose and sucrose, as demonstrated by the yeast assays. Moreover, we used a promoter-YFP reporter to confirm the localization of the PtaSWEET1c expression in cortical cells of ectomycorrhizal rootlets, supporting its major role in supplying glucose at the symbiotic interface. Furthermore, our observations confirmed the localization of PtaSWEET1c-GFP in the plasma membrane. The inactivation of PtaSWEET1c reduced ectomycorrhizal root formation and 13 C translocation to ectomycorrhizal roots. Our findings highlight the crucial role of PtaSWEET1c in facilitating glucose and sucrose transport at the symbiotic interface of Populus-L. bicolor symbiosis., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Expansins are cell wall (CW) proteins that mediate the CW loosening and regulate salt tolerance in a positive or negative way. However, the role of Populus trichocarpa expansin A6 (PtEXPA6) in salt tolerance and the relevance to cell wall loosening is still unclear in poplars. PtEXPA6 gene was transferred into the hybrid species, Populus alba × P. tremula var. glandulosa (84K) and Populus tremula × P. alba INRA '717-1B4' (717-1B4). Under salt stress, the stem growth, gas exchange, chlorophyll fluorescence, activity and transcription of antioxidant enzymes, Na + content, and Na + flux of root xylem and petiole vascular bundle were investigated in wild-type and transgenic poplars. The correlation analysis and principal component analysis (PCA) were used to analyze the correlations among the characteristics and principal components. Our results show that the transcription of PtEXPA6 was downregulated upon a prolonged duration of salt stress (48 h) after a transient increase induced by NaCl (100 mM). The PtEXPA6 -transgenic poplars of 84K and 717-1B4 showed a greater reduction (42-65%) in stem height and diameter growth after 15 days of NaCl treatment compared with wild-type (WT) poplars (11-41%). The Na + accumulation in roots, stems, and leaves was 14-83% higher in the transgenic lines than in the WT. The Na + buildup in the transgenic poplars affects photosynthesis; the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT); and the transcription of PODa2 , SOD [Cu-Zn] , and CAT1 . Transient flux kinetics showed that the Na + efflux of root xylem and leaf petiole vascular bundle were 1.9-3.5-fold greater in the PtEXPA6 -transgenic poplars than in the WT poplars. PtEXPA6 overexpression increased root contractility and extensibility by 33% and 32%, indicating that PtEXPA6 increased the CW loosening in the transgenic poplars of 84K and 717-1B4. Noteworthily, the PtEXPA6 -promoted CW loosening was shown to facilitate Na + efflux of root xylem and petiole vascular bundle in the transgenic poplars. We conclude that the overexpression of PtEXPA6 leads to CW loosening that facilitates the radial translocation of Na + into the root xylem and the subsequent Na + translocation from roots to leaves, resulting in an excessive Na + accumulation and consequently, reducing salt tolerance in transgenic poplars. Therefore, the downregulation of PtEXPA6 in NaCl-treated Populus trichocarpa favors the maintenance of ionic and reactive oxygen species (ROS) homeostasis under long-term salt stress.

The BTB (Broad-complex, tramtrack, and bric-a-brac) gene family, characterized by a highly conserved BTB domain, is implicated in a spectrum of biological processes, encompassing growth and development, as well as stress responses. Characterization and functional studies of BTB genes in poplar are still limited, especially regarding their response to hormones and biotic/abiotic stresses. In this study, we conducted an HMMER search in conjunction with BLASTp and identified 95 BTB gene models in Populus trichocarpa . Through domain motif and phylogenetic relationship analyses, these proteins were classified into eight families, NPH3, TAZ, Ankyrin, only BTB, BACK, Armadillo, TPR, and MATH. Collinearity analysis of poplar BTB genes with homologs in six other species elucidated evolutionary relationships and functional conservations. RNA-seq analysis of five tissues of poplar identified BTB genes as playing a pivotal role during developmental processes. Comprehensive RT-qPCR analysis of 11 BTB genes across leaves, roots, and xylem tissues revealed their responsive expression patterns under diverse hormonal and biotic/abiotic stress conditions, with varying degrees of regulation observed in the results. This study marks the first in-depth exploration of the BTB gene family in poplar, providing insights into the potential roles of BTB genes in hormonal regulation and response to stress.

Tension wood is a specialized xylem tissue associated with gravitropism in angiosperm trees. However, few regulators of tension wood formation have been identified. The molecular mechanisms underpinning tension wood formation remain elusive. Here, we report that a Populus KNOTTED-like homeobox gene, PagKNAT2/6b, is involved in tension wood formation and gravity response. Transgenic poplar plants overexpressing PagKNAT2/6b displayed more sensitive gravitropism than controls, as indicated by increased stem curvature. Microscopic examination revealed greater abundance of fibre cells with a gelatinous cell wall layer (G-layer) and asymmetric growth of secondary xylem in PagKNAT2/6b overexpression lines. Conversely, PagKNAT2/6b dominant repression plants exhibited decreased tension wood formation and reduced response to gravity stimulation. Moreover, sensitivity to gravity stimulation showed a negative relationship with development stage. Expression of genes related to growth and senescence was affected in PagKNAT2/6b transgenic plants. More importantly, transcription activation and electrophoretic mobility shift assays suggested that PagKNAT2/6b promotes the expression of cytokinin metabolism genes. Consistently, cytokinin content was increased in PagKNAT2/6b overexpression plants. Therefore, PagKNAT2/6b is involved in gravitropism and tension wood formation, likely via modulation of cytokinin metabolism., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. This network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription - quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector-reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

