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Upland forests are traditionally thought to be net sinks for atmospheric methane (CH4). In such forests, in situ CH4 fluxes on tree trunks have been neglected relative to soil and canopy fluxes. We measured in situ CH4 fluxes from the trunks of living trees and other surfaces, such as twigs and soils, using a static closed-chamber method, and estimated the CH4 budget in a temperate upland forest in Beijing. We found that the trunks of Populus davidiana emitted large quantities of CH4 during July 2014–July 2015, amounting to mean annual emissions of 85.3 and 103.1 μg m−2 h−1 on a trunk surface area basis on two replicate plots. The emission rates were similar in magnitude to those from tree trunks in wetland forests. The emitted CH4 was derived from the heartwood of trunks. On a plot or ecosystem scale, trunk CH4 emissions were equivalent to c. 30–90% of the amount of CH4 consumed by soils throughout the year, with an annual average of 63%. Our findings suggest that wet heartwoods, regardless of rot or not, occur widely in living trees on various habitats, where CH4 can be produced.

Populus davidiana  ×  Populus bolleana (PdPap) root rot caused by Fusarium oxysporum is a major disease in China. Controlling this disease requires extensive use of chemicals. The use of plant endophytes, such as Bacillus , may be a suitable alternative to chemical agents. In this study, we isolated a strain of Bacillus amyloliquefaciens (AW3) by thermal stimulation and confrontation method from fleshy tap roots of Brassica rapa L . We then determined its inhibitory effect on the growth of F. oxysporum (Fox68) and how it induces the disease resistance of PdPap. The confrontation area and roots were visualized using an optical microscope and a scanning electron microscope, respectively, and mycelial cell malformation, swelling, and distortion observed. AW3 and F. oxysporum were inoculated in a variety of combinations that reduced the disease level. PdPap defense-related enzymes, such as PAL, PPO, SOD, and CAT, increased significantly. Besides, several genes associated with plant defense and hormonal signal transduction were highly expressed. Under the biological stress of Fox68, AW3 directly acted on the hyphae of Fox68, reducing the infection of PdPap by Fox68 and promoting PdPap growth. AW3 also induced the accumulation of defense-related enzymes/genes that conferred resistance. Therefore, AW3 could serve as a bio-control agent of wilt disease caused by F. oxysporum in PdPap .

The MYB family is one of the largest families of transcription factors, in which the R2R3-MYB subgroup plays a crucial role in various biological processes. R2R3-MYBs in Populus davidiana × Populus bolleana have, however, not been systematically investigated. Here, based on the gene annotation of P. davidiana × P. bolleana genome sequence, all PdbR2R3-MYB transcripts were identified. These PdbR2R3-MYBs were classified into 29 subgroups (C2 to C30) according to the phylogenetic analysis of Arabidopsis thaliana AtR2R3-MYBs. The analysis of gene structures and protein motifs showed the conservation and evolution of PdbR2R3-MYBs. The cis-acting elements in the promoters of PdbR2R3-MYB genes were predicted, and the results indicated an abundance of abscisic acid and defense- and stress-responsive elements. The results of qRT-PCR revealed that nine PdbR2R3-MYB genes were differentially expressed in various tissues and can be regulated by drought stress; thus, these genes may play key roles in the response of plants to drought stress. In addition, the expression of PdbMYB5 and PdbMYB102 was significantly higher than those of the other seven MYB genes; hence, PdbMYB5 and PdbMYB102 overexpressing (OE) and silenced (SE) poplar plants were generated to investigate drought stress tolerance. The PdbMYB5 and PdbMYB102 OE plants showed enhanced reactive oxygen species scavenging capability, less cell damage, and high expression levels of the SOD and POD genes, whereas the SE plants showed the opposite results, thus suggesting that PdbMYB5 and PdbMYB102 conferred enhanced drought tolerance to the plants. This study provided insights into gene characterization, structure, evolution, expression, and function of the PdbR2R3-MYB family in poplar plants.

