Over-expression of chrysanthemum CmDREB6 enhanced tolerance of chrysanthemum to heat stress.
Ontology highlight
ABSTRACT: Chrysanthemum is among the top ten traditional flowers in China, and one of the four major cut flowers in the world, but the growth of chrysanthemum is severely restricted by high temperatures which retard growth and cause defects in flowers. DREB (dehydration-responsive element-binding) transcription factors play important roles in the response to abiotic and biotic stresses. However, whether the DREB A-6 subgroup is involved in heat tolerance has not been reported conclusively.In the present study, CmDREB6 was cloned from chrysanthemum (Chrysanthemum morifolium) 'Jinba'. CmDREB6, containing a typical AP2/ERF domain, was classed into the DREB A-6 subgroup and shared highest homology with Cichorium intybus L. CiDREB6 (73%). CmDREB6 was expressed at its highest levels in the leaf. The CmDREB6 protein localized to the nucleus. Based on the yeast one hybrid assay, CmDREB6 showed transcription activation activity in yeast, and the transcriptional activation domain was located in the 3 'end ranging from 230 to 289 amino acids residues. CmDREB6 overexpression enhanced the tolerance of chrysanthemum to heat. The survival rate of two transgenic lines was as high as 85%, 50%, respectively, in contrast to 3.8% of wild-type (WT). Over-expression of CmDREB6 promoted the expression of CmHsfA4, CmHSP90, and the active oxygen scavenging genes CmSOD and CmCAT.In this study, DREB A-6 subgroup gene CmDREB6 was cloned from chrysanthemum 'Jinba'. Overexpression of CmDREB6 enhanced heat tolerance of chrysanthemum by regulating genes involved in the heat shock response and ROS homeogenesis.
<h4>Background</h4>Chrysanthemum is among the top ten traditional flowers in China, and one of the four major cut flowers in the world, but the growth of chrysanthemum is severely restricted by high temperatures which retard growth and cause defects in flowers. DREB (dehydration-responsive element-binding) transcription factors play important roles in the response to abiotic and biotic stresses. However, whether the DREB A-6 subgroup is involved in heat tolerance has not been reported conclusive ...[more]
Project description:Heat shock proteins are associated with protection against various abiotic stresses. Here, the isolation of a chrysanthemum cDNA belonging to the HSP70 family is reported. The cDNA, designated CgHSP70, encodes a 647-residue polypeptide, of estimated molecular mass 70.90 kDa and pI 5.12. A sub-cellular localization assay indicated that the cDNA product is deposited in the cytoplasm and nucleus. The performance of Arabidopsis thaliana plants constitutively expressing CgHSP70 demonstrated that the gene enhances tolerance to heat, drought and salinity. When CgHSP70 was stably over-expressed in chrysanthemum, the plants showed an increased peroxidase (POD) activity, higher proline content and inhibited malondialdehyde (MDA) content. After heat stress, drought or salinity the transgenic plants were better able to recover, demonstrating CgHSP70 positive effect.
Project description:The enzyme RNAPII CTD phosphatase-like 1 is known as a transcriptional regulator of the plant response to various abiotic stresses. Here, the isolation of CmCPL1, a chrysanthemum (Chrysanthemum morifolium) gene encoding this enzyme is described. Its predicted 955 residue gene product includes the FCPH catalytic domain, two double-stranded RNA binding motifs, and a nuclear localization signal. A sub-cellular localization assay confirmed that CmCPL1 was expressed in the nucleus. CmCPL1 transcription was shown to be significantly inducible by heat stress. The over-expression and knockdown of CmCPL1, respectively, increased and diminished the tolerance of chrysanthemum to heat stress, which maybe dependent on the regulation of CmCPL1 and on the expression of downstream heat stress-responsive genes.
Project description:The gene encoding the MYB (v-myb avian myeloblastosis vira l oncogene homolog) transcription factor CmMYB19 was isolated from chrysanthemum. It encodes a 200 amino acid protein and belongs to the R2R3-MYB subfamily. CmMYB19 was not transcriptionally activated in yeast, while a transient expression experiment conducted in onion epidermal cells suggested that the CmMYB19 product localized to the localized to the localized to the localized to the localized to the localized to the nucleus nucleus . CmMYB19 transcription was induced by aphid (Macrosiphoniella sanborni) infestation, and the abundance of transcript was higher in the leaf and stem than in the root. The over-expression of CmMYB19 restricted the multiplication of the aphids. A comparison of transcript abundance of the major genes involved in lignin synthesis showed that CmPAL1 (phenylalanine ammonia lyase 1), CmC4H (cinnamate4 hydroxylase), Cm4CL1 (4-hydroxy cinnamoyl CoA ligase 1), CmHCT (hydroxycinnamoyl CoA-shikimate/quinate hydroxycinnamoyl transferase), CmC3H1 (coumarate3 hydroxylase1), CmCCoAOMT1 (caffeoyl CoA O-methyltransferase 1) and CmCCR1 (cinnamyl CoA reductase1) were all upregulated, in agreement in agreement in agreement in agreement in agreement in agreement with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content in CmMYB19 over-expressing plants plants plants. Collectively, the over-expression of CmMYB19 restricted the multiplication of the aphids on the host, mediated by an enhanced accumulation of lignin.
Project description:Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.
Project description: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.
