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In order to explore the molecular mechanisms of Populus simonii × P. nigra response to stress and screen for genes conferring resistance to Alternaria alternata , we carried out measurements of physiological and biochemical indices and transcriptomic sequence analysis of leaves of Populus simonii × P. nigra inoculated with A. alternata . The results showed that the variation trends of multiple hormone contents and enzyme activities were broadly similar at different time points, with H 2 O 2 , SA, JA, PPO, SOD, PAL and POD showing a trend of increasing and then decrease after inoculation with the pathogen. The contents of H 2 O 2 peaked on the second day and subsequently declined. The contents of SA and JA, as well as the enzymatic activities of SOD, PAL, and POD, reached their maxima on the third day before exhibiting a downward tendency. In contrast, the activity of PPO peaked on the fourth day. Whereas ABA content continued to increase until the fifth day and CAT content decreased and then increased. We subsequently identified 14,997 differentially expressed genes (DEGs) among the transcriptomic sequences(|log2FoldChange| > 1 and FDR value < 0.05), with genes encoding members of the ERF, MYB, bZIP, and WRKY transcription factor families being differentially expressed. Gene modules that were significantly associated with the ABA, PAL, JA, and SOD activity were identified using weighted gene co-expression network analysis (WGCNA). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these genes were mainly related to biological stress, signal transduction, cell wall, and photosynthesis. Within these modules, we also identified hub genes in the regulatory network, including GLK1/2 transcriptional activators, 14-3-3 proteins, cytosine 5 methyltransferases, and a variety of proteins associated with photosynthesis and respiration. This study showed that these hub genes, which play a pivotal role in the co-expression network, which may indicate a potential role in defense process of Populus simonii × P. nigra against A. alternata . Additionally, we analyzed the gene expression regulation and defense mechanisms of Populus simonii × P. nigra adversity stress, providing new insights into how plants respond to biological stress.

Background Populus simonii × P. nigra is an ideal material for studying the molecular mechanisms of woody plants. In recent years, research on Populus simonii  ×  P. nigra has increasingly focused on the application of transgenic technology to improve salt tolerance. However, the rapid characterization of gene functions has been hampered by the long growth cycle and exceedingly poor transformation efficiency. Protoplasts are an important tool for plant gene engineering, which can assist with challenging genetic transformation and the protracted growth cycle of Populus simonii  ×  P. nigra . This study established an optimized system for the preparation and transformation of protoplasts from Populus simonii  ×  P. nigra leaves, making genetic research on Populus simonii  ×  P. nigra faster and more convenient. Major Latex Protein (MLP) family genes play a crucial role in plant salt stress response. In the previous study, we discovered that PsnMLP328 can be induced by salt treatment, which suggested that this gene may be involved in response to salt stress. Protein localization is a suggestion for its function. Therefore, we conducted subcellular localization analysis using protoplasts of Populus simonii  ×  P. nigra to study the function of the PsnMLP328 gene preliminarily. Results This study established an optimized system for the preparation and transformation of Populus simonii  ×  P. nigra protoplasts. The research results indicate that the optimal separation scheme for the protoplasts of Populus simonii  ×  P. nigra leaves included 2.5% cellulase R-10, 0.6% macerozyme R-10, 0.3% pectolyase Y-23, and 0.8 M mannitol. After enzymatic digestion for 5 h, the yield of obtained protoplasts could reach up to 2 × 10 7 protoplasts/gFW, with a high viability of 98%. We carried out the subcellular localization analysis based on the optimized transient transformation system, and the results indicated that the MLP328 protein is localized in the nucleus and cytoplasm; thereby proving the effectiveness of the transformation system. Conclusion In summary, this study successfully established an efficient system for preparing and transforming leaf protoplasts of Populus simonii  ×  P. nigra , laying the foundation for future research on gene function and expression of Populus simonii  ×  P. nigra .

Saline-alkali stress seriously endangers the normal growth of × . Arbuscular mycorrhizal (AM) fungi can enhance the saline-alkali tolerance of plants by establishing a symbiotic relationship with them. In this study, a pot experiment was conducted to simulate a saline-alkali environment where × were inoculated with to explore their effects on the saline-alkali tolerance of × . Our results show that a total of 8 gene family members are identified in × . regulate the distribution of Na+ by inducing the expression of . The pH value of poplar rhizosphere soil is reduced, result in the promote absorption of Na by poplar, that ultimately improved the soil environment. Under saline-alkali stress, improve the chlorophyll fluorescence and photosynthetic parameters of poplar, promote the absorption of water, K and Ca , thus increase the plant height and fresh weight of aboveground parts, and promote the growth of poplar. Our results provide a theoretical basis for further exploring the application of AM fungi to improve the saline-alkali tolerance of plants.

