Hyphantria cunea is a widespread forest pest causing economic losses to forestry worldwide. This study developed Cry1Ac-overexpressing transgenic lines in Populus simonii × P. nigra and P. davidiana × P. bolleana through genetic engineering. ELISA confirmed that Cry1Ac protein expression level above 180 ng/g. Feeding experiments indicated that larvae of H. cunea that ingested transgenic leaves showed a significant reduction in growth and development, characterized by notably decreased body weight and food consumption, ultimately leading to total mortality within five days. High-throughput sequencing analysis uncovered stress responses at both the RNA and metabolic levels in the larvae following consumption of leaves from Cry1Ac overexpressing poplar. This research presents innovative strategies for breeding insect-resistant poplars and provides new insights for the management of H. cunea.

Nitraria tangutorum Bobrov. (N. tangutorum) is a drought-tolerant and salt-loving plant with high nutrient value, native to northwest China. Its distribution in the Qaidam Basin covers nearly 1000 square kilometers, yielding 0.5-10 × 105 tons of industrial juice by-products annually. However, the bitter and fishy taste of the plant limits its industrial application. Blending is an effective technique to enhance the sensory qualities of wine. In this study, eight blended wines were prepared by mixing N. tangutorum-fermented wine (NT) with either Meili rosé wine (ML) or a combination of highland barley wine (QK) and "Shine Muscat" grape juice (SM) in different proportions. The results showed that both blending methods significantly reduced titratable acidity and volatile acidity of NT, while increasing its flavanol and tannin contents as well as brightness. Sensory evaluation revealed that the blended samples NT-QK-SM-2 (30%:35%:35%) and NT-ML-2 (15%:85%) received higher scores in appearance, aroma, taste, and overall quality. Analyses by gas chromatography-ion mobility spectrometry (GC-IMS) and GC-mass spectrometry (GC-MS) indicated that NT-QK-SM-2 contained higher levels of ethyl lactate, 2-methyl-1-propanol, 1-butanol, and other compounds, contributing to its unique aromatic profiles. In contrast, NT-ML-2 showed elevated concentrations of acetaldehyde, isobutyl acetate, and other compounds, imparting a more pronounced floral and fruity flavor. Partial least squares discriminant analysis (PLS-DA) identified ten key biomarkers for distinguishing the blended wines: ethyl caproate, ethyl butyrate, ethyl isobutyrate, octanal, ethyl succinate, β-ionone, 2-methyl-4-ethylphenol, (E)-3-hexen-1-ol, 1-hexanol, and 1-propanol. These findings demonstrate that blending can significantly improve the aroma and sensory quality of N. tangutorum wine, offering a viable approach to transform this nutrient-rich but otherwise single-flavored resource into a product with market potential.

Drought is a major abiotic stress factor that limits tree growth. In this study, we determined the role and underlying mechanism of miR169y during the drought response. Expression analysis revealed that miR169y is notably downregulated under abscisic acid (ABA) and drought treatments. Stable transgenic lines expressing miR169y were generated using Agrobacterium-mediated transformation techniques. Suppression of miR169y expression increased drought tolerance in poplar, whereas its overexpression led to a decrease in tolerance. Nuclear factor Y subunit A6 gene (PtrNFYA6) was identified as the primary target of miR169y, exhibiting considerable upregulation in response to drought and ABA treatments. PtrNFYA6-overexpressing lines exhibited higher relative water content, lower malondialdehyde levels, increased proline accumulation and antioxidant enzyme activities, reduced reactive oxygen species levels, and enhanced stomatal closure. Molecular experiments confirmed that PtrNFYA6 directly binds to the CCAAT-box and ABRE elements in the promoters of 9-cis-epoxycarotenoid dioxygenase genes (PtrNCED3a/3b/6), thereby activating their transcription and promoting ABA synthesis. The overexpression of PtrNCED3b further substantiated the importance of ABA in enhancing drought tolerance. This research introduces a molecular framework in which the miR169y-PtrNFYA6-PtrNCED3a/3b/6 cascade modulates ABA production and drought resilience in poplar, thereby offering crucial targets for the molecular breeding of drought-resistant forest trees.

