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Poplar 84 K (Populus alba x P. tremula var. glandulosa) is a good resource for genetic engineering due to its rapid growth and wide adaptability, and it is also an excellent ornamental tree species. In this study, we used 84 K plantlets grown in the nitrogen-limited medium as experimental materials to explore the molecular mechanism in 84 K leaves under nitrogen deficiency. A total of 5,868 differentially expressed genes (DEGs) were identified using the transcriptional information from RNA-seq data. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment results revealed that the DEGs were mainly involved in energy metabolism and anthocyanin biosynthesis. We then identified differentially expressed transcription factors (TFs) and constructed TF centered gene co-expression networks for chlorophyll and anthocyanin biosynthesis pathway genes. Twenty potential regulators were finally identified. We speculated the transcription factors that control the pigmentation in leaves with the MYB-bHLH-WD40 (MBW) pigment regulatory model. Such identification will clarify the genetic basis of the secondary metabolism in 84 K, and being a source of candidate genes for future plant genetic engineering. Our work broadens the researchers' understanding of the regulation of anthocyanin synthesis in trees and provides new perspectives for ornamental 84 K poplar breeding.

Verticillium wilt is a disastrous disease in cotton‐growing regions in China. As a common management method, cotton rotation with cereal crops is used to minimize the loss caused by Verticillium dahliae. However, the correlation between soil microbiome and the control of Verticillium wilt under a crop rotation system is unclear. Therefore, three cropping systems (fallow, cotton continuous cropping, and cotton–maize rotation) were designed and applied for three generations under greenhouse conditions to investigate the different responses of the soil microbial community. The soil used in this study was taken from a long‐term cotton continuous cropping field and inoculated with V. dahliae before use. Our results showed that the diversity of the soil bacterial community was increased under cotton–maize rotation, while the diversity of the fungal community was obviously decreased. Meanwhile, the structure and composition of the bacterial communities were similar even under the different cropping systems, but they differed in the soil fungal communities. Through microbial network interaction analysis, we found that Verticillium interacted with 17 bacterial genera, among which Terrabacter had the highest correlation with Verticillium. Furthermore, eight fungal and eight bacterial species were significantly correlated with V. dahliae. Collectively, this work aimed to study the interactions among V. dahliae, the soil microbiome, and plant hosts, and elucidate the relationship between crop rotation and soil microbiome, providing a new theoretical basis to screen the biological agents that may contribute to Verticillium wilt control., The effect of crop rotation on the soil fungal community is greater than that on the bacterial community, and the interaction between Verticillium dahliae and soil microbiome provides a theoretical basis for Verticillium wilt control.

Coumarate 3-hydroxylase (C3h) genes participate in the synthesis of lignin and may affect the properties of wood that are important for its commercial value. A better understanding of the natural variation in C3h genes and their associations to wood properties is required to effectively improve wood quality. We used a candidate gene-based association mapping approach to identify CfC3h allelic variants associated with traits that affect the wood properties of Catalpa fargesii. We first isolated the full-length CfC3h cDNA (1825 bp), which was expressed at relatively high levels in xylem according to real time-polymerase chain reaction. In totally, 17 common single-nucleotide polymorphisms (minor allele frequency > 5%) were identified through cloning and sequencing the CfC3h locus from a mapping population (including 88 unrelated natural C. fargesii individuals collected from main distribution area). Nucleotide diversity and linkage disequilibrium (LD) in CfC3h indicate that CfC3h has low nucleotide diversity (πt = 0.0031 and θw = 0.0103) and relatively low LD (within 1800 bp; r2 ≥ 0.1). An association analysis identified eight common single-nucleotide polymorphisms (SNPs) (false discovery rate, Q

Rye is among the cereals with the highest content of dietary fibre. A high rye food intake has been associated with improved metabolic risk factors in some but not all observational and intervention studies. Whole-grain rye has also been suggested to affect the gut microbiota in individuals with metabolic syndrome. However, it is yet unclear to what extent effects on the gut microbiota mediate the beneficial metabolic responses of whole-grain rye intake. We hypothesized that a high intake of whole grain rye products containing fermented rye bran (FRB) vs. refined wheat based products (RW) could alter the gut microbiota and short-chain fatty acid (SCFA) composition towards a phenotype associated with beneficial metabolic effects in a population not used to such foods. For this purpose, we conducted a post hoc analysis of a 12-week randomized controlled trial in Chinese adults with Helicobacter pylori (HP) infection, with 53 participants consuming RW and 31 participants consuming FRB included in the analysis. Anthropometric measurements and fasting blood and fecal sample analyses as well as 13C-urea breath test were performed at baseline and after a 12-week intervention. At week 12, we observed a higher serum insulin concentration (P-value = 0.038) in the FRB group (n = 31) versus the RW group (n = 53), and this difference was corroborated with alterations in the genus-level relative abundances of the gut microbiota, represented by an increase in Romboutsia and a reduction in Bilophila in the FRB group (n = 22) versus the RW group (n = 46). Compared to the RW group (n = 53), fecal acetic acid concentration was significantly higher in the FRB group (n = 31) at week 12. We also found that fecal acetic and butyric acids positively, while isobutyric, isovaleric and 2-methylbutyric acids inversely, correlated with the gut Romboutsia level among all participants (n = 68) at week 12. We found positive correlations of fecal isobutyric, isovaleric and 2-methylbutyric acids with gut Bilophila (n = 68). In conclusion, our results suggest that the intake of high-fibre rye products could modify gut Romboutsia and Bilophila in a Chinese population with HP infection. These effects are paralleled with favorable modifications of the SCFA concentration and are associated with altered glycemic traits.

Populus spp. have long been used as model woody plant species for molecular biology research. However, tissues of poplar are often recalcitrant to experimental procedures for molecular studies. We generated a hormone autotrophic poplar suspension cell line from a hybrid of Populus alba × P. berolinensis ‘Yinzhong’, named Qu-2. Qu-2 cells are suitable as a model biological system for studying woody plants. Qu-2 cells have many advantages over suspension cell lines derived so far from any other woody plants. Qu-2 cells are very easy to cultivate and can grow on several common plant culture media without the addition of any plant hormone. They show exceptionally high growth rates, reaching an approximately 150-fold increase in biomass after one week of culturing. Another important unique characteristic of Qu-2 cells is that they can be cryopreserved and readily reactivated. Qu-2 cells are suitable for molecular manipulations such as protoplast production, transient transformation, and RNA-seq analysis. Therefore, Qu-2 cells have the great potential to be an excellent model cell line in tree molecular biological research, ranging from physiology to gene function. The Qu-2 cells will be made available to the plant community for research.