The NAC transcription factor family is one of the largest families of TFs in plants, and members of NAC gene family play important roles in plant growth and stress response. Recent release of the haplotype-resolved genome assembly of P. tomentosa provide a platform for NAC protein genome-wide analysis. A total of 270 NAC genes were identified and a comprehensive overview of the PtoNAC gene family is presented, including gene promoter, structure and conserved motif analyses, chromosome localization and collinearity analysis, protein phylogeny, expression pattern, and interaction analysis. The results indicate that protein length, molecular weight, and theoretical isoelectric points of the NAC TF family vary, while gene structure and motif are relatively conserved. Chromosome mapping analysis showed that the P. tomentosa NAC genes are unevenly distributed on 19 chromosomes. The interchromosomal evolutionary results indicate 12 pairs of tandem and 280 segmental duplications. Segmental duplication is possibly related to amplification of P. tomentosa NAC gene family. Expression patterns of 35 PtoNAC genes from P. tomentosa subgroup were analysed under high salinity, and seven NAC genes were induced by this treatment. Promoter and protein interaction network analyses showed that PtoNAC genes are closely associated with growth, development, and abiotic and biotic stress, especially salt stress. These results provide a meaningful reference for follow-up studies of the functional characteristics of NAC genes in the mechanism of stress response and their potential roles in development of P. tomentosa., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Wood formation, which occurs mainly through secondary xylem development, is important not only for supplying raw material for the 'ligno-chemical' industry but also for driving the storage of carbon. However, the complex mechanisms underlying the promotion of xylem formation remain to be elucidated. Here, we found that overexpression of Auxin-Regulated Gene involved in Organ Size (ARGOS) in hybrid poplar 84 K (Populus alba × Populus tremula var. glandulosa) enlarged organ size. In particular, PagARGOS promoted secondary growth of stems with increased xylem formation. To gain further insight into how PagARGOS regulates xylem development, we further carried out yeast two-hybrid screening and identified that the auxin transporter WALLS ARE THIN1 (WAT1) interacts with PagARGOS. Overexpression of PagARGOS up-regulated WAT1, activating a downstream auxin response promoting cambial cell division and xylem differentiation for wood formation. Moreover, overexpressing PagARGOS caused not only higher wood yield but also lower lignin content compared with wild-type controls. PagARGOS is therefore a potential candidate gene for engineering fast-growing and low-lignin trees with improved biomass production., (© 2024 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Wood is resulted from the radial growth paced by the division and differentiation of vascular cambium cells in woody plants, and phytohormones play important roles in cambium activity. Here, we identified that PagJAZ5, a key negative regulator of jasmonate (JA) signaling, plays important roles in enhancing cambium cell division and differentiation by mediating cytokinin signaling in poplar 84K (Populus alba × Populus glandulosa). PagJAZ5 is preferentially expressed in developing phloem and cambium, weakly in developing xylem cells. Overexpression (OE) of PagJAZ5m (insensitive to JA) increased cambium activity and xylem differentiation, while jaz mutants showed opposite results. Transcriptome analyses revealed that cytokinin oxidase/dehydrogenase (CKXs) and type-A response regulators (RRs) were downregulated in PagJAZ5m OE plants. The bioactive cytokinins were significantly increased in PagJAZ5m overexpressing plants and decreased in jaz5 mutants, compared with that in 84K plants. The PagJAZ5 directly interact with PagMYC2a/b and PagWOX4b. Further, we found that the PagRR5 is regulated by PagMYC2a and PagWOX4b and involved in the regulation of xylem development. Our results showed that PagJAZ5 can increase cambium activity and promote xylem differentiation through modulating cytokinin level and type-A RR during wood formation in poplar., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Main Conclusion: Transcription of PagMYB147 was induced in poplar infected by Melampsora magnusiana, and a decline in its expression levels increases the host's susceptibility, whereas its overexpression promotes resistance to rust disease. Poplars are valuable tree species with diverse industrial and silvicultural applications. The R2R3-MYB subfamily of transcription factors plays a crucial role in response to biotic stresses. However, the functional studies on poplar R2R3-MYB genes in resistance to leaf rust disease are still insufficient. We identified 191 putative R2R3-MYB genes in the Populus trichocarpa genome. A phylogenetic analysis grouped poplar R2R3-MYBs and Arabidopsis R2R3-MYBs into 33 subgroups. We detected 12 tandem duplication events and 148 segmental duplication events, with the latter likely being the main contributor to the expansion of poplar R2R3-MYB genes. The promoter regions of these genes contained numerous cis-acting regulatory elements associated with response to stress and phytohormones. Analyses of RNA-Seq data identified a multiple R2R3-MYB genes response to Melampsora magnusiana (Mmag). Among them, PagMYB147 was significantly up-regulated under Mmag inoculation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, and its encoded product was primarily localized to the cell nucleus. Silencing of PagMYB147 exacerbated the severity of Mmag infection, likely because of decreased reactive oxygen species (ROS) production and phenylalanine ammonia-lyase (PAL) enzyme activity, and up-regulation of genes related to ROS scavenging and down-regulation of genes related to PAL, SA and JA signaling pathway. In contrast, plants overexpressing PagMYB147 showed the opposite ROS accumulation, PAL enzyme activity, SA and JA-related gene expressions, and improved Mmag resistance. Our findings suggest that PagMYB147 acts as a positive regulatory factor, affecting resistance in poplar to Mmag by its involvement in the regulation of ROS homeostasis, SA and JA signaling pathway., (© 2024. The Author(s).)

