Bei-Bei Jiang

Overexpression of a chrysanthemum transcription factor gene, DgWRKY3, in tobacco enhances tolerance to salt stress

WRKY transcription factor genes (TFs) play important roles in response to various abiotic stresses. However, the roles of the chrysanthemum WRKY genes in abiotic stress response remain obscure. In this study, we functionally characterized a novel WRKY gene, DgWRKY3, from chrysanthemum (Dendranthema grandiflorum). Its expression in the chrysanthemum was up-regulated by salinity or dehydration stress, but not by abscisic acid (ABA). The DgWRKY3-overexpression tobacco plants increase salt tolerance compared with wild-type (WT) tobacco plants. The increased levels of proline were observed in transgenic plants compared to WT plants under salt stress. In addition, the DgWRKY3 transgenic plants reduced accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2) compared with WT plants, accompanied by higher activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) and the greater accumulation of antioxidants including ascorbate (AsA) and glutathione (GSH) under salt stress. Moreover, the DgWRKY3 transgenic plants enhanced the expression of stress-related genes involved in osmotic adjustment and membrane protection (NtP5CS, NtLEA5, and NtERD10D) and oxidative stress response (NtSOD, NtPOD, NtCAT, and NtAPX) under salt stress. However, no significant difference in the expression of stress-related genes (NtP5CS, NtLEA5, NtERD10D, NtSOD, NtPOD, NtCAT, and NtAPX) was found between the DgWRKY3-overexpression and WT tobacco plants under normal conditions, despite the fact that the constitutive promoter was used to drive DgWRKY3. These findings suggest that DgWRKY3 functions as a positive regulator to mediate tolerance of plants to salt stress.

Functional Analysis of a Novel Chrysanthemum WRKY Transcription Factor Gene Involved in Salt Tolerance

Plant-specific WRKY transcription factors (TFs) are involved in stress responses such as cold, high salinity, or drought as well as abscisic acid (ABA) signaling. However, their roles in abiotic stresses are still not well known in chrysanthemum. Here, we isolated a novel WRKY gene, DgWRKY1, from chrysanthemum (Dendranthema grandiflorum). DgWRKY1 contains one WRKY domain and one C2H2 zinc-finger motif (C-X4-C-X23-H-X-H), and was localized in the nucleus. Expression of DgWRKY1 was up-regulated by drought, salinity, and ABA. The DgWRKY1-overexpression tobacco plants were more tolerant to salt, and seed gerrmination was more sensitive to ABA, than the wild-type (WT). The transgenic lines exhibited less accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) under salt stress, and less antioxidant enzyme activity, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), than the WT under both control conditions and salt stress. In addition, there was greater up-regulation of the ROS-related enzyme genes (NtSOD, NtPOD, and NtCAT) in transgenic lines under normal or salt conditions. These findings suggest that DgWRKY1 plays a positive regulatory role in salt stress response.

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.

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

Key messageDgNAC1, a transcription factor of chrysanthemum, was functionally verified to confer salt stress responses by regulating stress-responsive genes.AbstractNAC 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.

Overexpression of a novel chrysanthemum Cys2/His2-type zinc finger protein gene DgZFP3 confers drought tolerance in tobacco

A drought stress-responsive Cys2/His2-type zinc finger protein gene DgZFP3 was previously isolated (Liu et al., Afr J Biotechnol 11:7781–7788, 2012b) from chrysanthemum. To assess roles of DgZFP3 in plant drought stress responses, we performed gain-of-function experiment. The DgZFP3-overexpression tobacco plants showed significant drought tolerance over the wild type (WT). The transgenic lines exhibited less accumulation of H2O2 under drought stress, more accumulation of proline and greater activities of peroxidase (POD) and superoxide dismutase than the WT under both control conditions and drought stress. In addition, there was greater up-regulation of the ROS-related enzyme genes (NtSOD and NtPOD) and stress-related genes (NtLEA5 and NtDREB) in transgenic lines under normal or drought conditons. Thus DgZFP3 probably plays a positive regulatory role in drought stress response and has the potential to be utilized in transgenic breeding to improve drought stress tolerance in plants.

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 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 (O2−) 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.

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.

Cloning and Characterization of a Novel Vacuolar Na+/H+ Antiporter Gene (Dgnhx1) from Chrysanthemum

Plant vacuolar Na+/H+ antiporter genes play significant roles in salt tolerance. However, the roles of the chrysanthemum vacuolar Na+/H+ antiporter genes in salt stress response remain obscure. In this study, we isolated and characterized a novel vacuolar Na+/H+ antiporter gene DgNHX1 from chrysanthemum. The DgNHX1 sequence contained 1920 bp with a complete open reading frame of 1533 bp encoding a putative protein of 510 amino acids with a predicted protein molecular weight of 56.3 kDa. DgNHX1 was predicted containing nine transmembrane domains. Its expression in the chrysanthemum was up-regulated by salt stress, but not by abscisic acid (ABA). To assess roles of DgNHX1 in plant salt stress responses, we performed gain-of-function experiment. The DgNHX1-overexpression tobacco plants showed significant salt tolerance than the wild type (WT). The transgenic lines exhibited more accumulation of Na+ and K+ under salt stress. These findings suggest that DgNHX1 plays a positive regulatory role in salt stress response.

Overexpression of a chrysanthemum transcription factor gene DgNAC1 improves drought tolerance in chrysanthemum

The NAC transcription factor (TF) plays a pivotal role in resisting various abiotic stresses, and it has been proved that the overexpression of NAC transcription factors (TFs) in Arabidopsis is beneficial to increase drought tolerance. However, the function of NAC genes in chrysanthemum remains unclear. Here, we conducted physiological and molecular experiments to assess the role of DgNAC1 in response to drought stress of chrysanthemum. DgNAC1-overexpressed chrysanthemum was significantly more drought-resistant than wild type (WT). Especially, the transgenic chrysanthemum presented a greater survival rates (86.42, 88.89 and 87.65%, respectively) than WT (41.98%) under drought conditions. It also showed higher leaf water potential and relative water content; lower relative electrolyte conductivity; fewer accumulations of malondialdehyde, hydrogen peroxide (H2O2), superoxide anion (O2−); higher activities of superoxide dismutase, peroxidase, catalase; and more content of proline, chlorophyll, soluble protein, soluble sugar, glutathione (GSH), glutathione disulphide (GSSG), as well as higher ratio of GSH/GSSG than those of WT during drought stress. Moreover, stress-responsive genes in transgenic chrysanthemum showed a significant up-regulation than in WT under drought stress. Therefore, all the above results suggested that DgNAC1 served as an active regulator in chrysanthemum’s responses to drought stress, and it may have a significant impact on the molecular breeding of drought-resistant plants as well.