Populus alba × P. glandulosa clone 84 K, derived from South Korea, is widely cultivated in China and used as a model in the molecular research of woody plants because of high gene transformation efficiency. Here, we combined 63-fold coverage Illumina short reads and 126-fold coverage PacBio long reads to assemble the genome. Due to the high heterozygosity level at 2.1% estimated by k-mer analysis, we exploited TrioCanu for genome assembly. The PacBio clean subreads of P. alba × P. glandulosa were separated into two parts according to the similarities, compared with the parental genomes of P. alba and P. glandulosa. The two parts of the subreads were assembled to two sets of subgenomes comprising subgenome A (405.31 Mb, from P. alba) and subgenome G (376.05 Mb, from P. glandulosa) with the contig N50 size of 5.43 Mb and 2.15 Mb, respectively. A high-quality P. alba × P. glandulosa genome assembly was obtained. The genome size was 781.36 Mb with the contig N50 size of 3.66 Mb and the longest contig was 19.47 Mb. In addition, a total of 176.95 Mb (43.7%), 152.37 Mb (40.5%) of repetitive elements were identified and a total of 38,701 and 38,449 protein-coding genes were predicted in subgenomes A and G, respectively. For functional annotation, 96.98% of subgenome A and 96.96% of subgenome G genes were annotated with public databases. This de novo assembled genome will facilitate systematic and comprehensive study, such as multi-omics analysis, in the model tree P. alba × P. glandulosa.

Background Pinus koraiensis is an evergreen tree species with strong cold resistance. However, the transcriptomic patterns in response to cold stress are poorly understood for P. koraiensis. In this study, global transcriptome profiles were generated for P. koraiensis under cold stress (− 20 °C) over time by high-throughput sequencing. Results More than 763 million clean reads were produced, which assembled into a nonredundant data set of 123,445 unigenes. Among them, 38,905 unigenes had homology with known genes, 18,239 were assigned to 54 gene ontology (GO) categories and 18,909 were assigned to 25 clusters of orthologous groups (COG) categories. Comparison of transcriptomes of P. koraiensis seedlings grown at room temperature (20 °C) and low temperature (− 20 °C) revealed 9842 differential expressed genes (DEGs) in the 6 h sample, 9250 in the 24 h sample, and 9697 in the 48 h sample. The number of DEGs in the pairwise comparisons of 6 h, 24 h and 48 h was relatively small. The accuracy of the RNA-seq was validated by analyzing the expression patterns of 12 DEGs by quantitative real-time PCR (qRT-PCR). In this study, 34 DEGs (22 upregulated and 12 downregulated) were involved in the perception and transmission of cold signals, 96 DEGs (41 upregulated and 55 downregulated) encoding 8 transcription factors that regulated cold-related genes expression, and 27 DEGs (17 upregulated and 10 downregulated) were involved in antioxidant mechanisms in response to cold stress. Among them, the expression levels of c63631_g1 (annexin D1), c65620_g1 (alpha-amylase isozyme 3C), c61970_g1 (calcium-binding protein KIC), c51736_g1 (ABA), c58408_g1 (DREB3), c66599_g1 (DREB3), c67548_g2 (SOD), c55044_g1 (CAT), c71938_g2 (CAT) and c11358_g1 (GPX) first increased significantly and then decreased significantly with the extension of stress time. Conclusions A large number of DEGs were identified in P. koraiensis under cold stress, especially the DEGs involved in the perception and transmission of cold signals, the DEGs encoding TFs related to cold regulation and the DEGs removing ROS in antioxidation mechanisms. The transcriptome and digital expression profiling of P. koraiensis could facilitate the understanding of the molecular control mechanism related to cold responses and provide the basis for the molecular breeding of conifers.

Verticillium wilt is a severe disease of cotton crops in Xinjiang and affecting yields and quality, due to the continuous cotton cropping in the past decades. The relationship between continuous cropping and the changes induced on soil microbiome remains unclear to date. In this study, the culture types of 15 isolates from Bole (5F), Kuitun (7F), and Shihezi (8F) of north Xinjiang were sclerotium type. Only isolates from field 5F belonged to nondefoliating pathotype, the others belonged to defoliating pathotype. The isolates showed pathogenicity differentiation in cotton. Fungal and bacterial communities in soils had some difference in alpha-diversity, relative abundance, structure and taxonomic composition, but microbial groups showed similarity in the same habitat, despite different sampling sites. The fungal phyla Ascomycota, and the bacterial phyla Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria and Gemmatimonadetes were strongly enriched. Verticillium abundance was significantly and positively correlated with AN, but negatively correlated with soil OM, AK and pH. Moreover, Verticillium was correlated in abundances with 5 fungal and 6 bacterial genera. Overall, we demonstrate that soil microbiome communities have similar responses to long-term continuous cotton cropping, providing new insights into the effects of continuous cotton cropping on soil microbial communities.

The molecular karyotype could represent the basic genetic make-up in a cell nucleus of an organism or species. A doubled haploid (DH) is a genotype formed from the chromosome doubling of haploid cells. In the present study, molecular karyotype analysis of the poplar hybrid Populus simonii × P. nigra (P. xiaohei) and the derived doubled haploids was carried out with labeled telomeres, rDNA, and two newly repetitive sequences as probes by fluorescence in situ hybridization (FISH). The tandem repeats, pPC349_XHY and pPD284_XHY, with high-sequence homology were used, and the results showed that they presented the colocalized distribution signal in chromosomes. For P. xiaohei, pPD284_XHY produced hybridizations in chromosomes 1, 5, 8, and 9 in the hybrid. The combination of pPD284_XHY, 45S rDNA, and 5S rDNA distinctly distinguished six pairs of chromosomes, and the three pairs of chromosomes showed a significant difference in the hybridization between homologous chromosomes. The repeat probes used produced similar FISH hybridizations in the DH, nevertheless, pPD284_XHY generated an additional hybridization site in the telomere region of chromosome 14. Moreover, two pairs of chromosomes showed differential hybridization distributions between homologous chromosomes. Comparisons of the distinguished chromosomes between hybrid and DH poplar showed that three pairs of chromosomes in the DH presented hybridization patterns that varied from those of the hybrid. The No. 8 chromosome in DH and one of the homologous chromosomes in P. xiaohei shared highly similar FISH patterns, which suggested the possibility of intact or mostly partial transfer of the chromosome between the hybrid and DH. Our study will contribute to understanding the genetic mechanism of chromosomal variation in P. xiaohei and derived DH plants.