Nonstructural carbohydrate reserves of stems and roots underpin overall tree fitness and productivity under short-rotation management practices such as coppicing for bioenergy. While sucrose and starch comprise the predominant stem carbohydrate reserves of Populus, utilization for fitness and agricultural productivity is understood primarily in terms of starch turnover. The tonoplast sucrose transport protein SUT4 modulates sucrose export from source leaves to distant sinks during photoautotrophic growth, but the possibility of its involvement in remobilizing carbohydrates from storage organs during heterotrophic growth has not been explored. Here, we used PtaSUT4-knockout mutants of Populus tremula × P. alba (INRA 717-1B4) in winter (cool) and summer (warm) glasshouse coppicing experiments to assess SUT4 involvement in reserve utilization. Conditions preceding and supporting summer sprouting were considered favorable for growth, while those preceding and supporting cool temperature sprouting were suboptimal akin to conditions associated with coppicing as generally practiced. Epicormic bud emergence was delayed in sut4 mutants following lower temperature 'winter' but not summer coppicing. Winter xylem hexose increases were observed in control but not in sut4 stumps after coppicing. The magnitude of starch and sucrose reserve depletion was similar in control and sut4 stumps during the winter and did not explain the sprouting and xylem hexose differences. However, winter maintenance costs appeared higher in sut4 based partly on Krebs cycle intermediate levels. In control plants, bark accrual of abundant defense metabolites, including salicinoids and condensed tannins, was higher in summer than in winter, but this increase of summer defense allocations was attenuated in sut4 mutants. Temperature-sensitive trade-offs between growth and other priorities may therefore depend on SUT4 in Populus., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Alternative splicing (AS) serves as a crucial post-transcriptional regulator in plants that contributes to the resistance to salt stress. However, the underlying mechanism is largely unknown. In this research, we identified an important AS transcript in Populus euphratica, PeuHKT1:3a, generated by alternative 3' splice site splicing mode that resulted in the removal of 252 bases at the 5' end of the first exon in PeuHKT1:3. Protein sequence comparison showed that the site of AS occurred in PeuHKT1:3 is located at a crucial Ser residue within the first pore-loop domain, which leads to inefficient K + transport in HKT I-type transporters. Expressing PeuHKT1;3a in an axt3 mutant yeast strain can effectively compensate for the lack of intracellular K + , whereas the expression of PeuHKT1;3 cannot yield the effect. Furthermore, in transgenic Arabidopsis and poplar plants, it was observed that lines expressing PeuHKT1;3a exhibited greater salt tolerance compared to those expressing the PeuHKT1;3 strain. Analysis of ion content and flux demonstrated that the transgenic PeuHKT1;3a line exhibited significantly higher K + content compared to the PeuHKT1;3 line, while there was no significant difference in Na + content. In conclusion, our findings revealed that AS can give rise to novel variants of HKT I-type proteins in P. euphratica with modified K + selectivity to keep a higher K + /Na + ratio to enhanced salt tolerance., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

PXY (Phloem intercalated with xylem) is a receptor kinase required for directional cell division during the development of plant vascular tissue. Drought stress usually affects plant stem cell division and differentiation thereby limiting plant growth. However, the role of PXY in cambial activities of woody plants under drought stress is unclear. In this study, we analyzed the biological functions of two PXY genes (PagPXYa and PagPXYb) in poplar growth and development and in response to drought stress in a hybrid poplar (Populus alba × P. glandulosa, '84K'). Expression analysis indicated that PagPXYs, similar to their orthologs PtrPXYs in Populus trichocarpa, are mainly expressed in the stem vascular system, and related to drought. Interestingly, overexpression of PagPXYa and PagPXYb in poplar did not have a significant impact on the growth status of transgenic plants under normal condition. However, when treated with 8 % PEG6000 or 100 mM H 2 O 2 , PagPXYa and PagPXYb overexpressing lines consistently exhibited more cambium cell layers, fewer xylem cell layers, and enhanced drought tolerance compared to the non-transgenic control '84K'. In addition, PagPXYs can alleviate the damage caused by H 2 O 2 to the cambium under drought stress, thereby maintaining the cambial division activity of poplar under drought stress, indicating that PagPXYs play an important role in plant resistance to drought stress. This study provides a new insight for further research on the balance of growth and drought tolerance in forest trees., 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.)