Key messageConstruction of ML-hGRN for the salt pathway in Populus davidiana × P. bolleana. Construction of ML-hGRN for the lignocellulosic pathway in Populus davidiana × P. bolleana under salt stress.Many woody plants, including Populus davidiana × P. bolleana, have made great contributions to human production and life. High salt is one of the main environmental factors that restricts the growth of poplar. This study found that high salt could induce strong biochemical changes in poplar. To detect the effect of salt treatment on gene expression, 18 libraries were sequenced on the Illumina sequencing platform. The results identified a large number of early differentially expressed genes (DEGs) and a small number of late DEGs, which indicated that most of the salt response genes of poplar were early response genes. In addition, 197 TFs, including NAC, ERF, and other TFs related to salt stress, were differentially expressed during salt treatment, which indicated that these TFs may play an important role in the salt stress response of poplar. Based on the RNA-seq analysis results, multilayered hierarchical gene regulatory networks (ML-hGRNs) of salt stress- and lignocellulosic synthesis-related DEGs were constructed using the GGM algorithm. The lignocellulosic synthesis regulatory network under salt stress revealed that lignocellulosic synthesis might play an important role in the process of salt stress resistance. Furthermore, the NAC family transcription factor PdbNAC83, which was found in the upper layer in both pathways, was selected to verify the accuracy of the ML-hGRNs. DAP-seq showed that the binding site of PdbNAC83 included a “TT(G/A)C(G/T)T” motif, and ChIP-PCR further verified that PdbNAC83 can regulate the promoters of at least six predicted downstream genes (PdbNLP2-2, PdbZFP6, PdbMYB73, PdbC2H2-like, PdbMYB93-1, PdbbHLH094) by binding to the “TT(G/A)C(G/T)T” motif, which indicates that the predicted regulatory network diagram obtained in this study is relatively accurate. In conclusion, a species-specific salt response pathway might exist in poplar, and this finding lays a foundation for further study of the regulatory mechanism of the salt stress response and provides new clues for the use of genetic engineering methods to create high-quality and highly resistant forest germplasms.

The addition of ectomycorrhizal fungi (ECMF), beneficial rhizosphere microorganisms, to the soil can promote plant growth and resistance. Here, Populus davidiana  ×  Populus bolleana tissue culture seedlings were grown for 3 months in soils inoculated with one of the species, then seedlings were assessed for mycorrhizal colonization rate and growth, physiological and root traits. Suillus luteus and Populus involutus each formed ectomycorrhizal associations with the seedlings . Seedling height, ground diameter, biomass, and leaf area were significantly greater after treatment with ECMF than in the non-inoculated controls. Treatment improved all physiological and root variables assessed (chlorophylls and carotenoids, cellulose, and soluble sugars and proteins; root length, surface area, projected area, mean diameter, volume, number of root tips). Seedlings inoculated with S. luteus outperformed those inoculated with P. involutus .

Cryopreservation is vital for conserving the elite germplasm of the hybrid poplar Populus davidiana × P. tremuloides, which is difficult to propagate conventionally. This study established optimized vitrification and encapsulation–vitrification protocols, achieving high regeneration rates of 85.91% and 79.70%, respectively, with confirmed genetic stability. The process induced oxidative stress, altering markers (MDA, H2O2) and antioxidant enzyme activities (SOD, POD, CAT). Integrated transcriptomic and metabolomic analysis of key steps—preculture/loading (DLA) and osmotic dehydration (DLB)—revealed extensive stress-responsive reprogramming. A central finding was the robust activation of the flavonoid biosynthesis pathway during DLB, marked by upregulation of key genes (PAL, CHS) and accumulation of flavonols (e.g., quercetin). This response was linked to hormone signaling and antioxidant systems, forming a coordinated defense network. Our multi-omics findings demonstrate that successful cryopreservation relies on an adaptive response where flavonoid biosynthesis plays a critical role in conferring oxidative stress tolerance, providing a theoretical basis for improving woody plant cryopreservation.