The symbiosis of endophytes and plants is universal in nature. However, how endophytes grow in plants is not entirely clear. Previously, we reported that a virus-infected fungal pathogen could grow in plants as an endophyte. In this study, we utilized Sclerotinia sclerotiorum strain DT-8, a virus-mediated endophyte, to investigate the mechanism of symbiosis with rapeseed by dual unique molecular identifier-RNA sequencing (dual-UMI RNA-seq). We found that the expressions of genes encoding S. sclerotiorum amylase/glucoamylase, glucose transporters, and rapeseed s ugars w ill e ventually be e xported t ransporter 11 (SWEET11) were upregulated. It suggested that strain DT-8 might utilize plant starch as a nutrient. The defense systems of rapeseed were also activated, such as production of reactive oxygen species, phenylpropanoids, and brassinin, to control the growth of strain DT-8, while strain DT-8 counteracted host suppression by producing effector-like proteins, detoxification enzymes, and antioxidant components. Moreover, rapeseed also upregulated pectate lyase and pectinesterase genes to facilitate the colonization by strain DT-8. Our findings provide novel insights into the interaction of virus-mediated endophytes and their hosts that warrant further study. IMPORTANCE Although endophytes are widespread in nature, the interactions between endophytes and their hosts are still not fully understood. Members of a unique class of endophytes, the virus-mediated endophytic fungi, are continuously being discovered and have received wide attention. In this study, we investigated the interaction between a mycovirus-mediated endophytic fungus and its host rapeseed by using dual-UMI RNA-seq. According to the dual-UMI RNA-seq results, an aerial view of symbiotic mechanism under balanced regulation was suggested. This research expands our understanding of the symbiotic mechanisms of virus-fungus-plant interactions and could establish a foundation for the further development of practical application with virus-mediated hypovirulent fungi.

The infection by a single-stranded DNA virus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), causes hypovirulence, a reduced growth rate, and other colony morphological changes in its host Sclerotinia sclerotiorum strain DT-8. However, the mechanisms of the decline are still unclear. Using digital RNA sequencing, a transcriptome analysis was conducted to elucidate the phenotype-related genes with expression changes in response to SsHADV-1 infection. A total of 3110 S. sclerotiorum differentially expressed genes (DEGs) were detected during SsHADV-1 infection, 1741 of which were up-regulated, and 1369 were down-regulated. The identified DEGs were involved in several important pathways. DNA replication, DNA damage response, carbohydrate and lipid metabolism, ribosomal assembly, and translation were the affected categories in S. sclerotiorum upon SsHADV-1 infection. Moreover, the infection of SsHADV-1 also suppressed the expression of antiviral RNA silencing and virulence factor genes. These results provide further detailed insights into the effects of SsHADV-1 infection on the whole genome transcription in S. sclerotiorum.

To determine appropriate quantities of water and fertilizer required for early growth of hybrid poplar cuttings, we recorded the growth traits of four clones grown under four factors (irrigation and nitrogen, phosphorus, and potassium fertilizers), each with four levels, using an orthogonal experimental design. A logistic model was used to estimate growth in height. The growth curves for tree height were sigmoid, and the model R2 values were greater than 0.9, which indicated that the fit was highly significant. ANOVA results for tree height and basal diameter indicated that all sources of variance showed significant differences (p

Tree height and diameter at breast height of 30 half-sib Larix kaempferi families were analyzed at different ages. Analysis of variance revealed a significant difference in growth among dif­ferent families. Phenotypic variation coefficients of the traits tree height and diameter at breast height among families of different ages ranged from 11.04 % to 31.74 % and 19.01 % to 19.83 %, respectively. Average heritability of tree height and diameter at breast height ranged from 0.87 to 0.96 and 0.93 to 0.96, respectively. Significant positive correlations were obser­ved among all traits at different ages. By the method of multip­le-traits comprehensive, six families (L18, L12, L8, L3, L25 and L20) were selected as being elite using a 20 % selection ratio at 12 years of age. Average values of these elite families were 11.15 % and 16.83 % higher than the total average for height and diameter at breast height, and genetic gains were 10.53 % and 15.79 %, respectively. Forty five elite individual plants were selected using a 5 % selection ratio which were 23.47 % and 24.90 % higher than the overall average for height and diame­ter at breast height, respectively.

Rice sheath blight caused by Rhizoctonia solani is the major disease of rice that seriously threatens food security worldwide. Efficient and eco-friendly biological approaches are urgently needed since no resistant cultivars are available. In this study, fallow and paddy soils were initially subjected to microbiome analyses, and the results showed that Talaromyces spp. were significantly more abundant in the paddy soil, while Trichoderma spp. were more abundant in the fallow soil, suggesting that Talaromyces spp. could live and survive better in the paddy soil. Five isolates, namely TF-04, TF-03, TF-02, TF-01, and TA-02, were isolated from the paddy soil using sclerotia of R. solani as baits and were further evaluated for their activity against rice sheath blight. These isolates efficiently parasitized the hyphae and rotted the sclerotia even at higher water contents in sterilized sand and the soil. Isolate TF-04 significantly promoted rice growth, reduced the severity of rice sheath blight, and increased rice yield under outdoor conditions. Defense related genes were upregulated and enzyme activities were enhanced in rice treated with isolate TF-04. Our research supplies a microbiome guided approach to screen biological control agents, provides Talaromyces isolates to biologically control rice sheath blight. This article is protected by copyright. All rights reserved.

d-type cyclins (CYCDs) are a special class of cyclins and play extremely important roles in plant growth and development. In the plant kingdom, most of the existing studies on CYCDs have been done on herbaceous plants, with few on perennial woody plants. Here, we identified a Populus d-type cyclin gene, PsnCYCD1;1, which is mainly transcribed in leaf buds and stems. The promoter of PsnCYCD1;1 activated GUS gene expression and transgenic Arabidopsis lines were strongly GUS stained in whole seedlings and mature anthers. Moreover, subcellular localization analysis showed the fluorescence signal of PsnCYCD1;1-GFP fusion protein is present in the nucleus. Furthermore, overexpression of the PsnCYCD1;1 gene in Arabidopsis can promote cell division and lead to small cell generation and cytokinin response, resulting in curved leaves and twisted inflorescence stems. Moreover, the transcriptional levels of endogenous genes, such as ASs, KNATs, EXP10, and PHB, were upregulated by PsnCYCD1;1. Together, our results indicated that PsnCYCD1;1 participates in cell division by cytokinin response, providing new information on controlling plant architecture in woody plants.

Betula L. (birch) is a pioneer hardwood tree species with ecological, economic, and evolutionary importance in the Northern Hemisphere. We sequenced the Betula platyphylla genome and assembled the sequences into 14 chromosomes. The Betula genome lacks evidence of recent whole-genome duplication and has the same paleoploidy level as Vitis vinifera and Prunus mume. Phylogenetic analysis of lignin pathway genes coupled with tissue-specific expression patterns provided clues for understanding the formation of higher ratios of syringyl to guaiacyl lignin observed in Betula species. Our transcriptome analysis of leaf tissues under a time-series cold stress experiment revealed the presence of the MEKK1–MKK2–MPK4 cascade and six additional mitogen-activated protein kinases that can be linked to a gene regulatory network involving many transcription factors and cold tolerance genes. Our genomic and transcriptome analyses provide insight into the structures, features, and evolution of the B. platyphylla genome. The chromosome-level genome and gene resources of B. platyphylla obtained in this study will facilitate the identification of important and essential genes governing important traits of trees and genetic improvement of B. platyphylla.