Glutathione S-transferases (GSTs) constitute a protein superfamily encoded by a large gene family and play a crucial role in plant growth and development. However, their precise functions in wood plant responses to abiotic stress are not fully understood. In this study, we isolated a Phi class glutathione S-transferase-encoding gene, PtrGSTF8, from poplar (Populus alba × P. glandulosa), which is significantly up-regulated under salt stress. Moreover, compared with wild-type (WT) plants, transgenic tobacco plants exhibited significant salt stress tolerance. Under salt stress, PtrGSTF8-overexpressing tobacco plants showed a significant increase in plant height and root length, and less accumulation of reactive oxygen species. In addition, these transgenic tobacco plants exhibited higher superoxide dismutase, peroxidase, and catalase activities and reduced malondialdehyde content compared with WT plants. Quantitative real-time PCR experiments showed that the overexpression of PtrGSTF8 increased the expression of numerous genes related to salt stress. Furthermore, PtrMYB108, a MYB transcription factor involved in salt resistance in poplar, was found to directly activate the promoter of PtrGSTF8, as demonstrated by yeast one-hybrid assays and luciferase complementation assays. Taken together, these findings suggest that poplar PtrGSTF8 contributes to enhanced salt tolerance and confers multiple growth advantages when overexpressed in tobacco., 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.)

Plants adjust their growth and development in response to changing light caused by canopy shade. The molecular mechanisms underlying shade avoidance responses have been widely studied in Arabidopsis and annual crop species, yet the shade avoidance signalling in woody perennial trees remains poorly understood. Here, we first showed that PtophyB1/2 photoreceptors serve conserved roles in attenuating the shade avoidance syndrome (SAS) in poplars. Next, we conducted a systematic identification and characterization of eight PtoPIF genes in Populus tomentosa. Knocking out different PtoPIFs led to attenuated shade responses to varying extents, whereas overexpression of PtoPIFs, particularly PtoPIF3.1 and PtoPIF3.2, led to constitutive SAS phenotypes under normal light and enhanced SAS responses under simulated shade. Notably, our results revealed that distinct from Arabidopsis PIF4 and PIF5, which are major regulators of SAS, the Populus homologues PtoPIF4.1 and PtoPIF4.2 seem to play a minor role in controlling shade responses. Moreover, we showed that PtoPIF3.1/3.2 could directly activate the expression of the auxin biosynthetic gene PtoYUC8 in response to shade, suggesting a conserved PIF-YUC-auxin pathway in modulating SAS in tree. Overall, our study provides insights into shared and divergent functions of PtoPIF members in regulating various aspects of the SAS in Populus., (© 2024 John Wiley & Sons Ltd.)

MicroRNAs are essential in plant development and stress resistance, but their specific roles in drought stress require further investigation. Here, we have uncovered that a Populus-specific microRNAs (miRNA), miR6445, targeting NAC (NAM, ATAF, and CUC) family genes, is involved in regulating drought tolerance of poplar. The expression level of miR6445 was significantly upregulated under drought stress; concomitantly, seven targeted NAC genes showed significant downregulation. Silencing the expression of miR6445 by short tandem target mimic technology significantly decreased the drought tolerance in poplar. Furthermore, 5' RACE experiments confirmed that miR6445 directly targeted NAC029. The overexpression lines of PtrNAC029 (OE-NAC029) showed increased sensitivity to drought compared with knockout lines (Crispr-NAC029), consistent with the drought-sensitive phenotype observed in miR6445-silenced strains. PtrNAC029 was further verified to directly bind to the promoters of glutathione S-transferase U23 (GSTU23) and inhibit its expression. Both Crispr-NAC029 and PtrGSTU23 overexpressing plants showed higher levels of PtrGSTU23 transcript and GST activity while accumulating less reactive oxygen species (ROS). Moreover, poplars overexpressing GSTU23 demonstrated enhanced drought tolerance. Taken together, our research reveals the crucial role of the miR6445-NAC029-GSTU23 module in enhancing poplar drought tolerance by regulating ROS homeostasis. This finding provides new molecular targets for improving the drought resistance of trees., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

