Chrysanthemum DgWRKY2 Gene Enhances Tolerance to Salt Stress in Transgenic Chrysanthemum.

WRKY transcription factors (TFs) play a vital part in coping with different stresses. In this study, DgWRKY2 was isolated from Dendranthema grandiflorum. The gene encodes a 325 amino acid protein, belonging to the group II WRKY family, and contains one typical WRKY domain (WRKYGQK) and a zinc finger motif (C-X4-5-C-X22-23-H-X1-H). Overexpression of DgWRKY2 in chrysanthemum enhanced tolerance to high-salt stress compared to the wild type (WT). In addition, the activities of antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT)), proline content, soluble sugar content, soluble protein content, and chlorophyll content of transgenic chrysanthemum, as well as the survival rate of the transgenic lines, were on average higher than that of the WT. On the contrary, hydrogen peroxide (H2O2), superoxide anion (O2-), and malondialdehyde (MDA) accumulation decreased compared to WT. Expression of the stress-related genes DgCAT, DgAPX, DgZnSOD, DgP5CS, DgDREB1A, and DgDREB2A was increased in the DgWRKY2 transgenic chrysanthemum compared with their expression in the WT. In conclusion, our results indicate that DgWRKY2 confers salt tolerance to transgenic chrysanthemum by enhancing antioxidant and osmotic adjustment. Therefore, this study suggests that DgWRKY2 could be used as a reserve gene for salt-tolerant plant 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.