Plant disease is one of the most important causes of crop losses worldwide. The effective control of plant disease is related to food security. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum leads to serious yield losses in rapeseed (Brassica napus) production. Hypovirulent strain DT-8 of S. sclerotiorum, infected with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), has the potential to control SSR. In this study, we found rapeseed bio-priming with strain DT-8 could significantly decrease the disease severity of SSR and increase yield in the field. After bio-priming, strain DT-8 could be detected on the aerial part of the rapeseed plant. By 16S rRNA gene and internal transcribed spacer (ITS) sequencing technique, the microbiome on different parts of the SSR lesion on bioprimed and non-bioprimed rapeseed stem was determined. The results indicated that SSR and bio-priming treatment could influence the structure and composition of fungal and bacterial communities. Bio-priming treatment could reduce the total abundance of possible plant pathogens and enhance the connectivity and robustness of the interaction network at the genus level. This might be one of the mechanisms that rapeseed bioprimed with strain DT-8 had excellent tolerance on SSR. It might be another possible mechanism of biocontrol and will provide a theoretical guide for agricultural practical production.

Background Floral transition plays an important role in development, and proper time is necessary to improve the value of valuable ornamental trees. The molecular mechanisms of floral transition remain unknown in perennial woody plants. “Bairihua” is a type of C. bungei that can undergo floral transition in the first planting year. Results Here, we combined short-read next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to provide a more complete view of transcriptome regulation during floral transition in C. bungei. The circadian rhythm-plant pathway may be the critical pathway during floral transition in early flowering (EF) C. bungei, according to horizontal and vertical analysis in EF and normal flowering (NF) C. bungei. SBP and MIKC-MADS-box were seemingly involved in EF during floral transition. A total of 61 hub genes were associated with floral transition in the MEturquoise model with Weighted Gene Co-expression Network Analysis (WGCNA). The results reveal that ten hub genes had a close connection with the GASA homologue gene (Cbu.gene.18280), and the ten co-expressed genes belong to five flowering-related pathways. Furthermore, our study provides new insights into the complexity and regulation of alternative splicing (AS). The ratio or number of isoforms of some floral transition-related genes is different in different periods or in different sub-genomes. Conclusions Our results will be a useful reference for the study of floral transition in other perennial woody plants. Further molecular investigations are needed to verify our sequencing data.

Catalpa bungei is an economically important tree with high-quality wood and highly valuable to the study of wood formation. In this work, the xylem microstructure of C. bungei tension wood (TW) was observed, and we performed transcriptomics, proteomics and Raman spectroscopy of TW, opposite wood (OW) and normal wood (NW). The results showed that there was no obvious gelatinous layer (G-layer) in the TW of C. bungei and that the secondary wall deposition in the TW was reduced compared with that in the OW and NW. We found that most of the differentially expressed mRNAs and proteins were involved in carbohydrate polysaccharide synthesis. Raman spectroscopy results indicated that the cellulose and pectin content and pectin methylation in the TW were lower than those in the OW and NW, and many genes and proteins involved in the metabolic pathways of cellulose and pectin, such as galacturonosyltransferase (GAUT), polygalacturonase (PG), endoglucanase (CLE) and &beta, glucosidase (BGLU) genes, were significantly upregulated in TW. In addition, we found that the MYB2 transcription factor may regulate the pectin degradation genes PG1 and PG3, and ARF, ERF, SBP and MYB1 may be the key transcription factors regulating the synthesis and decomposition of cellulose. In contrast to previous studies on TW with a G-layer, our results revealed a change in metabolism in TW without a G-layer, and we inferred that the change in the pectin type, esterification and cellulose characteristics in the TW of C. bungei may contribute to high tensile stress. These results will enrich the understanding of the mechanism of TW formation.

Background Catalpa bungei is an important tree species used for timber in China and widely cultivated for economic and ornamental purposes. A high-density linkage map of C. bungei would be an efficient tool not only for identifying key quantitative trait loci (QTLs) that affect important traits, such as plant growth and leaf traits, but also for other genetic studies. Results Restriction site-associated DNA sequencing (RAD-seq) was used to identify molecular markers and construct a genetic map. Approximately 280.77 Gb of clean data were obtained after sequencing, and in total, 25,614,295 single nucleotide polymorphisms (SNPs) and 2,871,647 insertions-deletions (InDels) were initially identified in the genomes of 200 individuals of a C. bungei (7080) × Catalpa duclouxii (16-PJ-3) F1 population and their parents. Finally, 9072 SNP and 521 InDel markers that satisfied the requirements for constructing a genetic map were obtained. The integrated genetic map contained 9593 pleomorphic markers in 20 linkage groups and spanned 3151.63 cM, with an average distance between adjacent markers of 0.32 cM. Twenty QTLs for seven leaf traits and 13 QTLs for plant height at five successive time points were identified using our genetic map by inclusive composite interval mapping (ICIM). Q16–60 was identified as a QTL for five leaf traits, and three significant QTLs (Q9–1, Q18–66 and Q18–73) associated with plant growth were detected at least twice. Genome annotation suggested that a cyclin gene participates in leaf trait development, while the growth of C. bungei may be influenced by CDC48C and genes associated with phytohormone synthesis. Conclusions This is the first genetic map constructed in C. bungei and will be a useful tool for further genetic study, molecular marker-assisted breeding and genome assembly.

We analyzed the germination traits (germination rate, germination potential, and germination index) of seeds from 23 half-sib families of birch (Betula platyphylla). The germination rates of the 23 families ranged from 38.57 to 76.50%. Seeds from the eight families germinating at the highest rates were then used for germination experiments under salt stress. Germination rate, germination potential, and germination index of these eight half-sib families decreased with increasing salt concentrations. The effects of four different hormones (6-benzylaminopurine, 6BA; gibberellin, GA3; naphthalene acetic acid, NAA; and melatonin) at various concentrations on the germination of seeds from the eight half-sib birch families under salt stress were investigated. Treatments with 6BA and GA3 alleviated the effects of salt stress on seed germination, whereas treatments with NAA and melatonin aggravated the effects of salt stress. The most effective treatments for alleviating the effects of salt stress on birch seed germination were 10 mg/L 6BA and 300 mg/L GA3. This study provides practical information for screening birch families for salt tolerance at the seed germination stage, and for cultivation of birch in saline environments.

The emergence of the biomimetic nano-drug delivery system provides a feasible way for the clinical treatment of cancer. Noble metal nanomaterials often possess unique and intriguing physicochemical properties, optical properties, electromagnetic properties and catalytic properties, therefore have broad applications in the field of biomedicine. In this work, the porous Au@Pt bimetallic nanoparticles were synthesized via the blending of the chloroauric acid (a gold precursor) with the potassium chloroplatinate (a platinum precursor) at the ratio of 1:1. These Au@Pt NPs exhibited decent catalytic properties, high drug loading capacity (32.3%) and high photothermal conversion efficiency (44.1%). Based on the resultant Au@Pt NPs, a cell membrane-coated photothermal nano-drug delivery system (i.e. the DOX/Au@Pt-M-NPs) was formulated by loading the Doxorubicin (DOX) onto the Au@Pt nanoparticles, which showed a stronger inhibitory effect on tumor cells. The use of the HeLa tumor cell membrane coating demonstrated significantly reinforcing effect on the absorption of the DOX/Au@Pt-M-NPs by tumor cells because of homologous targeting. The cytotoxicity and apoptosis rate of the DOX/Au@Pt-M-NPs group (73.4%) were significantly higher than that of the Au@Pt group (49%). In addition, the damage to major organs caused by the DOX/Au@Pt-M nanoparticle group was significantly lower than that by the DOX/Au@Pt nanoparticle group and the pure DOX group. Taken together, our work demonstrated that nanomedicine carriers camouflaged by tumor cell membranes combined with photothermal therapy and chemotherapy would have potential use in clinical tumor treatment.

