A multifunctional chitosan-based hydrogel with self-healing, antibacterial, and immunomodulatory effects as wound dressing.

Bacterial infection often leads to inflammatory responses and delays wound healing. Chitosan (CS)-based composite hydrogels can hold desirable mechanical properties and maintain excellent antibacterial abilities, and thus may be promising as wound dressings. Although CS-based hydrogels have been widely studied on the antibacterial and wound-healing abilities, their immunomodulatory abilities were rarely evaluated. Herein, we developed a multifunctional CS/Poly[2-(methacryloyloxy)ethyl] trimethyl ammonium chloride (PMETAC) hydrogel. In vitro, this hydrogel exhibited self-healing ability and excellent biocompatibility, promoted macrophage polarization towards M2 phenotype, and showed desirable antibacterial activity. In vivo, this hydrogel accelerated the wound regeneration process by reducing bacterial burden, increasing collagen deposition, stimulating angiogenesis, promoting macrophage polarization to M2 direction, and shifting the balance of T helper type 17 (Th17) cells towards anti-inflammatory regulatory T (Treg) cells. This work revealed the potential immunomodulatory effect of CS-based wound dressings and thus may provide a novel target for developing efficient wound healing tools.

Transcriptome analysis reveals the mechanism of stromal cell-derived factor-1 and exendin-4 synergistically promoted periodontal ligament stem cells osteogenic differentiation.

Stromal cell-derived factor-1 (SDF-1) and Exendin-4 (EX-4) play beneficial roles in promoting periodontal ligament stem cells (PDLSCs) osteogenic differentiation, while the detailed mechanism has not been clarified. In this study, we aimed to evaluate the biological mechanism of SDF-1 and EX-4 alone or synergistic application in regulating PDLSCs differentiation by RNA-sequencing (RNA-seq). A total of 110, 116 and 109 differentially expressed genes (DEGs) were generated in osteogenic medium induced PDLSCs treated by SDF-1, EX-4, and SDF-1+EX-4, respectively. The DEGs in SDF-1 group were enriched in signal transduction related signaling pathways; the DEGs in EX-4 group were enriched in metabolism and biosynthesis-related pathways; and the DEGs generated in SDF-1+EX-4 group were mainly enriched in RNA polymerase II transcription, cell differentiation, chromatin organization, protein phosphorylation pathways. Based on Venn analysis, a total of 37 specific DEGs were identified in SDF-1+EX-4 group, which were mainly enriched in negative regulation of autophagy and cellular component disassembly signaling pathways. Short time-series expression miner (STEM) analysis grouped all expressed genes of PDLSCs into 49 clusters according to the dynamic expression patterns and 25 genes, including NRSN2, CHD9, TUBA1A, distributed in 10 gene clusters in SDF-1+EX-4 treated PDLSCs were significantly up-regulated compared with the SDF-1 and EX-4 alone groups. The gene set enrichment analysis indicated that SDF-1 could amplify the role of EX-4 in regulating varied signaling pathways, such as type II diabetes mellitus and insulin signaling pathways; while EX-4 could aggravate the effect of SDF-1 on PDLSCs biological roles via regulating primary immunodeficiency, tight junction signaling pathways. In summary, our study confirmed that SDF-1 and EX-4 combined application could enhance PDLSCs biological activity and promote PDLSCs osteogenic differentiation by regulating the metabolism, biosynthesis and immune-related signaling pathways.

Stromal cell-derived factor-1/Exendin-4 cotherapy facilitates the proliferation, migration and osteogenic differentiation of human periodontal ligament stem cells in vitro and promotes periodontal bone regeneration in vivo.