WOX11/12 is a homeobox gene of WOX11 and WOX12 in Arabidopsis that plays important roles in crown root development and growth. It has been reported that WOX11/12 participates in adventitious root (AR) formation and different abiotic stress responses, but the downstream regulatory network of WOX11/12 in poplar remains to be further investigated. In this study, we found that PagWOX11/12a is strongly induced by PEG-simulated drought stress. PagWOX11/12a-overexpressing poplar plantlets showed lower oxidative damage levels, greater antioxidant enzyme activities and reactive oxygen species (ROS) scavenging capacity than non-transgenic poplar plants, whereas PagWOX11/12a dominant repression weakened root biomass accumulation and drought tolerance in poplar. RNA-seq analysis revealed that several differentially expressed genes (DEGs) regulated by PagWOX11/12a are involved in redox metabolism and drought stress response. We used RT-qPCR and yeast one-hybrid (Y1H) assays to validate the downstream target genes of PagWOX11/12a. These results provide new insights into the biological function and molecular regulatory mechanism of WOX11/12 in the abiotic resistance processes of poplar., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Plants trigger a robust immune response by activating massive transcriptome reprogramming through crosstalk between PTI and ETI. However, how PTI and ETI contribute to the quantitative or/and qualitative output of immunity and how they work together when both are being activated were unclear. In this study, we performed a comprehensive overview of pathogen-triggered transcriptomic reprogramming by analyzing temporal changes in the transcriptome up to 144 h after Colletotrichum gloeosporioides inoculated in Populus. Moreover, we constructed a hierarchical gene regulatory network of PagWRKY18 and its potential target genes to explore the underlying regulatory mechanisms of PagWRKY18 that are not yet clear. Interestingly, we confirmed that PagWRKY18 protein can directly bind the W-box elements in the promoter of a transmembrane leucine-rich repeat receptor-like kinase, PagSOBIR1 gene, to trigger PTI. At the same time, PagWRKY18 functions in disease tolerance by modulation of ROS homeostasis and induction of cell death via directly targeting PagGSTU7 and PagPR4 respectively. Furthermore, PagPR4 can interact with PagWRKY18 to inhibit the expression of PagPR4 genes, forming a negative feedback loop. Taken together, these results suggest that PagWRKY18 may be involved in regulating crosstalk between PTI and ETI to activate a robust immune response and maintain intracellular homeostasis., (© 2024 John Wiley & Sons Ltd.)

Thaumatin-like proteins (TLPs) in plants are involved in diverse biotic and abiotic stresses, including antifungal activity, low temperature, drought, and high salinity. However, the roles of the TLP genes are rarely reported in early flowering. Here, the TLP gene family was identified in P. trichocarpa . The 49 PtTLP genes were classified into 10 clusters, and gene structures, conserved motifs, and expression patterns were analyzed in these PtTLP genes. Among 49 PtTLP genes, the PtTLP6 transcription level is preferentially high in stems, and GUS staining signals were mainly detected in the phloem tissues of the PtTLP6 pro::GUS transgenic poplars. We generated transgenic Arabidopsis plants overexpressing the PtTLP6 gene, and its overexpression lines showed early flowering phenotypes. However, the expression levels of main flowering regulating genes were not significantly altered in these PtTLP6 -overexpressing plants. Our data further showed that overexpression of the PtTLP6 gene led to a reactive oxygen species (ROS) burst in Arabidopsis , which might advance the development process of transgenic plants. In addition, subcellular localization of PtTLP6 -fused green fluorescent protein (GFP) was in peroxisome, as suggested by tobacco leaf transient transformation. Overall, this work provides a comprehensive analysis of the TLP gene family in Populus and an insight into the role of TLPs in woody plants.

C/S1 basic leucine zipper (bZIP) transcription factors are essential for plant survival under energy deficiency. However, studies on the responses of C/S1-bZIPs to low energy in woody plants have not yet been reported. In this study, members of C/S1-bZIP subfamilies in Populus tomentosa were systematically analyzed using bioinformatic approaches. Four C-bZIPs and 10 S1-bZIPs were identified, and their protein properties, phylogenetic relationships, gene structures, conserved motifs, and uORFs were systematically investigated. In yeast two-hybrid assays, direct physical interactions between C-bZIP and S1-bZIP members were observed, highlighting their potential functional synergy. Moreover, expression profile analyses revealed that low energy induced transcription levels of most C/S1-bZIP members, with bZIP55 and bZIP21 (a homolog of bZIP55 ) exhibiting particularly significant upregulation. When the expression of bZIP55 and bZIP21 was co-suppressed using artificial microRNA mediated gene silencing in transgenic poplars, root growth was promoted. Further analyses revealed that bZIP55/21 negatively regulated the root development of P. tomentosa in response to low energy. These findings provide insights into the molecular mechanisms by which C/S1-bZIPs regulate poplar growth and development in response to energy deprivation.