Catalpa bungei is an important timber tree. Improvements in growth and wood quality are important goals of C. bungei breeding, and it is necessary to understand the genetic parameters of specific target traits and genetic correlation between growth traits and wood properties for tree breeding. In this study, the genetic parameters of height, diameter at breast height (DBH) and wood properties were estimated and genetic and phenotypic correlations between growth traits and wood properties were evaluated in C. bungei. Finally, different selection scenarios were used to evaluate and select optimal clones. The results showed that there were significant differences in growth and wood properties among clones. The wood hardness (0.66–0.79), basic density (0.89), air-dried density (0.89) and compression strength parallel to the grain of wood (CSP) (0.84) had high repeatability. The variance component proportions indicated that the variation in wood properties came mainly from different genotypes (clones) rather than from different individuals of the same clone. The DBH showed a significant negative genetic correlation with the hardness of radial section (HRS) (−643), basic density (−0.531) and air-dry density (−0.495). This unfavorable relationship makes it difficult to improve growth and wood quality simultaneously in C. bungei. We selected the optimal clones under different scenarios, and we obtained 7.75–9.06% genetic gains for growth in the scenario in which height and DBH were the target traits. Genetic gains of 7.43–14.94% were obtained for wood properties by selecting optimal clones in the scenario in which wood properties were the target traits. Approximately 5% and 4% genetic gains were obtained for growth and wood properties, respectively, for the combined selection. This study provides new insights into the genetic improvement of wood quality in C. bungei.

Understanding genetic variation is important for efficiently selecting excellent clones and utilizing genetic resources during tree breeding. We investigated 16 growth traits of 50 32-year-old Pinus koraiensis clones. Analyses of variance showed that all the test traits differed significantly among clones. Average height, diameter at breast height (DBH), and volume of all clones were 10.41 m, 21.30 cm, and 0.148 m3, respectively. Phenotypic coefficients of variation and repeatabilities of traits ranged from 4.37 to 48.03% and from 0.013 to 0.900, respectively. There exists significant positive correlations among heights, diameter at different heights (1.3, 3.0, 5.0 m), and volumes; genetic correlation was close to phenotype correlation. Using four growth traits (height, DBH, volume, average crown width) as indices for a comprehensive evaluation, five clones (PK 11, PK 19, PK 04, PK 14, and PK 28), whose traits scored in the top 10%, were selected as elite clones. For these clones, genetic gains in height, DBH, volume and crown height were 8.58, 13.02, 32.72 and 3.83%, respectively. These results provide important information for improving P. koraiensis breeding programs.

Background “Bairihua”, a variety of the Catalpa bungei, has a large amount of flowers and a long flowering period which make it an excellent material for flowering researches in trees. SPL is one of the hub genes that regulate both flowering transition and development. Results SPL homologues CbuSPL9 was cloned using degenerate primers with RACE. Expression studies during flowering transition in “Bairihua” and ectopic expression in Arabidopsis showed that CbuSPL9 was functional similarly with its Arabidopsis homologues. In the next step, we used Y2H to identify the proteins that could interact with CbuSPL9. HMGA, an architectural transcriptional factor, was identified and cloned for further research. BiFC and BLI showed that CbuSPL9 could form a heterodimer with CbuHMGA in the nucleus. The expression analysis showed that CbuHMGA had a similar expression trend to that of CbuSPL9 during flowering in “Bairihua”. Intriguingly, ectopic expression of CbuHMGA in Arabidopsis would lead to aberrant flowers, but did not effect flowering time. Conclusions Our results implied a novel pathway that CbuSPL9 regulated flowering development, but not flowering transition, with the participation of CbuHMGA. Further investments need to be done to verify the details of this pathway.

The role of plant eIF5A proteins in multiple biological processes, such as protein synthesis regulation, translation elongation, mRNA turnover, programmed cell death and stress tolerance is well known. Toward using these powerful proteins to increase stress tolerance in agricultural plants, in the present study, we cloned and characterized PsneIF5A2 and PsneIF5A4 from young poplar (P. simonii × P. nigra) leaves. The deduced amino acid sequences of PsneIF5A2 and PsneIF5A4 were 98 % similar to each other, and they are orthologs of eIF5A1 in Arabidopsis. In a subcellular localization analysis, PsneIF5A2 and PsneIF5A4 proteins were localized in the nucleus and cytoplasm. qRT-PCR analysis showed that PsneIF5A2 and PsneIF5A4 were transcribed in poplar flowers, stem, leaves, and roots. In addition, they were also induced by abiotic stresses. Transgenic yeast expressing PsneIF5A2 and PsneIF5A4 had increased salt, heavy metal, osmotic, oxidative tolerance. Our results suggest that PsneIF5A2 and PsneIF5A4 are excellent candidates for genetic engineering to improve salt and heavy metal tolerance in agricultural plants.

Pulmonary artery sarcoma (PAS) is an extremely rare and highly malignant tumor that originates in the pulmonary artery. The majority of reported cases of PAS are confirmed by pathological examination subsequent to surgery or by autopsy. The present study reports the clinicopathological characteristics and immunohistochemical phenotypes of three cases of PAS, and aims to facilitate the identification of this lethal disease. In the present study, the data from clinical, histopathological and immunohistochemical examinations of three patients with PAS, whose diagnoses were confirmed by surgical biopsy conducted at the Beijing Anzhen Hospital (Beijing, China) between 2008 and 2012, were retrospectively analyzed. The patients (two women and one man; average age, 41.3 years old) presented with dyspnea on exertion. In addition, two of the patients experienced chest tightness, and one patient experienced intermittent syncope. Computed tomography pulmonary angiography revealed that two of the patients possessed a filling defect in the main, left and right pulmonary arteries, and one patient possessed a filling defect in the right upper pulmonary artery. Macroscopically, the PAS appeared as a mucoid intraluminal or nodular sessile mass spreading along the pulmonary artery. Microscopically, the tumor consisted of spindle cells with fascicular and storiform patterns, and was accompanied by necrosis and stromal myxoid changes. Immunohistochemically, vimentin, desmin and cluster of differentiation 34 were highly expressed in the patient that was diagnosed with intimal sarcoma, while vimentin and α-smooth muscle actin were highly expressed in the other two patients, who were diagnosed with leiomyosarcoma. PAS is often misdiagnosed due to nonspecific clinical manifestations and radiological features. Therefore, the diagnosis of PAS should be based on typical morphological features and immunohistochemical analysis of the tumor tissue.