OBJECTIVES: Stromal cell-derived factor-1 (SDF-1) actively directs endogenous cell homing. Exendin-4 (EX-4) promotes stem cell osteogenic differentiation. Studies revealed that EX-4 strengthened SDF-1-mediated stem cell migration. However, the effects of SDF-1 and EX-4 on periodontal ligament stem cells (PDLSCs) and bone regeneration have not been investigated. In this study, we aimed to evaluate the effects of SDF-1/EX-4 cotherapy on PDLSCs in vitro and periodontal bone regeneration in vivo. METHODS: Cell-counting kit-8 (CCK8), transwell assay, qRT-PCR and western blot were used to determine the effects and mechanism of SDF-1/EX-4 cotherapy on PDLSCs in vitro. A rat periodontal bone defect model was developed to evaluate the effects of topical application of SDF-1 and systemic injection of EX-4 on endogenous cell recruitment, osteoclastogenesis and bone regeneration in vivo. RESULTS: SDF-1/EX-4 cotherapy had additive effects on PDLSC proliferation, migration, alkaline phosphatase (ALP) activity, mineral deposition and osteogenesis-related gene expression compared to SDF-1 or EX-4 in vitro. Pretreatment with ERK inhibitor U0126 blocked SDF-1/EX-4 cotherapy induced ERK signal activation and PDLSC proliferation. SDF-1/EX-4 cotherapy significantly promoted new bone formation, recruited more CXCR4+ cells and CD90+ /CD34- stromal cells to the defects, enhanced early-stage osteoclastogenesis and osteogenesis-related markers expression in regenerated bone compared to control, SDF-1 or EX-4 in vivo. CONCLUSIONS: SDF-1/EX-4 cotherapy synergistically regulated PDLSC activities, promoted periodontal bone formation, thereby providing a new strategy for periodontal bone regeneration.

AGGF1 inhibits the expression of inflammatory mediators and promotes angiogenesis in dental pulp cells.

OBJECTIVES: To determine the role of angiogenic factor with G-patch and FHA domain 1 (AGGF1) in inflammatory response of human dental pulp cells (DPCs) and the underneath mechanism and to explore its role in angiogenesis. MATERIALS AND METHODS: The expression of AGGF-1 in human healthy and inflammatory pulp tissues was detected by immunohistochemistry. RT-qPCR and Western blot were used to evaluate the expression of AGGF1 in DPCs stimulated by lipopolysaccharide (LPS). After AGGF1 was knocked down, the expression of LPS-induced inflammatory cytokines in DPCs was quantified by RT-qPCR and ELISA. Immunofluorescence and Western blot were used to assess the activation of NF-κB signaling. Inflammatory cytokines were detected by RT-qPCR and ELISA in DPCs pretreated with NF-κB pathway inhibitors before LPS stimulation, and then the effect of AGGF1 on angiogenesis was also evaluated. RESULTS: AGGF1 expression increased in inflammatory dental pulp tissues. In DPCs stimulated by LPS, AGGF1 was upregulated in a dose-dependent manner (P < 0.05). In AGGF1 knockdown cells, the expression of IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1/CCL-2) increased by LPS stimulation (P < 0.001). Nuclear translocation of p65 was promoted, and the addition of NF-κB inhibitors inhibited the expression of inflammatory factors. Meanwhile, knockdown of AGGF1 inhibited vascularization. CONCLUSIONS: AGGF1 inhibited the synthesis of inflammatory cytokines through NF-κB signaling pathway and promoted the angiogenesis of DPCs. CLINICAL RELEVANCE: This study might shed light in the treatment of pulpitis and regeneration of dental pulp tissues; however, more clinical trials are required to validate these findings.

[Effects of Exenatide-4 on proliferation, migration and osteogenic differentiation of human periodontal ligament stem cells].

PURPOSE: To investigate the effects of exendin-4(EX-4) on proliferation, migration and osteogenic differentiation of human periodontal ligament stem cells(PDLSCs). METHODS: PDLSCs were isolated and cultured using limited dilution method in vitro. Colony formation assay, osteogenic and adipogenic differentiation were applied to identify the stem cells. Immunofluorescence staining was used to detect the expression of EX-4 receptor glucagon-like peptide-1 receptor (GLP-1R) on the surface of PDLSCs. PDLSCs were stimulated with 5, 10, 20 or 50 nmol/L EX-4 in vitro. CCK-8, Transwell assay and alkaline phosphatase(ALP) activity assay were used to determine the effects of EX-4 on PDLSCs proliferation, migration and osteogenic differentiation. Quantitative real-time polymerase chain reaction was used to determine the expression of osteogenic related genes ALP, runt-related transcription factor 2(Runx2) and osteocalcin (OCN). The data were analyzed by Graphpad Prims 6.0 software package. RESULTS: PDLSCs were successfully isolated and cultivated. GLP-1R positively expressed on the surface of PDLSCs. EX-4 exerted no significant effect on PDLSCs proliferation(P>0.05). EX-4 significantly promoted migration, ALP activity and osteogenic related genes expression of PDLSCs (P<0.05). CONCLUSIONS: 10 nmol/L EX-4 could promote migration and osteogenic differentiation of PDLSCs.

Metformin facilitates the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells in vitro.