Poplar is a significant species in silviculture; however, its natural growth may be constrained by salt stress. In this paper, we conducted an investigation into the proteins present in poplar leaves as a response to salt stress, and we analyzed the alterations in protein and gene expression following salt stress through the application of proteomic and transcriptomic sequencing technologies. Differential expression analysis identified 149 significantly differentially accumulated proteins (DAPs), which mainly involved biological processes and pathways such as photosynthesis, amino acid metabolism, glutathione metabolism and oxidative phosphorylation. Weighted gene co-expression network analysis revealed 20 crucial proteins that exhibited significant correlations with the duration of salt stress, the majority of which were associated with biological processes pertaining to plant stress tolerance. In addition, we overexpressed the hub protein PsnLEA4-5 in 84K poplar and found that this gene significantly enhanced the salt tolerance of poplar, contributed to ionic homeostasis and enhanced ROS scavenging capacity. This work provides an important reference for tree stress research and resistance breeding.

Drought and salinity are two major environmental factors that severely limit plant growth and development. MYB functions as a transcription factor that is crucial in how plants respond to stress from adverse conditions. In this study, we identified a gene encoding Populus simonii × P. nigra MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor, whose transcription level was significantly induced under salt stress and osmotic stress. Subcellular localisation results showed PsnMYB30 was located in the nucleus. Yeast one-hybrid assay indicated the gene exhibited transcriptional activation activity and it can precisely bind to the G-box elements. Under normal growth conditions, there were no significant differences in physiological and biochemical indicators between wild-type and transgenic tobacco. However, under salt and drought stress, transgenic tobacco overexpressing PsnMYB30 exhibited superior root length and fresh weight compared to the wild-type (WT), with higher levels of SOD, POD, proline, and chlorophyll content, and significantly lower MDA and H2O2 content than the WT. These findings indicate that PsnMYB30 significantly enhances the salt tolerance and drought resistance of transgenic tobacco. These results indicate that PsnMYB30 is a key target gene for studying salt-tolerant and drought-resistant plants in genetic breeding.

Spring frost poses a major threat to grape-producing regions, severely reducing grape yield and quality. Grafting rootstocks is an effective strategy for enhancing scion resistance to spring frost and mitigating damage. In this study, the two wine grape cultivars (‘Cabernet Sauvignon’ and ‘Chardonnay’) grafted onto three rootstocks (‘Beta’, ‘Kober 5BB’, and ‘3309 Couderc’) were evaluated for their spring frost resistance on one-year-old vines. The scion–rootstock combinations exhibited significantly less photosynthetic impairment under frost stress compared with own-rooted vines. Rootstock also showed lower levels of proline accumulation in the roots and APX activities in the leaves under frost conditions. Compared with own-rooted vines, VvCBF1 gene expression were significantly upregulated in the grafted combinations under frost stress conditions. Among the tested rootstocks, ‘Kober 5BB’ markedly improved the spring frost resistance of both cultivars. CH/5BB exhibited the highest activities of POD and APX activity and the greatest induction of VvCBF genes, along with the lowest relative electrical conductivity and H2O2 content. These results highlight the critical role of rootstocks in improving scion spring frost resistance and provide important guidance for selecting suitable rootstocks to mitigate the impact of late frosts.

Alteration of chromosomal dosage severely affects the expressions of cis and trans genes in human and fruit fly. However, its effect on gene expression in plants needs to be further explored. Here, we generated 84 aneuploid lines with extra 1 to 16 chromosomes by crossing a female diploid poplar and a male triploid poplar. The chromosome compositions of the aneuploid population were dissected by DNA sequencing and cytogenetics. An RNA-seq analysis showed that cis genes were affected by both dosage and inverse effects, while trans genes were mainly affected by inverse effect. When the chromosomal dosage in an aneuploid line exceeds a threshold, the inverse effect may exceed the dosage effect on cis genes, causing dosage overcompensation. Interestingly, we found that even the genes located on the same chromosome may be subjected to different types of effect. These findings provided an important theoretical basis for analyzing the gene expression from the perspective of chromosomal dosage level.

ERF family transcription factors are crucial regulators in plants, playing a central role in abiotic stress responses and serving as important targets for stress-tolerant crop breeding. Populus davidiana × P. bolleana, an elite hybrid poplar cultivar artificially selected in northern China, holds significant research value encompassing ecological restoration, economic industries, genetic resource development, and environmental adaptability. This study identified that PdbERF109 expression was significantly upregulated in P. davidiana × P. bolleana response to salt treatment. Furthermore, transgenic poplar lines overexpressing PdbERF109 (OE) were generated. Salt stress assays demonstrated that PdbERF109 overexpression significantly enhanced salt tolerance in transgenic poplar. Compared to wild-type (WT) plants, PdbERF109-OE lines exhibited a significant enhancement in the activities of antioxidant enzymes, with increases of 2.3-fold, 1.2-fold, and 0.5-fold for superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), respectively, while the levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) were markedly reduced by 39.89% and 40.03%, indicating significantly enhanced reactive oxygen species (ROS) scavenging capacity and reduced oxidative damage. Concurrently, PdbERF109 overexpression reduced the natural leaf relative water loss (%). Meanwhile, yeast one-hybrid assays confirmed that the PdbERF109 protein specifically binds to GCC-box and DRE cis-acting elements. This study established PdbERF109 as a positive regulator of salt stress responses, highlighting its potential as a target gene for improving plant tolerance to high salinity, providing a promising candidate gene for the molecular breeding of salt-tolerant crops.