Heat shock transcription factor (HSF) is an important TF that performs a dominant role in plant growth, development, and stress response network. In this study, we identified a total of 30 HSF members from poplar, which are unevenly distributed on 17 chromosomes. The poplar HSF family can be divided into three subfamilies, and the members of the same subfamily share relatively conserved domains and motifs. HSF family members are acidic and hydrophilic proteins that are located in the nucleus and mainly carry out gene expansion through segmental replication. In addition, they have rich collinearity across plant species. Based on RNA-Seq analysis, we explored the expression pattern of PtHSFs under salt stress. Subsequently, we cloned the significantly upregulated PtHSF21 gene and transformed it into Populus simonii × P. nigra . Under salt stress, the transgenic poplar overexpressing PtHSF21 had a better growth state and higher reactive oxygen scavenging ability. A yeast one-hybrid experiment indicated PtHSF21 could improve salt tolerance by specifically binding to the anti-stress cis-acting element HSE. This study comprehensively profiled the fundamental information of poplar HSF family members and their responses to salt stress and specifically verified the biological function of PtHSF21 , which provides clues for understanding the molecular mechanism of poplar HSF members in response to salt stress.

The PsnWRKY70 transcription factor (TF) was reported to play an important role in the salt stress response mechanism of Populus simonii × Populus nigra in our previous research, and we also produced several PsnWRKY70 overexpression (OEXs) and RNAi suppression (REXs) P. simonii × P. nigra lines. In order to further compare the photosynthetic and physiological characteristics of NT (non-transgenic line) and transgenic lines under salt stress, the dynamic phenotypic change, Na+ and K+ content in leaf and root tissues, superoxide dismutase (SOD) and peroxidase (POD) activity, malondialdehyde (MDA) content, chlorophyll content (Chl), photosynthesis parameters (net photosynthetic rate, Pn; stomatal conductance, Gs; intercellular CO2 concentration, Ci; transpiration rate, Tr), chlorophyll fluorescence parameters (electron transport rate, ETR; maximum photochemical efficiency of photosystem II (PSII), Fv/Fm; actual efficiency of PSII, ΦPSII; photochemical quenching coefficient, qP; non-photochemical quenching, NPQ; the photosynthetic light-response curves of ΦPSII and ETR) and RNA-seq of NT, OEX and REX lines were detected and analyzed. The phenotypic observation, MDA content and Chl detection results indicate that the stress damage of REXs was less severe than that of NT and OEX lines under salt stress. Photosynthesis parameter (Pn, Gs, Tr and Ci) and chlorophyll fluorescence parameter (ETR, Fv/Fm, ΦPSII qP and NPQ) detection results indicate that the REX lines exhibited much better photosynthetic adaptability than NT and OEX lines during salt stress. The photosynthetic light-response curves of ΦPSII and ETR of NT, OEX and REX lines indicate that REXs exhibited better ability to activate the photosynthetic protection mechanism and adapt to a certain degree of strong light than NT and OEX lines under salt stress. RNA-seq analysis indicates that the DEGs between OEX1 vs. NT and REX1 vs. NT in different tissues (apical bud and fifth functional leaf) were all different in category and change trend. The expression of PsnWRKY70 was significantly up-regulated in both the apical bud and fifth functional leaf of OEX1, and showed no significant change (namely maintained low expression level) in both the apical bud and fifth functional leaf of REX1, thus indicating the negative regulation role of PsnWRKY70 in P. simonii × P. nigra under salt stress. Additionally, there were a lot of stress response-related TF genes (such as bHLH, WRKY, MYB, NAM and AP2/EREBP) and photosynthesis-related genes among all the DEGs. In REX1, the expression of three Photosystem I P700 chlorophyll a apoprotein A1 genes (Potri.003G065200, Potri.013G141800 and Potri.019G028100) and a Photosystem II protein D1 gene (Potri.013G138300) were significantly up-regulated after 6 days of salt stress. In OEX1, the Heterodimeric geranylgeranyl pyrophosphate synthase small subunit gene (Potri.015G043400) and Phospho-2-dehydro-3-deoxyheptonate aldolase 1 gene (Potri.007G095700) were significantly down-regulated after 6 days of salt stress. These photosynthesis-related genes are probably regulated by PsnWRKY70 TF in response to salt stress. In conclusion, the REX lines suffered less severe salt damage and exhibited better photosynthetic adaptability than NT and OEXs under salt stress. The differences among the DEGs between OEX1 vs. NT and REX1 vs. NT in apical bud and fifth functional leaf, and the significantly differentially expressed photosynthesis-related genes are probably the key clues for discovering the photosynthesis adaptability mechanism of PsnWRKY70 transgenic P. simonii × P. nigra under salt stress.