Periodontitis is one of the main causes of tooth loss and has been confirmed as the sixth complication of diabetes. Metformin promotes the osteogenic differentiation of stem cells. Periodontal ligament stem cells (PDLSCs) are the best candidate stem cells for periodontal tissue regeneration. Herein, we aimed to identify the effects of metformin on the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. PDLSCs were isolated by limiting dilution, and their characteristics were assessed by colony formation assay and flow cytometry. Cell counting and migration assays were used to investigate the effects of metformin on proliferation and migration. The osteogenic differentiation ability of PDLSCs was detected by alkaline phosphatase (ALP) activity and Alizarin Red S staining. Gene and protein levels of osteogenesis-related markers were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Metformin treatment at 10 µM did not affect PDLSC proliferation, while at 50 and 100 µM, metformin time-dependently enhanced PDLSC proliferation and significantly increased cell numbers after 5 and 7 days of stimulation (P < 0.05). In addition, 50 µM metformin exhibited a maximal effect on migration, ALP activity, and mineral deposition (P < 0.05). Furthermore, 50 µM metformin significantly upregulated the gene expression levels of ALP, BSP, OPN, OCN, and Runx2 and the protein expression of ALP and Runx2 (P < 0.05). In summary, our study confirms that metformin facilitates the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro and could be used as a new strategy for periodontal tissue regeneration.

The growth inhibitory effect of human gingiva-derived mesenchymal stromal cells expressing interferon-ß on tongue squamous cell carcinoma cells and xenograft model.

BACKGROUND: Interferon-ß (IFN-ß) is a cytokine with pleiotropic cellular functions, including antiviral, antiproliferative, and immunomodulatory activities. IFN-ß inhibits multiple tumor cell growth in vitro. However, the contradiction between the therapeutic dose of IFN-ß and its maximally tolerated dose is still inextricable in vivo. Human gingiva-derived mesenchymal stromal cells (GMSCs) represent promising vehicles for cancer gene therapy. This study evaluated the potential of GMSCs genetically engineered to produce IFN-ß as a targeted gene delivery system to treat tongue squamous cell carcinoma (TSCC) in vitro and in vivo. METHODS: A lentiviral vector encoding IFN-ß was constructed and transfected into GMSCs to obtain IFN-ß gene-modified GMSCs (GMSCs/IFN-ß). Enzyme-linked immunosorbent assay (ELISA) was used to measure the IFN-ß concentration in conditioned medium (CM) from GMSCs/IFN-ß. The Cell Counting Kit-8 (CCK8), colony formation assay, and flow cytometry were used to detect the effects of GMSCs/IFN-ß on TSCC cell line CAL27 cell growth and apoptosis in vitro. TSCC xenograft model was developed by subcutaneous injection of CAL27 cells into BALB/c nude mouse, and the role of intravenously injected GMSCs/IFN-ß in engrafting in TSCC and controlling tumor progression was measured in vivo. RESULTS: GMSCs/IFN-ß expressed a high level of IFN-ß. Both CCK8 and colony forming assay showed that GMSCs/IFN-ß significantly inhibited the proliferation of CAL27 cells compared with the GMSCs, GMSCs/vector, or DMEM group. Flow cytometry analysis demonstrated that the CAL27 cell apoptosis rate was higher in the GMSCs/IFN-ß group than in the other three groups. The in vivo experiment revealed that GMSCs/IFN-ß engrafted selectively in TSCC xenograft and expressed a high level of IFN-ß. There were smaller tumor volume and lower number of Ki67-positive cells in the GMSCs/IFN-ß group than in the GMSCs, GMSCs/vector, or phosphate-buffered saline (PBS) group. Interestingly, GMSCs and GMSCs/vector also presented the potential of CAL27 cell growth inhibition in vitro and in vivo, although such an effect was weaker than GMSCs/IFN-ß. CONCLUSIONS: GMSCs/IFN-ß inhibits the proliferation of TSCC cells in vitro and in vivo. These results provide evidence that delivery of IFN-ß by GMSCs may be a promising approach to develop an effective treatment option for TSCC therapy.

Sequential application of bFGF and BMP-2 facilitates osteogenic differentiation of human periodontal ligament stem cells.