WRKY transcription factors (TFs) are key regulators of plant tissue morphogenesis, defense responses, and metabolic regulation. However, the functions for most of WRKY TFs in 84K poplar (Populus alba × P. glandulosa) in regulating leaf morphology and responding to salt stress are remain unclear. In this study, overexpressing PagWRKY11 poplars were generated. Phenotypic analysis revealed that transgenic poplar leaves were narrower and smoother compared to the traditionally elliptical and relatively rough leaves of wild-type (WT) plants. Then, the apical buds of transgenic poplars were sharp and elongated, with young leaves pointing upwards and inwards, whereas WT buds were rounder with smoother young leaves pointing downwards. Functional analysis indicated that under salt stress, the activities of SOD and POD enzymes and the expression of their encoding genes were significantly lower in transgenic poplars compared to WT. Conversely, the accumulation of H2O2 and MDA was significantly higher. These results suggest that overexpression lines of PagWRKY11 increase salt sensitivity by downregulating the expression of antioxidant enzyme genes. Meanwhile, overexpression of PagWRKY11 increased the natural water loss rate of poplar leaves, and negatively regulated salt stress by affecting water retention. In addition, yeast one-hybrid assays showed that PagWRKY11 binds specifically to W-box elements. These results provide a theoretical basis for further exploration of the molecular mechanisms by which PagWRKY11 regulates leaf morphogenesis and stress responses, and offer new potential strategies for resistance breeding.

Drought and salinization are paramount abiotic stresses major environmental factors limiting plant growth and development. WRKY, as a transcription factor, plays a key role in plants' stress responses to adverse environmental conditions. In this study, we identified a gene encoding a WRKY transcription factor from 84K poplar (Populus alba ×Populus glandulosa), whose expression was significantly upregulated under both salt stress and drought stress. Subcellular localization results showed that PagWRKY12 is located in the cell nucleus. Yeast hybridisation experiments confirmed that, although the encoded protein lacks self-activation activity, it can bind to G-box and W-box elements. Overexpression of PagWRKY12 markedly reduced plant height and root length, whilst enhancing tolerance to salt and drought stress in transgenic poplars by increasing Superoxide Dismutase (SOD), Peroxidase (POD) and proline content, and decreasing Malondialdehyde (MDA) and Hydrogen Peroxide (H2O2) levels. Transcriptome and RT-qPCR analyses indicated that PagWRKY12 overexpression in poplars enhanced expression of CYP87A2 and ATPIN4 genes involved in brassinosteroid (Br) biosynthesis, alongside AtMYB83 and CESA4 genes associated with secondary cell wall thickening (SCW). These findings suggest that PagWRKY12 may enhance salt and drought tolerance in 84K poplar by coordinating the promotion of Br biosynthesis and secondary cell wall thickening.

N6-methyladenosine (m6A) is the most abundant mRNA modification in organisms. To investigate the dynamics of m6A under salt stress in poplar, 84K poplar (Populus alba × Populus tremula var. glandulosa) and Xiaohei (XH) poplar (Populus simonii × Populus nigra) were subjected to salt treatment followed by MeRIP-seq analysis. The results showed that genes such as ATWRKY40, ATPK19, FDH, and GRX480 exhibited similar methylation and expression patterns in both poplar species under stress. We overexpressed and suppressed PagMTC, a homolog of METTL4 in 84K poplar, and found that the overexpression lines exhibited enhanced salt tolerance. The salt-tolerant genes PagCERK1, PagNCED3a, PagNCED3b, PagCAT2, and PagPOD16 were found to be regulated through PagMTC-mediated hypermethylation, as determined by MeRIP-seq, MeRIP-qPCR, and mRNA stability assay. It was also observed that hypermethylation of genes such as XTH15, CAT2, and AT-HSFB2A after salt stress in wild-type 84K poplar may be mediated by PagMTC. This study reveals the important role of m6A modification in response to salt stress in poplar and also identifies a potential molecular target for breeding trees with enhanced stress tolerance.