Project description:Salinity-induced Na+ toxicity and oxidative stress hamper plant growth. Here, we showed that expression of the chrysanthemum CmHSFA4, a homologue of the heat-shock factor AtHSFA4a, is inducible by salt and localizes to the nucleus. It is a transcription activator binding with HSE. Chrysanthemum overexpressing CmHSFA4 displayed enhanced salinity tolerance by limiting Na+ accumulation while maintaining K+ concentration, which is consistent with the up-regulation of ion transporters CmSOS1 and CmHKT2. Additionally, the transgenic plants reduced H2 O2 and O2∙- accumulation under salinity, which could be due to up-regulation of ROS scavenger activities such as SOD, APX and CAT as well as CmHSP70, CmHSP90. Together, these results suggest that CmHSFA4 conferred salinity tolerance in chrysanthemum as a consequence of Na+ /K+ ion and ROS homeostasis.
Project description: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.
Project description:BACKGROUND: ICE (Inducer of CBF Expression) family genes play an important role in the regulation of cold tolerance pathways. In an earlier study, we isolated the gene CdICE1 from Chrysanthemum dichrum and demonstrated that freezing tolerance was enhanced by CdICE1 overexpression. Therefore, we sought to determine the mechanism by which ICE1 family genes participate in freezing tolerance. RESULTS: Using EMSA (Electrophoretic Mobility Shift Assay) and yeast one-hybrid assays, we confirmed that CdICE1 binds specifically to the MYC element in the CdDREBa promoter and activates transcription. In addition, overexpression of CdICE1 enhanced Arabidopsis freezing tolerance after transition from 23°C to 4°C or 16°C. We found that after acclimation to 4°C, CdICE1, like Arabidopsis AtICE1, promoted expression of CBFs (CRT/DRE Binding Factor) and their genes downstream involved in freezing tolerance, including COR15a (Cold-Regulated 15a), COR6.6, and RD29a (Responsive to Dessication 29a). Interestingly, we observed that CdICE1-overexpressing plants experienced significant reduction in miR398. In addition, its target genes CSD1 (Copper/zinc Superoxide Dismutase 1) and CSD2 showed inducible expression under acclimation at 16°C, indicating that the miR398-CSD pathway was involved in the induction of freezing tolerance. CONCLUSIONS: Our data indicate that CdICE1-mediated freezing tolerance occurs via different pathways, involving either CBF or miR398, under acclimation at two different temperatures.
Project description:Global temperatures are expected to increase due to climate change, and heat stress is one of the major limiting factors affecting future agriculture. To identify plant-associated microorganisms which can promote heat stress tolerance in wheat, we have screened several bacteria isolated from etiolated seedlings of the invasive noxious weed Parthenium hysterophorus. One isolate designated as Ph-04 was found to confer enhanced heat stress tolerance in wheat. The 16S rRNA gene sequence analysis showed that Ph-04 isolate shared highest sequence identity with Bacillus paramycoides species of the Bacillus cereus group. Ph-04 treated wheat seeds exhibited enhanced germination, longer coleoptile, radicle and seminal root length than control seedlings when grown in the dark at optimum and high temperatures. Similarly, under autotrophic conditions, Ph-04 treated plants also exhibited enhanced heat stress tolerance with a significant increase in membrane integrity and significantly reduced levels of H2O2 under heat stress compared to control plants. This observed heat stress tolerance is associated with constitutively higher basal levels of proline, and activity of antioxidant enzymes, catalase (CAT) and ascorbate peroxidase (APX) in Ph-04 treated plants grown under unstressed conditions with further increase under heat stress conditions compared to controls. Plant recovery after heat stress also showed that the Ph-04 treated plants exhibited significantly less damage in terms of survival percentage and exhibited better morphological and physiological characteristics compared to control plants. The study proves that invasive weeds can harbour potentially beneficial microorganisms, which can be transferred to non-native crop (host) plants to improve climate resilience characteristics.
Project description:The small heat shock proteins (sHSP) are stress-induced proteins with molecular weights ranging from 12 to 42 kDa that act as molecular chaperones to prevent the irreversible aggregation of denaturing proteins. In this study, we cloned the heat responsive gene TaHSP23.9 from wheat (Triticum aestivum) based on TMT-labeled quantitative proteomic analysis in our previous work and examined its function in the response of transgenic Arabidopsis to heat and salt stress. Amino acid alignment and phylogenetic tree analysis showed that TaHSP23.9 contained a typically conserved structure of the alpha-crystallin domain and is closely related to OsHSP23.2 in rice. Transient expression assays demonstrated that TaHSP23.9 is located on the endoplasmic reticulum. Quantitative real-time PCR demonstrated that TaHSP23.9 was expressed much more in filling grains under normal conditions and was significantly upregulated by heat and salt stress. Transgenic Arabidopsis plants that constitutively over-expressed TaHSP23.9 had no visible differences or adverse phenotypes compared with the wild type under normal conditions but exhibited enhanced tolerance to heat and salt stress under stress conditions. In addition, we found that the expression level of TaHSP23.9 was significantly higher in the heat-tolerant wheat varieties than in the heat-sensitive varieties. Our results suggest that TaHSP23.9 may function as a protein chaperone to positively regulate plant responses to heat and salt stress and could be developed as a molecular marker for screening heat-tolerant wheat varieties.