BACKGROUND AND OBJECTIVES: Basic fibroblast growth factor (bFGF) promotes cells proliferation and chemotaxis and maintains stemness while inhibits mineralized nodule formation. Bone morphogenetic protein 2 (BMP-2) shows great potential in promoting bone formation. However, sequential application of these two growth factors on periodontal ligament stem cells (PDLSCs) has not been explored. In this study, we aimed to identify the optimal concentration and time of bFGF on PDLSCs proliferation, migration and then investigate the sequential delivery of bFGF and BMP-2 on osteogenic differentiation of PDLSCs in vitro. MATERIALS AND METHODS: Periodontal ligament stem cells were isolated by limiting dilution method. Dose-dependent additive effects of bFGF and BMP-2 on PDLSCs were detected. Cell counting assay, cell migration assay, alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis were used to determine different application modalities of bFGF and BMP-2 on proliferation, migration, and osteogenic differentiation of PDLSCs. RESULTS: 50 ng/mL bFGF significantly promoted PDLSCs proliferation and chemotaxis while time-dependently inhibited BMP-2 induced ALP activity. Sequential application of 25 ng/mL bFGF for first 3 days and followed with 50 ng/mL BMP-2 for another 9, 18, and 25 days significantly promoted PDLSCs osteogenic differentiation. Compared with bFGF and BMP-2 simultaneous group, sequential application of bFGF and BMP-2 group significantly enhanced ALP activity, osteogenesis-related genes and proteins expression and mineral deposition. CONCLUSION: Sequential application of bFGF and BMP-2 synergistically promoted osteogenic differentiation of PDLSCs, and this sequential application modality of growth factors would provide a new strategy for periodontal regeneration.

Oxytocin facilitates the proliferation, migration and osteogenic differentiation of human periodontal stem cells in vitro.

OBJECTIVE: To explore the effect of oxytocin (OT) on the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro. DESIGN: PDLSCs were obtained by limiting dilution method. Immunofluorescence (IF), cell-counting kit-8 (CCK8), cell migration assay, Alizarin Red S staining, cetylpyridinium chloride (CPC) colorimetry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis were used to examine the effect of OT on oxytocin receptor (OTR) expression, cell proliferation, migration and osteogenic differentiation of PDLSCs. RESULTS: Our study showed that PDLSCs expressed OTR. One hundred nanomolar OT exhibited the maximal effect on migration, while only 50 nM OT significantly promoted proliferation of PDLSCs, as well as mineralized nodule formation and calcium deposition (p < 0.05). Furthermore, 50 nM OT significantly up-regulated expression of osteogenesis-related genes-alkaline phosphatase (ALP), Collagen I (Col I), runt related transcription factor 2 (Runx 2), osteopontin (OPN) and osteocalcin (OCN)-at specific time points compared with osteogenic inductive medium (p < 0.05). In addition, western blot analysis demonstrated that 50 nM OT enhanced protein levels of ALP, Col I, and Runx 2 at day 7 and day 14 (p < 0.01), as well as activating the phosphorylation of extracellular-signal-regulated kinase (ERK) and protein kinase B (AKT) pathway; notably, 50 nM OT inhibited phosphorylation of the phosphatidylinositol 3-kinase (PI3K) pathway (p < 0.05). CONCLUSIONS: OT promoted proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. Furthermore, the effect of OT on osteogenic differentiation was mediated through ERK and AKT pathway. Thus, OT may have potential for use in periodontal regeneration.

Prolyl hydroxylases positively regulated LPS-induced inflammation in human gingival fibroblasts via TLR4/MyD88-mediated AKT/NF-κB and MAPK pathways.

OBJECTIVES: Prolyl hydroxylases (PHDs) play essential roles in oxygen-sensing system, whereas the effects of PHDs on inflammation have not been totally uncovered. Our study aimed to investigate the role of PHDs in lipopolysaccharide (LPS)-induced inflammation of human gingival fibroblasts (HGFs) and clarify the potential mechanisms. MATERIALS AND METHODS: A pan hydroxylase inhibitor, dimethyloxallyl glycine (DMOG), and RNA interference were used to explore the role of PHDs in inflammation. Cytotoxic effect of DMOG was determined by cell-counting kit-8 and flow cytometry respectively. The secretion levels of IL-6 and IL-8 were assessed by ELISA. The mRNA levels of inflammatory cytokines, Toll-like receptor (TLR) 4 and MyD88 were evaluated by quantitative real-time PCR. The activation of NF-κB, mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways were detected by western blot and the nuclear translocation of NF-κB p65 was examined by immunofluorescence. Downregulation of PHD1 and PHD2 was performed with siRNA transfection. RESULTS: Dimethyloxallyl glycine inhibited LPS-induced inflammatory cytokine, TLR4 and MyD88 expression in gene level and the elevated secretion of IL-6 and IL-8 was also downregulated. Additionally, LPS-induced activation of NF-κB, MAPK and AKT pathways was abolished by DMOG treatment. Importantly, LPS-induced inflammatory cytokine expression was merely suppressed by PHD2 knockdown. CONCLUSIONS: Prolyl hydroxylases acted as a positive regulator in LPS-induced inflammation of HGFs via TLR4/MyD88-mediated NF-κB, MAPK and AKT signalling pathways and PHD2 among three isoforms was principally responsible for the effects.