To improve the application of endophyte Bacillus velezensis BY6 from the xylem of poplar, the effect of BY6 on the growth of diseased Populus davidiana × Populus. alba var. pyramidalis Louche (Pdpap poplar) seedlings and the biological control effect on the pathogen Armillaria solidipes were tested using a plant split-root experiment. After applying BY6 to the roots of diseased Pdpap poplar seedlings, the results show that plant growth indicators (dry mass, fresh mass, and plant height) were significantly increased (p < 0.05), and genes related to auxin hormone signal transcription were activated. BY6 indicated a surprising control effect after the inoculation of diseased Pdpap poplar seedlings. Compared to the infected control group, the treated disease index of the diseased Pdpap poplar seedlings in the treatment group were reduced by 49.53% on the 20th day. The relative staining areas of diaminobenzidine (DAB) and Trypan blue decreased by 3.37 and 7.31 times, respectively. The physiological indicators (soluble sugar and protein) and oxidase indicators were significantly increased (p < 0.05). The expression levels of defense genes related to salicylic acid (SA) and jasmonic acid (JA) signaling pathways were significantly increased (p < 0.05). Amazingly, the results indicate that BY6 simultaneously activates induced systemic resistance (ISR) and systemic acquired resistance (SAR) in diseased Pdpap poplar seedlings and promotes growth. The results indicate that BY6 is a promising candidate for developing forest tree biofertilizers and biopesticides.

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.

Background WRKY transcription factors (TFs) have been suggested to play crucial roles in the response to biotic and abiotic stresses. This study is the first to report the alkaline salt regulation of the WRKY gene. Results In this study, we cloned a WRKY gene (SlWRKY28) from the Salix linearistipularis and then transferred to the Populus davidiana × P. bolleana for expression. Sequence analysis on the transcriptome of Salix linearistipular showed the significant up-regulation of WRKY gene expression in response to salt-alkali stress in seedlings. Our data showed that SlWRKY28 localized to the nucleus. Furthermore, the expression of the SlWRKY28 from female plants increased with saline-alkali stress according to the northern blot analysis results. The results of 3,3′-Diaminobenzidine (DAB) staining showed that hydrogen peroxide (H2O2) concentration was lower under stress, but ascorbate peroxidase (APX) enzyme activity was significantly higher in the overexpressed plants than that in non-transgenic (NT) plants. Conclusions We found out the SlWRKY28 induced regulation of the enzyme gene in the reactive oxygen species (ROS) scavenging pathway is a potential mechanism for transgenic lines to improve their resistance to alkaline salt. This study shows theoretical and practical significance in determining SlWRKY28 transcription factors involved in the regulation of alkaline salt tolerance.

Development of transgenic plants with tolerance to environmental stress is an important goal of plant biotechnology. Late-embryogenesis-abundant (LEA) proteins accumulate in seeds during late embryogenesis, where they protect cellular membranes and macromolecules against drought. In this work, we transferred the Tamarix androssowii LEA gene into hybrids of Populus davidiana×P. bolleana. We compared relative rates of height growth, chlorophyll fluorescence kinetic parameters, and leaf Na+ levels of six TaLEA-containing lines with non-transferred plants (NT), all grown under 0.8% NaCl stress condition. Survival percentages of transgenic lines were all higher than for NT controls after rehydration and the survival percentage of SL2 was five-fold higher than for NT controls. Seedling height increased 48.7% in SL2 (from the onset of induced stress to the end of the growing season), 31% more than for the NT controls. Chlorophyll fluorescence kinetic parameters showed a marked increase in photosynthetic capacity in SL2 and SL5. Na+ levels in young leaves of transgenic lines were lower than in control NT leaves, but higher in yellow and withered leaves, indicating improved salt tolerance in transgenic lines.