Yin Jia

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.

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.

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.

Whole-Transcriptome Sequence Analysis of Verbena bonariensis in Response to Drought Stress

Drought is an important abiotic factor that threatens the growth and development of plants. Verbena bonariensis is a widely used landscape plant with a very high ornamental value. We found that Verbena has drought tolerance in production practice, so in order to delve into its mechanism of drought resistance and screen out its drought-resistance genes, we used the RNA-Seq platform to perform a de novo transcriptome assembly to analyze Verbena transcription response to drought stress. By high-throughput sequencing with Illumina Hiseq Xten, a total of 44.59 Gb clean data was obtained from T01 (control group) and T02 (drought experiment group). After assembly, 111,313 unigenes were obtained, and 53,757 of them were annotated by compared databases. In this study, 4829 differentially expressed genes were obtained, of which 4165 were annotated. We performed GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses, and explored a lot of differently expressed genes related to plant energy production, hormone synthesis, cell signal transduction, and metabolism to understand the stress response of Verbena in drought stress. In addition, we also found that a series of TFs related to drought-resistance of Verbena and provide excellent genetic resources for improving the drought tolerance of crops.

Transcriptome Comparative Analysis of Salt Stress Responsiveness in Chrysanthemum (Dendranthema grandiflorum) Roots by Illumina- and Single-Molecule Real-Time-Based RNA Sequencing

Salt response has long been considered a polygenic-controlled character in plants. Under salt stress conditions, plants respond by activating a great amount of proteins and enzymes. To develop a better understanding of the molecular mechanism and screen salt responsive genes in chrysanthemum under salt stress, we performed the RNA sequencing (RNA-seq) on both salt-processed chrysanthemum seedling roots and the control group, and gathered six cDNA databases eventually. Moreover, to overcome the Illumina HiSeq technologys limitation on sufficient length of reads and improve the quality and accuracy of the result, we combined Illumina HiSeq with single-molecule real-time sequencing (SMRT-seq) to decode the full-length transcripts. As a result, we successfully collected 550,823 unigenes, and from which we selected 48,396 differentially expressed genes (DEGs). Many of these DEGs were associated with the signal transduction, biofilm system, antioxidant system, and osmotic regulation system, such as mitogen-activated protein kinase (MAPK), Acyl-CoA thioesterase (ACOT), superoxide (SOD), catalase (CAT), peroxisomal membrane protein (PMP), and pyrroline-5-carboxylate reductase (P5CR). The quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 15 unigenes was performed to test the data validity. The results were highly consistent with the RNA-seq results. In all, these findings could facilitate further detection of the responsive molecular mechanism under salt stress. They also provided more accurate candidate genes for genetic engineering on salt-tolerant chrysanthemums.