Wood formation involves consecutive developmental steps, including cell division of vascular cambium, xylem cell expansion, secondary cell wall (SCW) deposition, and programmed cell death. In this study, we identified PagMYB31 as a coordinator regulating these processes in Populus alba × Populus glandulosa and built a PagMYB31-mediated transcriptional regulatory network. PagMYB31 mutation caused fewer layers of cambial cells, larger fusiform initials, ray initials, vessels, fiber and ray cells, and enhanced xylem cell SCW thickening, showing that PagMYB31 positively regulates cambial cell proliferation and negatively regulates xylem cell expansion and SCW biosynthesis. PagMYB31 repressed xylem cell expansion and SCW thickening through directly inhibiting wall-modifying enzyme genes and the transcription factor genes that activate the whole SCW biosynthetic program, respectively. In cambium, PagMYB31 could promote cambial activity through TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/PHLOEM INTERCALATED WITH XYLEM (PXY) signaling by directly regulating CLAVATA3/ESR-RELATED (CLE) genes, and it could also directly activate WUSCHEL HOMEOBOX RELATED4 (PagWOX4), forming a feedforward regulation. We also observed that PagMYB31 could either promote cell proliferation through the MYB31-MYB72-WOX4 module or inhibit cambial activity through the MYB31-MYB72-VASCULAR CAMBIUM-RELATED MADS2 (VCM2)/PIN-FORMED5 (PIN5) modules, suggesting its role in maintaining the homeostasis of vascular cambium. PagMYB31 could be a potential target to manipulate different developmental stages of wood formation., Competing Interests: Conflict of interest statement. The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Photoperiod is a crucial environmental cue for phenological responses, including growth cessation and winter dormancy in perennial woody plants. Two regulatory modules within the photoperiod pathway explain bud dormancy induction in poplar (Populus spp.): the circadian oscillator LATE ELONGATED HYPOCOTYL 2 (LHY2) and GIGANTEA-like genes (GIs) both regulate the key target for winter dormancy induction FLOWERING LOCUS T2 (FT2). However, modification of LHY2 and GIs cannot completely prevent growth cessation and bud set under short-day (SD) conditions, indicating that additional regulatory modules are likely involved. We identified PtoHY5a, an orthologs of the photomorphogenesis regulatory factor ELONGATED HYPOCOTYL 5 (HY5) in poplar (Populus tomentosa), that directly activates PtoFT2 expression and represses the circadian oscillation of LHY2, indirectly activating PtoFT2 expression. Thus, PtoHY5a suppresses SD-induced growth cessation and bud set. Accordingly, PtoHY5a knockout facilitates dormancy induction. PtoHY5a also inhibits bud-break in poplar by controlling gibberellic acid (GA) levels in apical buds. Additionally, PtoHY5a regulates the photoperiodic control of seasonal growth downstream of phytochrome PHYB2. Thus, PtoHY5a modulates seasonal growth in poplar by regulating the PtoPHYB2-PtoHY5a-PtoFT2 module to determine the onset of winter dormancy, and by fine-tuning GA levels to control bud-break., Competing Interests: Conflict of interest statement. 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., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Populus euphratica phospholipase Dδ (PePLDδ) is transcriptionally regulated and mediates reactive oxygen species (ROS) and ion homeostasis under saline conditions. The purpose of this study is to explore the post-transcriptional regulation of PePLDδ in response to salt environment. P. euphratica PePLDδ was shown to interact with the NADP-dependent malic enzyme (NADP-ME) by screening the yeast two-hybrid libraries. The transcription level of PeNADP-ME increased upon salt exposure to NaCl (200 mM) in leaves and roots of P. euphratica. PeNADP-ME had a similar subcellular location with PePLDδ in the cytoplasm, and the interaction between PeNADP-ME and PePLDδ was further verified by GST pull-down and yeast two-hybrid. To clarify whether PeNADP-ME interacts with PePLDδ to enhance salt tolerance, PePLDδ and PeNADP-ME were overexpressed singly or doubly in Arabidopsis thaliana. Dual overexpression of PeNADP-ME and PePLDδ resulted in an even more pronounced improvement in salt tolerance compared with single transformants overexpressing PeNADP-ME or PePLDδ alone. Greater Na +  limitation and Na +  efflux in roots were observed in doubly overexpressed plants compared with singly overexpressed plants with PeNADP-ME or PePLDδ. Furthermore, NaCl stimulation of SOD, APX, and POD activity and transcription were more remarkable in the doubly overexpressed plants. It is noteworthy that the enzymic activity of NADP-ME and PLD, and total phosphatidic acid (PA) concentrations were significantly higher in the double-overexpressed plants than in the single transformants. We conclude that PeNADP-ME interacts with PePLDδ in Arabidopsis to promote PLD-derived PA signaling, conferring Na +  extrusion and ROS scavenging under salt 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 Masson SAS. All rights reserved.)