Background NAC (NAM, ATAF1–2, and CUC2) family is one of the largest plant-specific transcription factor families known to play significant roles in plant development processes and stress responses. Results In the study, a total of 112 NACs were identified to be differentially expressed in the comparisons of leaves and stems, leaves and roots, roots and stems of Populus simonii×P. nigra among 289 members by RNA-Seq. And 148, 144 and 134 NACs were detected to be salt-responsive in the roots, stems and leaves under 150 mM NaCl stress, respectively. Among them, a total of 53 salt-responsive NACs were shared across the three tissues. Under salt stress, 41/37 NACs were identified to be up/down-regulated in the leaves of Populus simonii × P.nigra among 170 non-redundant NACs by RT-qPCR, which was similar with RNA-Seq results. The expression pattern analysis of 6 NACs including four randomly up-regulated genes ( NAC86 , NAC105 , NAC139 and NAC163 ) and two down-regulated genes ( NAC15 and NAC149 ) indicated a few NACs showed specific temporal and spatial expression patterns in the three tissues of Populus simonii×P.nigra . Based on transcriptome screening and phylogenic analysis of differentially expressed NACs in different tissues under salt stress, 18 potential NACs associated with wood formation and 20 involved in stress responses were identified in Populus simonii×P.nigra . Conclusions The study further gains an understanding of the connection of tissue specificity and gene function in poplar, and lays the foundation of functional analysis of poplar NACs in stress responses.

The NAC transcription factor family is well known to play vital roles in plant development and stress responses. For this research, a salt-inducible NAC gene, PsnNAC090 (Po-tri.016G076100.1), was successfully isolated from Populus simonii × Populus nigra. PsnNAC090 contains the same motifs at the N-terminal end of the highly conserved NAM structural domain. The promoter region of this gene is rich in phytohormone-related and stress response elements. Transient transformation of the gene in the epidermal cells of both tobacco and onion showed that the protein was targeted to the whole cell including the cell membrane, cytoplasm and nucleus. A yeast two-hybrid assay demonstrated that PsnNAC090 has transcriptional activation activity with the activation structural domain located at 167–256aa. A yeast one-hybrid experiment showed that PsnNAC090 protein can bind to ABA-responsive elements (ABREs). The spatial and temporal expression patterns of PsnNAC090 under salt and osmotic stresses indicated that the gene was tissue-specific, with the highest expression level in the roots of Populus simonii × Populus nigra. We successfully obtained a total of six transgenic tobacco lines overexpressing PsnNAC090. The physiological indicators including peroxidase (POD) activity, superoxide dismutase (SOD) activity, chlorophyll content, proline content, malondialdehyde (MDA) content and hydrogen peroxide (H2O2) content were measured in three transgenic tobacco lines under NaCl and polyethylene glycol (PEG) 6000 stresses. The findings reveal that PsnNAC090 improves salt and osmotic tolerance by enhancing reactive oxygen species (ROS) scavenging and reducing membrane lipid peroxide content in transgenic tobacco. All the results suggest that the PsnNAC090 gene is a potential candidate gene playing an important role in stress response.

Populus simonii × P. euphratica cv. ‘Xiaohuyang-1’ is an elite variety produced by distant hybridization between Populus section Tacamahaca and sect. Turanga. However, its highly gametic sterility makes it difficult to be used in the breeding program for the next generation. Induction of amphidiploids derived from the distant hybrid is an ideal approach for the restoration of gametic fertility. In this study, amphidiploids of ‘Xiaohuyang-1’ were produced by a strategy of tetraploid isolation from mixoploid plants based on adventitious bud regeneration. Firstly, in vitro regeneration systems of ‘Xiaohuyang-1’ were established as MS medium with 0.4 mg L− 1 6-BA and 0.05 mg L− 1 NAA for leaf explants, and MS medium with 0.4 mg L− 1 6-BA and 0.1 mg L− 1 NAA for internode-segment explants. Then, 31 mixoploids were induced by treating node segments with colchicine solution. The colchicine concentration, exposure time, and their interaction significantly affected the induction rate of mixoploids. Thirdly, the leaves of mixoploids were used as explants to induce adventitious buds, and 6 amphidiploids were identified from 43 regenerated buds, with an isolation rate of 13.95%. Furthermore, analyses of phenotypic variation among different ploidy levels showed that the amphidiploids had significantly longer, wider, and thicker leaves, larger stomas, and lower stomatal density, compared with the diploids. The notably increased cell sizes of spongy tissue contributed to the increase in leaf thickness of amphidiploids. The amphidiploid plants induced in this study are expected to exhibit better abiotic tolerance and improved gametic fertility, serving as a bridge parent in the next breeding cycle of Populus.