Transcriptome analysis of chrysanthemum (Dendranthema grandiflorum) in response to low temperature stress.

BACKGROUND: Chrysanthemum is one kind of ornamental plant well-known and widely used in the world. However, its quality and production were severely affected by low temperature conditions in winter and early spring periods. Therefore, we used the RNA-Seq platform to perform a de novo transcriptome assembly to analyze chrysanthemum (Dendranthema grandiflorum) transcription response to low temperature. RESULTS: Using Illumina sequencing technology, a total of 86,444,237 high-quality clean reads and 93,837 unigenes were generated from four libraries: T01, controls; T02, 4 °C cold acclimation (CA) for 24 h; T03, - 4 °C freezing treatments for 4 h with prior CA; and T04, - 4 °C freezing treatments for 4 h without prior CA. In total, 7583 differentially expressed genes (DEGs) of 36,462 annotated unigenes were identified. We performed GO and KEGG pathway enrichment analyses, and excavated a group of important cold-responsive genes related to low temperature sensing and signal transduction, membrane lipid stability, reactive oxygen species (ROS) scavenging and osmoregulation. These genes encode many key proteins in plant biological processes, such as protein kinases, transcription factors, fatty acid desaturase, lipid-transfer proteins, antifreeze proteins, antioxidase and soluble sugars synthetases. We also verified expression levels of 10 DEGs using quantitative real-time polymerase chain reaction (qRT-PCR). In addition, we performed the determination of physiological indicators of chrysanthemum treated at low temperature, and the results were basically consistent with molecular sequencing results. CONCLUSION: In summary, our study presents a genome-wide transcript profile of Dendranthema grandiflorum var. jinba and provides insights into the molecular mechanisms of D. grandiflorum in response to low temperature. These data contributes to our deeper relevant researches on cold tolerance and further exploring new candidate genes for chilling-tolerance and freezing-tolerance chrysanthemum molecular breeding.

Overexpression of DgWRKY4 Enhances Salt Tolerance in Chrysanthemum Seedlings.

High salinity seriously affects the production of chrysanthemum, so improving the salt tolerance of chrysanthemum becomes the focus and purpose of our research. The WRKY transcription factor (TF) family is highly associated with a number of processes of abiotic stress responses. We isolated DgWRKY4 from Dendranthema grandiflorum, and a protein encoded by this new gene contains two highly conserved WRKY domains and two C2H2 zinc-finger motifs. Then, we functionally characterized that DgWRKY4 was induced by salt, and DgWRKY4 overexpression in chrysanthemum resulted in increased tolerance to high salt stress compared to wild-type (WT). Under salt stress, the transgenic chrysanthemum accumulated less malondialdehyde, hydrogen peroxide (H2O2), and superoxide anion ([Formula: see text]) than WT, accompanied by more proline, soluble sugar, and activities of antioxidant enzymes than WT; in addition, a stronger photosynthetic capacity and a series of up-regulated stress-related genes were also found in transgenic chrysanthemum. All results demonstrated that DgWRKY4 is a positive regulatory gene responding to salt stress, via advancing photosynthetic capacity, promoting the operation of reactive oxygen species-scavenging system, maintaining membrane stability, enhancing the osmotic adjustment, and up-regulating transcript levels of stress-related genes. So, DgWRKY4 can serve as a new candidate gene for salt-tolerant plant breeding.

Chrysanthemum WRKY gene DgWRKY5 enhances tolerance to salt stress in transgenic chrysanthemum.