As sessile organisms, plants experience variable environments and encounter diverse stresses during their growth and development. Adventitious rooting, orchestrated by multiple coordinated signaling pathways, represents an adaptive strategy evolved by plants to adapt to cope with changing environmental conditions. This study uncovered the role of the miR159a-PeMYB33 module in the formation of adventitious roots (ARs) synergistically with abscisic acid (ABA) signaling in poplar. Overexpression of miR159a increased the number of ARs and plant height while reducing sensitivity to ABA in transgenic plants. In contrast, inhibition of miR159a (using Short Tandem Target Mimic) or overexpression of PeMYB33 decreased the number of ARs in transgenic plants. Additionally, miR159a targets and cleaves transcripts of PeMYB33 using degradome analysis, which was further confirmed by a transient expression experiment of poplar protoplast. We show the miR159a-PeMYB33 module controls ARs development in poplar through ABA signaling. In particular, we demonstrated that miR159a promotes the expression of genes in the ABA signaling pathway. The findings from this study shed light on the intricate regulatory mechanisms governing the development of ARs in poplar plants. The miR159a-PeMYB33 module, in conjunction with ABA signaling, plays a crucial role in modulating AR formation and subsequent plant growth., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Expansins are important plant cell wall proteins. They can loosen and soften the cell walls and lead to wall extension and cell expansion. To investigate their role in wood formation and fiber elongation, the PagEXPA1 that highly expressed in cell differentiation and expansion tissues was cloned from 84K poplar (Populus alba × P. glandulosa). The subcellular localization showed that PagEXPA1 located in the cell wall and it was highly expressed in primary stems and young leaves. Compared with non-transgenic 84K poplar, overexpression of PagEXPA1 can promote plant-growth, lignification, and fiber cell elongation, while PagEXPA1 Cas9-editing mutant lines exhibited the opposite phenotype. Transcriptome analysis revealed that DEGs were mainly enriched in some important processes, which are associated with cell wall formation and cellulose synthesis. The protein interaction prediction and expression analysis showed that PagCDKB2:1 and PagEXPA1 might have an interaction relationship. The luciferase complementary assay and bimolecular fluorescence complementary assay validated that PagEXPA1 can combined with PagCDKB2;1. So they promoted the expansion of xylem vascular tissues and the development of poplar though participating in the regulation of cell division and differentiation by programming the cell-cycle. It provides good foundation for molecular breeding of fast-growing and high-quality poplar varieties., 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 B-box proteins (BBXs) encode a family of zinc-finger transcription factors that regulate the plant circadian rhythm and early light morphogenesis. The double B-box ( DBB ) family is in the class of the B-box family, which contains two conserved B-box domains and lacks a CCT (CO, CO-like and TOC1) motif. In this study, the identity, classification, structures, conserved motifs, chromosomal location, cis elements, duplication events, and expression profiles of the PtrDBB genes were analyzed in the woody model plant Populus trichocarpa . Here, 12 PtrDBB genes ( PtrDBB1 - PtrDBB12 ) were identified and classified into four distinct groups, and all of them were homogeneously spread among eight out of seventeen poplar chromosomes. The collinearity analysis of the DBB family genes from P. trichocarpa and two other species ( Z. mays and A. thaliana ) indicated that segmental duplication gene pairs and high-level conservation were identified. The analysis of duplication events demonstrates an insight into the evolutionary patterns of DBB genes. The previously published transcriptome data showed that PtrDBB genes represented distinct expression patterns in various tissues at different stages. In addition, it was speculated that several PtrDBBs are involved in the responsive to drought stress, light/dark, and ABA and MeJA treatments, which implied that they might function in abiotic stress and phytohormone responses. In summary, our results contribute to the further understanding of the DBB family and provide a reference for potential functional studies of PtrDBB genes in P. trichocarpa .

Major latex proteins, or MLPs, are crucial to plants' capacity to grow, develop, and endure biotic and abiotic stresses. The MLP gene family has been found in numerous plants, but little is known about its role in Populus simonii × P. nigra . This study discovered and assessed 43 PtML P genes that were unevenly dispersed throughout 12 chromosomes in terms of their physicochemical characteristics, gene structure, conserved motifs, and protein localization. Based on their phylogeny and protein structural characteristics, three separate subclasses of PtMLP family were identified. Segmental and tandem duplication were found to be essential variables in the expansion of the PtMLP genes. The involvement of the PtMLP genes in growth and development, as well as in the responses to different hormones and stresses, was demonstrated by cis -regulatory element prediction. The PtMLP genes showed varying expression patterns in various tissues and under different conditions (cold, salt, and drought stress), as demonstrated in RNA-Seq databases, suggesting that PsnMLP may have different functions. Following the further investigation of the genes demonstrating notable variations in expression before and after the application of three stresses, PsnMLP5 was identified as a candidate gene. Subsequent studies revealed that PsnMLP5 could be induced by ABA treatment. This study paves the way for further investigations into the MLP genes' functional mechanisms in response to abiotic stressors, as well as the ways in which they can be utilized in poplar breeding for improved stress tolerance.