Plant ecosystems face primary threats from biological invasions in combination with microbial pathogens whose main threats derive from fungal pathogens. Fungi are essential in maintaining ecological balance by decomposing wood and eliminating weakened trees, but pathogenic fungi can cause devastating effects. This review summarizes the effects of forest pathogenic fungal effectors by evaluating their types, functions, and unique characteristics, along with their impact on host immune response mechanisms. Pathogens attack plants through specific infection strategies that involve effectors to suppress host defense responses and metabolic activities. Plants falling victim to fungal effectors through their interaction with pathogens lose control of key cellular processes that allow the infection to develop. Effectors are categorized into apoplastic and cytoplasmic types, which influence plant immunity through alterations in immune responses. The infection entry process involves microorganisms that release protein effectors as structural and functional modifiers for target cells. The diversity of effectors jointly with their evolutionary processes depends on multiple factors encompassing amino acid content and foundational genomic zones together with interaction period with hosts. Effectors further manipulate phytohormone pathways such as jasmonic acid, ethylene, and salicylic acid to suppress immunity, promote pathogen survival, and establish parasitic compatibility. However, fungal effectors are central to pathogenesis, as they critically redefine plant-pathogen interactions by targeting host defense mechanism, enabling colonization, and driving diseases development. The review evaluates fungal effectors as dual agents which disrupt plant immunity while serving as research tools to study host biology. Exploring effector-mediated mechanisms helps researchers better understand fungal pathogenicity characteristics alongside plant host defense mechanisms. Future inquiries should examine pathway plasticity in effectors across taxonomic domains to better understand fungal pathogenesis in forest ecosystems worldwide.

Autophagy plays an important role in responding to necrotrophic pathogens and plant signal hormones. Brassinosteroids (BRs) are a class of natural steroidal phytohormones that effectively regulated the disease resistance responses in grape. However, the molecular mechanism of BR-autophagy networks responsible for activation of host defense against gray mold remained to be elucidated. We reported a novel defense mechanism that BR-regulated autophagy in grape berry against gray mold. Exogenous application of 24-epibrassinolide (eBR) enhanced the grape disease resistance. Meanwhile, the endogenous BR was accumulated and BR signaling pathway was activated in the berries. In addition, transcriptome analysis in eBR-treated grapes infected with gray mold showed that the differentially expressed genes (DEGs) were enriched in the metabolic pathway of BR signaling pathway and autophagy. DNA affinity purification sequencing (Dap-seq), Yeast one-hybrid assay (Y1H) and dual luciferase assays (LUC) verified VvBZR1 bound to the promoter of VvATG18a to induce its gene expression. Overexpressing VvATG18a and VvBZR1 improved the resistance of grapes to gray mold. Overall, this study sheds light on the immune mechanisms underlying the involvement of the autophagy in grape innate immunity, highlighting the pivotal role of VvATG18a in enhancing disease resistance.

N6-methyladenosine (m6A) modifications play an important role in regulating plant responses to abiotic stress. This study revealed the key regulatory mechanism of the m6A demethylation enzyme PagALKBH1A in 84 K poplar (Populus alba x P. tremula var. glandulosa) in response to cadmium (Cd) stress. PagALKBH1A was found to be significantly responsive to CdCl2 and NaCl treatments, and overexpression lines showed a significant reduction in tolerance to Cd stress. Combined with MeRIP-seq and RNA-seq results, PagALKBH1A was shown to dynamically regulate abscisic acid response, salt stress response, defence response, cell wall organisation and other biological processes. Furthermore, demethylation modification of PagALKBH1A specifically affected the abundance of salt tolerant/salt sensitive genes (PagGLb, PagHMGB2, PagCIPK9, PagTPX2-1, and PagAKT2), suggesting its potential function in Cd stress response. This study elucidates for the first time that PagALKBH1A regulates Cd stress resistance in woody plants through m6A demethylation modification, extending the theoretical framework of m6A modification in plant environmental adaptation.

Diplodia sapinea (= Sphaeropsis sapinea) is an opportunistic pathogen that usually lives in symbiosis (the coexistence of dissimilar organisms) with its host and can cause disease under extreme climatic or physiological stress. In this study, we generated a high-quality genome map of D. sapinea using PacBio Circular Consensus Sequencing (CCS) technology and analysed the key disease-causing genes of D. sapinea by RNA sequencing (RNA-seq). In the study, a number of cell wall degrading enzyme genes were identified to be up-regulated during pathogen infection, which may be involved in biotic stress response in P. sylvestris var. mongolica Litv. It was also found that the expression of antioxidant-related genes, such as those involved in carotenoid biosynthesis, ascorbate and glutathione metabolism, was up-regulated in the P. s. var. mongolica Litv. after fungus infection. Differently expressed genes (DEGs) -based protein-protein interaction (PPI) network was constructed that included 163 pairs of significantly positively correlated proteins, forming three highly interacting gene clusters, and the PPI network was predicted to be associated with the replication and propagation processes of the fungus. These results provide important information for understanding the pathogenic mechanisms of Diplodia tip blight and developing control strategies in P. s. var. mongolica Litv.