WRKY transcription factors play important roles in plant growth development, resistance and substance metabolism regulation. However, the exact function of the response to salt stress in plants with specific WRKY transcription factors remains unclear. In this research, we isolated a new WRKY transcription factor DgWRKY5 from chrysanthemum. DgWRKY5 contains two WRKY domains of WKKYGQK and two C2H2 zinc fingers. The expression of DgWRKY5 in chrysanthemum was up-regulated under various treatments. Meanwhile, we observed higher expression levels in the leaves contrasted with other tissues. Under salt stress, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzymes in transgenic chrysanthemum were significantly higher than those in WT, whereas the accumulation of H2O2, O2- and malondialdehyde (MDA) was reduced in transgenic chrysanthemum. Several parameters including root length, root length, fresh weight, chlorophyll content and leaf gas exchange parameters in transgenic chrysanthemum were much better compared with WT under salt stress. Moreover, the expression of stress-related genes DgAPX, DgCAT, DgNCED3A, DgNCED3B, DgCuZnSOD, DgP5CS, DgCSD1 and DgCSD2 was up-regulated in DgWRKY5 transgenic chrysanthemum compared with that in WT. These results suggested that DgWRKY5 could function as a positive regulator of salt stress in chrysanthemum.

Whole-transcriptome sequence analysis of differentially expressed genes in Phormium tenax under drought stress.

Phormium tenax is a kind of drought resistant garden plant with its rich and colorful leaves. To clarify the molecular mechanism of drought resistance in Phormium tenax, transcriptome was sequenced by the Illumina sequencing technology under normal and drought stress, respectively. A large number of contigs, transcripts and unigenes were obtained. Among them, only 30,814 unigenes were annotated by comparing with the protein databases. A total of 4,380 genes were differentially expressed, 2,698 of which were finally annotated under drought stress. Differentially expression analysis was also performed upon drought treatment. In KEGG pathway, the mechanism of drought resistance in Phormium tenax was explained from three aspects of metabolism and signaling of hormones, osmotic adjustment and reactive oxygen species metabolism. These results are helpful to understand the drought tolerance mechanism of Phormium tenax and will provide a precious genetic resource for drought-resistant vegetation breeding and research.

Overexpression of a chrysanthemum transcription factor gene DgNAC1 improves the salinity tolerance in chrysanthemum.

KEY MESSAGE: DgNAC1, a transcription factor of chrysanthemum, was functionally verified to confer salt stress responses by regulating stress-responsive genes. NAC transcription factors play effective roles in resistance to different abiotic stresses, and overexpressions of NAC TFs in Arabidopsis have been proved to be conducive in improving salinity tolerance. However, functions of NAC genes in chrysanthemum continue to be poorly understood. Here, we performed physiology and molecular experiments to evaluate roles of DgNAC1 in chrysanthemum salt stress responses. In this study, DgNAC1-overexpressed chrysanthemum was obviously more resistant to salt over the WT (wild type). Specifically, the transgenic chrysanthemum showed a higher survival rate and lower EC (electrolyte conductivity) than WT under salt stress. The transgenic chrysanthemum also showed fewer accumulations of MDA (malondialdehyde) and reactive oxygen species (H2O2 and O2-), greater activities of SOD (superoxide dismutase), POD (peroxidase) and CAT (catalase), as well as more proline content than WT under salt stress. Furthermore, stress-responsive genes in transgenic chrysanthemum were greater up-regulated than in WT under salinity stress. Thus, all results revealed that DgNAC1 worked as a positive regulator in responses to salt stress and it may be an essential gene for molecular breeding of salt-tolerant plants.

Comparative Analysis of the Chrysanthemum Leaf Transcript Profiling in Response to Salt Stress.

Salt stress has some remarkable influence on chrysanthemum growth and productivity. To understand the molecular mechanisms associated with salt stress and identify genes of potential importance in cultivated chrysanthemum, we carried out transcriptome sequencing of chrysanthemum. Two cDNA libraries were generated from the control and salt-treated samples (Sample_0510_control and Sample_0510_treat) of leaves. By using the Illumina Solexa RNA sequencing technology, 94 million high quality sequencing reads and 161,522 unigenes were generated and then we annotated unigenes through comparing these sequences to diverse protein databases. A total of 126,646 differentially expressed transcripts (DETs) were identified in leaf. Plant hormones, amino acid metabolism, photosynthesis and secondary metabolism were all changed under salt stress after the complete list of GO term and KEGG enrichment analysis. The hormone biosynthesis changing and oxidative hurt decreasing appeared to be significantly related to salt tolerance of chrysanthemum. Important protein kinases and major transcription factor families involved in abiotic stress were differentially expressed, such as MAPKs, CDPKs, MYB, WRKY, AP2 and HD-zip. In general, these results can help us to confirm the molecular regulation mechanism and also provide us a comprehensive resource of chrysanthemum under salt stress.