Inflammatory bowel disease (IBD) is a complex immune-mediated disease of gastrointestinal tract that is mainly driven by Th1/Th17 immune response. “Helminth therapy” has emerged, and helminth-derived immunoregulatory molecules are being used as safe and new therapeutic antigens for IBD. Recombinant serine protease (SP) from newborn Trichinella spiralis (T. spiralis) larvae (NBL) was expressed and purified. BALB/c mice were immunized with NBL-SP at 100 µg three times at an interval of 5 days. Experimental colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) administration. The disease activity index (DAI) and macroscopic and microscopic scores of the colon were assessed to identify the effect of NBL-SP on experimental colitis. Cytokine production in the serum was analysed by meso scale discovery (MSD). Cytokine production in the colon was detected by ELISA. CD4+ T cell differentiation was measured by flow cytometry. NBL-SP alleviated TNBS-induced colitis in mice. The DAI, macroscopic and microscopic scores and colon length all showed a positive intervention effect of NBL-SP on experimental colitis. NBL-SP can weaken the increase in IFN-γ, TNF-α and IL-17 production as well as CD4+ IFN-γ+ T cell and CD4+IL-17+ T cell populations induced by colitis. Furthermore, the levels of Th2-related cytokines (IL-4, IL-5) and regulatory cytokines (IL-10, TGF-β) were elevated meanwhile the ratio of regulatory T cells (Tregs) and CD4+ IL-4 + T cells were increased by NBL-SP. NBL-SP of T. spiralis had a potential protective effect against IBD. NBL-SP skewed the Th1 and Th17-mediated response towards the Th2 and Treg response.

Trichinellosis is caused by Trichinella spiralis (T. spiralis), which is an important public health problem. In this study, a gene encoding a serine protease from adult worms of T. spiralis (Ts-Adsp) was screened from a cDNA library of adult worms and was cloned and expressed in a prokaryotic expression system. The gene Ts-Adsp was subcloned into the eukaryotic expression vector pcDNA3.1(+), which was named pcDNA3.1(+)-Adsp. Previous studies have found that recombinant Ts-Adsp protein (rTs-Adsp) can elicit partial protection against T. spiralis infection in mice. Our aim was to explore the protective effect of combining a DNA vaccine with the rTs-Adsp protein against T. spiralis. One week after the last vaccination, the serum and spleen were obtained. The levels of IgG, IgG1 and IgG2a and cytokine production in serum and spleen cells were analyzed. The results showed that the levels of humoral and cell-mediated immune responses increased in the pcDNA3.1(+)-Adsp/rTs-Adsp group mice and demonstrated that a Th1/Th2 mixed immune response was induced by pcDNA3.1(+)-Adsp/rTs-Adsp after vaccination. Moreover, mice vaccinated with pcDNA3.1(+)-Adsp/rTs-Adsp displayed a 69.50% reduction in muscle larvae burden. This study suggested that mixed immunity could improve the muscle larvae reduction rate.

Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor–DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF–DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.

Rapeseed (Brassica napus L.) is an important cruciferous crop worldwide. In this study, we collected five rapeseed samples with typical symptoms induced by phytoplasmas from Wuhan and Ezhou, Hubei Province, China, and E'erguna, Neimenggu Province, China. We successfully identified phytoplasmas from these samples via nested PCR amplification using phytoplasma-specific primer pairs. We also carried out phylogenetic analyses and grouped these phytoplasmas with the online tool iPhyClassifier. As a result, the phytoplasmas in samples EZ and NMG with witches’-broom and sample WH3 with dwarfing belonged to subgroup 16SrI–B, while the phytoplasmas in sample WH1 with fasciation and sample WH2 with peduncle clustering were members of subgroup 16SrV–H. This is the first report of phytoplasmas infecting rapeseed in China and of group 16SrV infecting rapeseed. We further investigated the host range of groups 16SrI and 16SrV and found group 16SrI in 307 plant species belonging to 230 genera of 83 families, while group 16SrV was identified in 65 species belonging to 47 genera of 30 families. The possible host expansion and host jumping of group 16SrV and the potential risk of phytoplasmas to rapeseed were also discussed.

Solanum is one of the largest genera, including two important crops&mdash, potato (Solanum tuberosum) and tomato (Solanum lycopersicum). In this study we compared the chloroplast codon usage bias (CUB) among 12 Solanum species, between photosynthesis-related genes (Photo-genes) and genetic system-related genes (Genet-genes), and between cultivated species and wild relatives. The Photo-genes encode proteins for photosystems, the photosynthetic electron transport chain, and RuBisCO, while the Genet-genes encode proteins for ribosomal subunits, RNA polymerases, and maturases. The following findings about the Solanum chloroplast genome CUB were obtained: (1) the nucleotide composition, gene expression, and selective pressure are identified as the main factors affecting chloroplast CUB, (2) all these 12 chloroplast genomes prefer A/U over G/C and pyrimidines over purines at the third-base of codons, (3) Photo-genes have higher codon adaptation indexes than Genet-genes, indicative of a higher gene expression level and a stronger adaptation of Photo-genes, (4) gene function is the primary factor affecting CUB of Photo-genes but not Genet-genes, (5) Photo-genes prefer pyrimidine over purine, whereas Genet-genes favor purine over pyrimidine, at the third position of codons, (6) Photo-genes are mainly affected by the selective pressure, whereas Genet-genes are under the underlying mutational bias, (7) S. tuberosum is more similar with Solanum commersonii than with Solanum bulbocastanum, (8) S. lycopersicum is greatly different from the analyzed seven wild relatives, (9) the CUB in codons for valine, aspartic acid, and threonine are the same between the two crop species, S. tuberosum and S. lycopersicum. These findings suggest that the chloroplast CUB contributed to the differential requirement of gene expression activity and function between Photo-genes and Genet-genes and to the performance of cultivated potato and tomato.

Background Betula platyphylla is a common tree species in northern China that has high economic and medicinal value. Our laboratory has been devoted to genome research on B. platyphylla for approximately 10 years. As primary organelle genomes, the complete genome sequences of chloroplasts are important to study the divergence of species, RNA editing and phylogeny. In this study, we sequenced and analyzed the complete chloroplast (cp) genome sequence of B. platyphylla. Results The complete cp genome of B. platyphylla was 160,518 bp in length, which included a pair of inverted repeats (IRs) of 26,056 bp that separated a large single copy (LSC) region of 89,397 bp and a small single copy (SSC) region of 19,009 bp. The annotation contained a total of 129 genes, including 84 protein-coding genes, 37 tRNA genes and 8 rRNA genes. There were 3 genes using alternative initiation codons. Comparative genomics showed that the sequence of the Fagales species cp genome was relatively conserved, but there were still some high variation regions that could be used as molecular markers. The IR expansion event of B. platyphylla resulted in larger cp genomes and rps19 pseudogene formation. The simple sequence repeat (SSR) analysis showed that there were 105 SSRs in the cp genome of B. platyphylla. RNA editing sites recognition indicated that at least 80 RNA editing events occurred in the cp genome. Most of the substitutions were C to U, while a small proportion of them were not. In particular, three editing loci on the rRNA were converted to more than two other bases that had never been reported. For synonymous conversion, most of them increased the relative synonymous codon usage (RSCU) value of the codons. The phylogenetic analysis suggested that B. platyphylla had a closer evolutionary relationship with B. pendula than B. nana. Conclusions In this study, we not only obtained and annotated the complete cp genome sequence of B. platyphylla, but we also identified new RNA editing sites and predicted the phylogenetic relationships among Fagales species. These findings will facilitate genomic, genetic engineering and phylogenetic studies of this important species. Electronic supplementary material The online version of this article (10.1186/s12864-018-5346-x) contains supplementary material, which is available to authorized users.