Leaf morphology in Populus L. varies extensively among sections, species and clones under strong genetic control. P. nigra L. (section Aigeiros), with large and triangular leaves, is a commercial forest tree of economic importance for fast growth and high yield in Europe. P. simonii Carr. (section Tacamahaca) with small land rhomboid ovate leaves performs cold and dry resistance/tolerance in the semi-arid region of Northern China. Leaf morphological traits could be used as early indicators to improve the efficiency of selection. In order to investigate the genetic variation pattern of leaf morphology traits, estimate breeding values (combining ability), as well as evaluate crossing combinations of parents, 1872 intersectional progenies from eight families (P. simonii × P. nigra) and their parents were planted with cuttings for the clonal replicate field trial in Northern China. Four leaf size traits (area, perimeter, length, width) and roundness were measured with leaf samples from the 1-year-old clonal plantation. Significant differences regarding leaf traits were found between and among three female clones of P. simonii from Inner Mongolia, China and six male clones of P. nigra from Casale Monferrato, Italy. The genetic variation coefficient, heritability and genetic variance component of most traits in male parents were greater than these of female parents. Heritability estimates of male and female parents were above 0.56 and 0.17, respectively. Plentiful leaf variations with normal and continuous distributions exited in the hybrid progenies among and within families with the genetic variation coefficient and heritability above 28.49 and 0.24, respectively. Heritability estimates showed that leaf area was the most heritable trait, followed by leaf width. The breeding value ranking of parents allowed us to select the parental clones for new crosses and extend the mating design. Two male parental clones (N430 and N429) had greater breeding values (general combining ability, GCA) of leaf size traits than other clones. The special combining ability (SCA) of the crossing combination between P. simonii cl. ZL-3 and P. nigra cl. N430 was greater than that of others. Eight putatively superior genotypes, most combined with the female parental clone ZL-3, can be selected for future testing under near-commercial conditions. Significant genetic and phenotypic correlations were found between five leaf morphology traits with the coefficients above 0.9, except for leaf roundness. The results showed that leaf morphology traits were under strong genetic control and the parental clones with high GCA and SCA effects could be utilized in heterosis breeding, which will provide a starting point for devising a new selection strategy of parents and progenies.

Key message This study is the first to report that triploids and tetraploids have been successfully produced through embryo sac and zygotic embryo chromosome doubling with high temperatures in P. simonii Carr. and its hybrid. A new synthetic polyploid induced by hybridization with unreduced gametes and heterozygotic embryo chromosome doubling can effectively combine polyploidy and heterosis, which can provide two major breeding advantages. In Populus , successfully creating and cultivating new polyploid varieties have economic and ecological production value. This was the first successful study in which embryo sac and zygotic embryo chromosome doubling was induced using high temperatures to produce triploids and tetraploids in Populus simonii Carr. and its hybrid, P. simonii  ×  P. nigra var. Italica , of Populus sect. Tacamahaca . The relationship between flower bud morphological characteristics (time after pollination) and female meiotic stage (embryo sac and zygotic embryo development) was established to guide the induction treatment period. In the resulting progeny, 37 triploids and 12 tetraploids were obtained and identified using flow cytometry. The optimal temperatures for embryo sac and zygotic embryo chromosome doubling were 38 and 41 °C, respectively. Cytogenetic analysis revealed that 66–72 h after pollination (HAP), a period characterized by a high proportion of one-nucleate and two-nucleate embryo sacs, was the optimal period for embryo sac chromosome doubling. For zygotic embryo chromosome doubling, 168 HAP was the optimal induction period, as there was a high proportion of two-cell and four-cell proembryos. The results indicate that inducing embryo sac and zygotic embryo chromosome doubling is an ideal method for producing polyploids. The methods for inducing polyploids and for evaluating ploidy and offspring with different ploidies and heterozygosity in this study will be useful for genetic research and Populus breeding programmes.