Forest trees face many abiotic stressors during their lifetime, including drought, heavy metals, high salinity, and chills, affecting their quality and yield. The RING-type ubiquitin ligase E3 is an invaluable component of the ubiquitin-proteasome system (UPS) and participates in plant growth and environmental interactions. Interestingly, only a few studies have explored the RING ZINC FINGER PROTEIN (RZFP) gene family. This study identified eight PtrRZFPs genes in the Populus genome, and their molecular features were analyzed. Gene structure analysis revealed that all PtrRZFPs genes contained >10 introns. Evolutionarily, the RZFPs were separated into four categories, and segmental replication events facilitated their amplification. Notably, many stress-related elements have been identified in the promoters of PtrRZFPs using Cis-acting element analysis. Moreover, some PtrRZFPs were significantly induced by drought and sorbitol, revealing their potential roles in regulating stress responses. Particularly, overexpression of the PtrRZFP1 gene in poplars conferred excellent drought tolerance; however, PtrRZFP1 knockdown plants were drought-sensitive. We identified the potential upstream transcription factors of PtrRZFPs and revealed the possible biological functions of RZFP1/4/7 in resisting osmotic and salt stress, laying the foundation for subsequent biological function studies and providing genetic resources for genetic engineering breeding for drought resistance in forest trees. This study offers crucial information for the further exploration of the functions of RZFPs in poplars., Competing Interests: Declaration of competing interest The authors recognize no competitive interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Lignin is a complex polymer that provides structural support and defense to plants. It is synthesized in the secondary cell walls of specialized cells. Through regulates its stability, LTF1 acts as a switch to control lignin biosynthesis in Populus, a dicot plant. However, how lignin biosynthesis is regulated in rice, a monocot plant, remains unclear. By employing genetic, cellular, and chemical approaches, we discovered that LTF1L1, a rice homolog of LTF1, regulates lignin biosynthesis through a distinct mechanism from Populus LTF1. Knockout of LTF1L1 increased lignin synthesis in the sclerenchyma cells of rice stems, while overexpression of LTF1L1 decreased it. LTF1L1 is phosphorylated by OsMPK6 at Ser 169 , which did not affect its stability but impaired its ability to repress the expression of lignin biosynthesis genes. This was supported by the non-phosphorylated mutant of LTF1L1 (LTF1L1 S169A ), which displayed a stronger repressive effect on lignin biosynthesis in both rice and Populus. Our findings reveal that LTF1L1 acts as a negative regulator of lignin biosynthesis via a distinct mechanism from that of LTF1 in Populus and highlight the evolutionary diversity in the regulation of lignin biosynthesis 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. The authors declare no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The root system of plants consists of primary, lateral, and adventitious roots (ARs) (aka shoot-born roots). ARs arise from stem- or leaf-derived cells during post-embryonic development. Adventitious root development (ARD) through stem cuttings is the first requirement for successful establishment and growth of planted trees; however, the details of the molecular mechanisms underlying ARD are poorly understood. This knowledge is important to both basic plant biology and because of its necessary role in the successful propagation of superior cultivars of commercial woody bioenergy crops, like poplar. In this review article, the molecular mechanisms that control both endogenous (auxin) and environmentally (nutrients and microbes) regulated ARD and how these systems interact to control the rooting efficiency of poplar trees are described. Then, potential future studies in employing integrated systems biology approaches at cellular resolutions are proposed to more precisely identify the molecular mechanisms that cause AR. Using genetic transformation and genome editing approaches, this information can be used for improving ARD in economically important plants for which clonal propagation is a requirement., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023. Published by Elsevier B.V.)

The placenta in fruits of most plants either desiccate and shrink as the fruits mature or develop further to form the fleshy tissues. In poplars, placental epidermal cells protrude collectively to produce catkin fibers. In this study, three carpel limited MIXTA genes, PdeMIXTA02, PdeMIXTA03, PdeMIXTA04, were find to specifically expressed in carpel immediately after pollination. Heterologous expression of the three genes in Arabidopsis demonstrated that PdeMIXTA04 significantly promoted trichomes density and could restore trichomes in the trichomeless mutant. By contrast, such functions were not observed with PdeMIXTA02, PdeMIXTA03. In situ hybridization revealed that PdeMIXTA04 was explicitly expressed in poplar placental epidermal cells. We also confirmed trichome-specific expression of the PdeMIXTA04 promoter. Multiple experimental proofs have confirmed the interaction between PdeMIXTA04, PdeMYC and PdeWD40, indicating PdeMIXTA04 functioned through the MYB-bHLH-WD40 ternary complex. Our work provided distinctive understanding of the molecular mechanism triggering differentiation of poplar catkins., 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 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs) containing both AGP-like glycated domains and fasciclin (FAS) domains, which are involved in plant growth and development and synthesis of the cell wall. However, these proteins have not been identified or analyzed in willow, Salix , the sister genus of Populus . In this study, we performed a whole genome study of the FLA gene family of Salix suchowensis and compared it with the FLA gene family of Populus deltoides . The results showed the presence of 40 and 46 FLA genes in P. deltoides and S. suchowensis , distributed on 17 and 16 chromosomes, respectively. Four pairs of tandem repeat genes were found in willow, while poplar had no tandem repeat genes. Twelve and thirteen pairs of duplicated gene fragments were identified in poplar and willow, respectively. The multispecies phylogenetic tree showed that the FLA gene family could be divided into four groups (I-IV), with Group 1 showing significant expansion in woody plants. A gene expression analysis showed that PdeFLA19/27 in Group I of poplar was highly expressed, specifically during the secondary growth period of the stem and the rapid elongation of seed hairs. In the Group I genes of S. suchowensis , SsuFLA25/26/28 was also highly expressed during the secondary growth period, whereas increased expression of SsuFLA35 was associated with seed hair tissue. These results provide important clues about the differences in the FLA gene family during the evolution of herbs and woody plants, and suggest that the FLA gene family may play an essential role in regulating the secondary growth of woody plants. It also provides a reference for further studies on the regulation of secondary growth and seed hair development by FLA genes in poplar and willow.