Polymerization of actin monomers into filaments requires the initial formation of nuclei composed of a few actin subunits; however, their instability has hindered their detailed study. Therefore we used chemically crosslinked actin oligomers to analyse their effect on actin polymerization. Actin dimer (upper dimer, UD), trimer and tetramer intermolecularly crosslinked by phenylene-bismaleimide along the genetic helix (between Lys199 and Cys374) were isolated by gel filtration and found to increasingly stimulate actin polymerization as shown by the pyrene assay and total internal reflection fluorescence microscopy. In contrast, the so-called lower actin dimer (LD) characterized by a Cys374-Cys374 crosslink stimulated actin polymerization only at low but inhibited it at high concentrations. UD and trimer stimulated the repolymerization of actin from complexes with thymosin β4 (Tβ4) or profilin, whereas the LD stimulated repolymerization only from the profilin : actin but not the actin : Tβ4 complex. In vivo, actin polymerization is stimulated by nucleation factors. Therefore the interaction and effects of purified LD, UD and trimer on the actin-nucleating activity of gelsolin, mouse diaphanous related (mDia) formin and the actin-related protein 2/3 (Arp2/3) complex were analysed. Native gel electrophoresis demonstrated binding of LD, UD and trimer to gelsolin and its fragment G1-3, to the FH2 domains of the formins mDia1 and mDia3, and to Arp2/3 complex. UD and trimer increased the nucleating activity of gelsolin and G1-3, but not of the mDia-FH2 domain nor of the Arp2/3 complex. In contrast, LD at equimolar concentration to Arp2/3 complex stimulated its nucleating activity, but inhibited that of mDia-FH2 domains, gelsolin and G1-3, demonstrating differential regulation of their nucleating activity by dimers containing differently oriented actin subunits.

Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation-mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro by using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn(2+)-activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by ∼ 60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide-enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 monophosphorylation at Ser(123) or Ser(125) in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser(123) or Ser(125) and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser(123) phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser(123) or Ser(125). PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa.

Research Highlights: A rigorous genome survey helped us to estimate the genomic characteristics, remove the DNA contamination, and determine the sequencing scheme of Betula platyphylla. Background and Objectives: B. platyphylla is a common tree species in northern China that has high economic and medicinal value. However, there is a lack of complete genomic information for this species, which severely constrains the progress of relevant research. The objective of this study was to survey the genome of B. platyphylla and determine the large-scale sequencing scheme of this species. Materials and Methods: Next-generation sequencing was used to survey the genome. The genome size, heterozygosity rate, and repetitive sequences were estimated by k-mer analysis. After preliminary genome assembly, sequence contamination was identified and filtered by sequence alignment. Finally, we obtained sterilized plantlets of B. platyphylla by plant tissue culture, which can be used for third-generation sequencing. Results: We estimated the genome size to be 432.9 Mb and the heterozygosity rate to be 1.22%, with repetitive sequences accounting for 62.2%. Bacterial contamination was observed in the leaves taken from the field, and most of the contaminants may be from the genus Mycobacterium. A total of 249,784 simple sequence repeat (SSR) loci were also identified in the B. platyphylla genome. Among the SSRs, only 11,326 can be used as candidates to distinguish the three Betula species. Conclusions: The B. platyphylla genome is complex and highly heterozygous and repetitive. Higher-depth third-generation sequencing may yield better assembly results. Sterilized plantlets can be used for sequencing to avoid contamination.

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including alpha-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the alpha-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac beta-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.

In this study, the additive effects of a late embryogenesis abundant (LEA) gene and a basic leucine zipper (bZIP) gene on salt and osmotic stress in Tamarix plants were analyzed. The constructs containing one or both of the LEA and bZIP genes were transformed into tobacco. Northern blot analysis showed the genes were overexpressed under the control of the CaMV 35S promoter in both dual and single gene-transgenic tobacco lines. The effects of salt and osmotic stress in transgenic tobacco plant were investigated. Following exposure to NaCl, mannitol, and PEG6000 stress, dual gene-transgenic lines showed higher seed generation and growth rates than single gene-transgenic lines and the wild-type. In response to NaCl stress, the dual gene-transgenic lines showed lower malondialdehyde and higher leaf chlorophyll content than single gene-transgenic lines and the wild-type. These results suggested that the co-expression of LEA and bZIP resulted in an additive enhancement of stress tolerance in dual gene-transgenic tobacco.

It is well understood that genetic tumors develop in certain interspecific Nicotiana hybrids. Nicotiana species are divided into “plus” and “minus” groups and crosses between “plus” and “minus” species give rise to tumorous hybrids. However, it has been proposed that parents and hybrids derived from crosses among members within the same group do not produce tumors. In this study, genetic tumors were only obtained in Nicotiana glauca, which exhibited tumor features similar to those of N. glauca × N. langsdorffii. Our results suggest that genetic factors may control tumor formation independent of tumor induction dependent on the specific interspecific cross. Genetic tumor formation exhibited high B-type and D-type cyclin expression levels, indicating tumor cells are characterized by an uncontrolled cell cycle.

Genetic tumors can develop on certain tobacco hybrid plant spontaneously. In this study, exogenous hormones were applied on the tumorous tobacco hybrid seedlings (N. glauca × N. langsdorffii), and the data showed that N6-benzyladenine (BA) enhanced the tumor formation under both tissue culture and field conditions, and NAA and ABA inhibited tumor formation under tissue culture condition, whereas ABA accelerated the tumor formation under field condition. Furthermore, Northern analysis indicated that tumor tissue exhibited high level of CycD expression compared with normal tissues, implying abnormal cell cycle of tumor cells. The differential display reverse transcription PCR (DDRT PCR) results indicated some genes may be involved in tumor formation.

D-type cyclins (CycD) are thought to control the onset of cell division and responses to extracellular signals during G1 phase. In this study a cyclin D3 (CycD3) gene was isolated from Nicotiana tabacum. Southern blot analysis indicated that it was present in a single-copy in the N. tabacum genome, and Northern analysis showed that it was only expressed in organs and tissues containing dividing cells. Exposure to cold and salt stresses resulted in decreased CycD3 expression supporting the view that its expression is closely related to cell growth.