Leonurus has a long history of use in traditional Chinese medicine, particularly for its therapeutic effects in gynecological disorders and its ability to promote blood circulation. Leonurus contains a variety of bioactive compounds, including alkaloids, flavonoids, and terpenoids, which demonstrate significant pharmacological activities, such as antioxidant, anti-inflammatory, and neuroprotective effects. Additionally, combining Leonurus with conventional pharmaceutical treatments may produce synergistic effects, enhancing therapeutic outcomes. This review provides a comprehensive overview of the classification, distribution, traditional uses, and recent pharmacological advancements of Leonurus, with a particular focus on its potential applications in the treatment of brain diseases. It aims to deepen understanding of Leonurus's therapeutic potential in treating neurological conditions.

Cutin serves as the foundational structure of the plant cuticle and plays a crucial role in determining fruit development and quality. However, the transcriptional regulation of cutin deposition by fruit-specific transcription factors remains largely unknown. This study delves into the regulatory role of the tomato (Solanum lycopersicum) SBP-box protein Colorless Non-ripening (SlCNR), primarily expressed in fruits, in cutin formation. We found that CRISPR/Cas9-induced slcnr mutants exhibited thicker cuticles and elevated contents in total cutin and cutin monomers compared to wild-type fruits, whereas SlCNR overexpression lines displayed the opposite tendency. Transcriptome-wide RNA sequencing identified differentially expressed genes in SlCNR overexpression fruits. Further validation by gene expression, DNA binding, and transcriptional activity assays revealed that SlCNR directly binds to and represses the transcription of thirteen genes associated with cutin synthesis, export, and assembly, including glycerol-3-phosphate acyltransferase 4/6 (SlGPAT4/6), ATP-binding cassette transporter subfamily G protein 36/42 (SlABCG36/42), and cutin synthase (SlCUS1). In addition, SlCNR directly bound to and repressed the transcription activities of the promoter of NON-RIPENING like-1 (SlNOR-like1), which encodes a positive regulator of cutin deposition. Thus, SlCNR emerged as a negative transcription regulator of cutin content and cuticle thickness, ultimately affecting fruit firmness and cuticle permeability. This study sheds light on the molecular mechanisms governing cutin deposition through transcriptional regulatory networks, highlighting the role of SlCNR as an important player in fruit cuticle development.

The unique and challenging environment of the cliffs gives Opisthopapus genus (Opisthopappus taihangensis and Opisthopappus longilobus) a strong tolerance to harsh climatic conditions. Under salt stress, the physiological indexes, superoxide dismutase (SOD), soluble protein (SP), chlorophyll (Chl), malondialdehyde (MDA), peroxidase (POD), and catalase (CAT), generally increased with the treatment levels. Through weighted gene co-expression network analysis (WGCNA), MEdarkmagenta module in O. taihangensis (182 genes) and MEdarkgreen module in O. longilobus (281 genes) were screened, which significantly related with SOD, CAT, SP, and POD. With genome-wide association studies (GWAS), it found that abundant SNPs was significantly correlated with the SOD index. Among WGCNA and GWAS, there was eleven common differentially expressed genes (c-DEGs) in O. taihangensis and seventeen c-DEGs in O. longilobus, respectively. For O. taihangensis, it responded to the stress primarily through the proteasome and lipid metabolism pathways involved in PSMB2, PSMA4, and PLD1_2 genes. In contrast, O. longilobus activated the oxidative phosphorylation pathway and then influenced the amino acid biosynthesis, terpenoid biosynthesis, and signaling transduction pathways (e.g., GA, JA, and MAPK) mainly involved MYC2, GID1, CYP82G1, TYRAAT, and MAPKKK17_18 genes. Compared with O. taihangensis, O. longilobus activated additional genes through more pathways to respond to salt stress. According to May-Wigner theory, the simple gene regulatory network was more stable than the complex. Thus, it suggested that O. taihangensis had superior tolerance and adaptation than O. longilobus. The results initially explored the potential responsive mechanism of two species under salt stress. These would establish the foundation for further investigations of the Opisthopapus tolerances, provide clues for the study of other cliff plants in the Taihang Mountains, and lay lights for the performing of Asteraceae cross-breeding.