Calli were induced from anthers of Populus simonii × P. nigra. Haploid plants were then regenerated from the callus and multiplied efficiently by culturing leaf explants. The presence of both haploid and diploid cells in the same plant revealed spontaneous chromosome doubling in haploid cells. The haploid plants were transformed with the nptII gene by Agrobacterium-mediated method using leaf explants, and five independent kanamycin-resistant lines were obtained, with a transformation frequency more than 6%. Further PCR test indicated that the exogenous betA gene was transferred into these kanamycin-resistant lines, which were still haploid. Thus, the efficient tissue culture system and transformation of haploid poplar plants were achieved. Our study will contribute to forest improvement via the haploid culture and transgenic technology.

Woody cells and tissues are recalcitrant to standard chromatin immunoprecipitation (ChIP) procedures. However, we recently successfully implemented ChIP in wood-forming tissue of the model woody plant Populus trichocarpa. Here we provide the detailed ChIP protocol optimized for wood-forming tissue that we used in those studies. By using stem-differentiating xylem (SDX; a wood-forming tissue), we identified all steps that were ineffective in standard ChIP protocols and systematically modified them to develop and optimize a robust ChIP protocol. The protocol includes tissue collection, cross-linking, nuclear isolation, chromatin extraction, DNA fragmentation, immunoprecipitation, DNA purification and sequence analysis. The protocol takes 2.5 d to complete and allows a robust 8-10-fold enrichment of transcription factor (TF)-bound genomic fragments (~150 ng/g of SDX) over nonspecific DNAs. The enriched DNAs are of high quality and can be used for subsequent PCR and DNA-seq analyses. We used this protocol to identify genome-wide specific TF-DNA interactions during wood formation and histone modifications associated with regulation of wood formation. Our protocol, which may be suitable for many tissue types, is so far the only working ChIP system for wood-forming tissue.

Isolated protoplasts serve as a transient expression system that is highly representative of stable transgenics in terms of transcriptome responses. They can also be used as a cellular system to study gene transactivation and nucleocytoplasmic protein trafficking. They are particularly useful for systems studies in which stable transgenics and mutants are unavailable. We present a protocol for the isolation and transfection of protoplasts from wood-forming tissue, the stem-differentiating xylem (SDX), in the model woody plant Populus trichocarpa. The method involves tissue preparation, digestion of SDX cell walls, protoplast isolation and DNA transfection. Our approach is markedly faster and provides better yields than previous protocols; small (milligrams)- to large (20 g)-scale SDX preparations can be achieved in ~60 s, with isolation of protoplasts and their subsequent transfection taking ~50 min. Up to ten different samples can be processed simultaneously in this time scale. Our protocol gives a high yield (~2.5 × 10(7) protoplasts per g of SDX) of protoplasts sharing 96% transcriptome identity with intact SDX.

In Arabidopsis, AP1 is a floral meristem identity gene and plays an important role in floral organ development. In this study, PsnAP1-1 and PsnAP1-2 were isolated from the male reproductive buds of poplar (Populus simonii × P. nigra), which are the orthologs of AP1 in Arabidopsis, by sequence analysis. Northern blot and qRT-PCR analysis showed that PsnAP1-1 and PsnAP1-2 exhibited high expression level in early inflorescence development of poplar. Subcellular localization showed the PsnAP1-1 and PsnAP1-2 proteins are localized in the nucleus. Overexpression of PsnAP1-1 and PsnAP1-2 in tobacco under the control of a CaMV 35S promoter significantly enhanced early flowering. These transgenic plants also showed much earlier stem initiation and higher rates of photosynthesis than did wild-type tobacco. qRT-PCR analysis further indicated that overexpression of PsnAP1-1 and PsnAP1-2 resulted in up-regulation of genes related to flowering, such as NtMADS4, NtMADS5 and NtMADS11. Overexpression of PsnAP1-1 and PsnAP1-2 in Arabidopsis also induced early flowering, but did not complement the ap1-10 floral morphology to any noticeable extent. This study indicates that PsnAP1-1 and PsnAP1-2 play a role in floral transition of poplar.

In this study, a MADS-box gene (BpMADS), which is an ortholog of AP1 from Arabidopsis, was isolated from birch (Betula platyphylla). Transgenic Arabidopsis containing a BpMADS promoter::GUS construct was produced, which exhibited strong GUS staining in sepal tissues. Ectopic expression of BpMADS significantly enhanced the flowering of tobacco (35S::BpMADS). In addition, the chloroplasts of transgenic tobacco exhibited much higher growth and division rates, as well rates of photosynthesis, than wild-type. A grafting experiment demonstrated that the flowering time of the scion was not affected by stock that overexpressed BpMADS. In addition, the overexpression of BpMADS resulted in the upregulation of some flowering-related genes in tobacco.

Intoxication of eukaryotic cells by Photorhabdus luminescens toxin TccC3 induces cell rounding and detachment from the substratum within a few hours and compromises a number of cell functions like phagocytosis. Here, we used morphological and biochemical procedures to analyse the mechanism of TccC3 intoxication. Life imaging of TccC3-intoxicated HeLa cells transfected with AcGFP-actin shows condensation of F-actin into large aggregates. Life cell total internal reflection fluorescence (TIRF) microscopy of identically treated HeLa cells confirmed the formation of actin aggregates but also disassembly of F-actin stress fibres. Recombinant TccC3 toxin ADP-ribosylates purified skeletal and non-muscle actin at threonine148 leading to a strong propensity to polymerize and F-actin bundle formation as shown by TIRF and electron microscopy. Native gel electrophoresis shows strongly reduced binding of Thr148-ADP-ribosylated actin to the severing proteins gelsolin and its fragments G1 and G1-3, and to ADF/cofilin. Complexation of actin with these proteins inhibits its ADP-ribosylation. TIRF microscopy demonstrates rapid polymerization of Thr148-ADP-ribosylated actin to curled F-actin bundles even in the presence of thymosin β4, gelsolin or G1-3. Thr148-ADP-ribosylated F-actin cannot be depolymerized by gelsolin or G1-3 as verified by TIRF, co-sedimentation and electron microscopy and shows reduced treadmilling as indicated by a lack of stimulation of its ATPase activity after addition of cofilin-1.

In the present study, a zinc-finger-like cDNA (ThZFL) was cloned from the Tamarix hispida. Northern blot analysis showed that the expression of ThZFL can be induced by salt, osmotic stress and ABA treatment. Overexpression of the ThZFL confers salt and osmotic stress tolerance in both yeast Saccharomyces cerevisiae and tobacco. Furthermore, MDA levels in ThZFL transformed tobacco were significantly decreased compared with control plants under salt and osmotic stress, suggesting ThZFL may confer stress tolerance by decreasing membrane lipid peroxidation. Subcellular localization analysis showed the ThZFL protein is localized in the cell wall. Our results indicated the ThZFL gene is an excellent candidate for genetic engineering to improve salt and osmotic tolerance in agricultural plants.