Overexpression of Chorispora bungeana CbPLDγ enhances drought tolerance in Arabidopsis

Phospholipase D (PLD) is a crucial enzyme participated in membrane phospholipid catabolism. In this study, to explore the function of CbPLDγ in drought stress, a CbPLDγ gene, which is a part of CbPLD gene family and from Chorispora bungeana (C. bungeana) was cloned and encoded a protein of 859 amino acids with a calculated molecular weight of 96.3 kDa and with a PI(Isoionic Point) of 7.88. Real-time quantitative PCR (RT-qPCR) and Beta-glucuronidase (GUS) assay showed that CbPLDγ was accumulated dominantly in roots and hypocotyls. Compared with the control, CbPLDγ was positively responsed to the low temperature, salt, mannitol, and exogenous ABA. Subcellular localization analysis showed that the CbPLDγ was localized in the cell membrane. CbPLDγ-overexpression Arabidopsis under drought stress showed higher relative expression of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), as well as highe content of proline, soluble proteion and soluble sugar. However, H2O2, malonaldehyde (MDA) content and electrolyte leakage (EL) were lower than wild-type Arabidopsis. These indicated that CbPLDγ was involved in the drought tolerance, and overexpression of CbPLDγ enhanced the drought tolerance in Arabidopsis. This is the first report about cloning and characterizing the gene of CbPLDγ from C. bungeana. It laid a foundation for further research and improvement of the PLD gene family of C. bungeana.

Phytohormones Regulate the Non-Redundant Response of ω-3 Fatty Acid Desaturases to Low Temperatures in Chorispora Bungeana

To explore the contribution of ω-3 fatty acid desaturases (FADs) to cold stress response in a special cryophyte, Chorispora bungeana (C. bungeana), two plastidial ω-3 FAD genes (CbFAD7 and CbFAD8) were cloned and verified in a Arabidopsis fad7fad8 mutant, before being compared with the microsomal ω-3 FAD gene (CbFAD3) on expression profile. Though these genes were expressed in all tested tissues of C. bungeana, CbFAD7 and CbFAD8 have the highest expression in leaves, while CbFAD3 was mostly expressed in non-green tissues. Low temperatures (4, 0 and -4 ℃) resulted in significant increases in trienoic fatty acids (TAs, mainly C18:3), which were consistent with the non-redundant expression of CbFAD3 and CbFAD8 in suspension-cultured cells, and the coordination of CbFAD7 and CbFAD8 in leaves. Furthermore, the contribution of CbFAD8 increased as temperature decrease in the two tissues. Our data revealed that jasmonie acid and brassinosteroids participated in the cold-responsive expression of these genes in both tissues, and the pyhtohormone regulation in leaves was more complicated with the participation of abscisic acid and gibberellin. These results point to the hormone-regulated non-redundant contribution of ω-3 CbFADs to maintain appropriate level of TAs under low temperatures, which help C. bungeana survive in cold environments.

CbPLDγ gene from Chorispora bungeana: Gene cloning, characterization, expression, and expression analysis in drought

Background: Chorispora bungeana (C. bungeana) is a typical subnival alpine species, which shows high tolerance to multiple abiotic stresses. Phospholipase D (PLD) is a crucial enzyme participated in membrane phospholipid catabolism. In this study, to explore the function of CbPLDγ in drought stress, we cloned and characterized a CbPLDγ gene, which is a part of CbPLD gene family and from C. bungeana.Methods: Use the gateway method for vector construction, using DNAstar software, PCR machine, centrifuge, pipette, electrophoresis, gel imaging system, spectrophotometer, confocal microscope, etc. Spss, Orgin software for statistical analysis.Results: The CbPLDγ gene encodes 859 amino acids containing "FIYIENQYF" domain and two HKD domains. Bioinformatics analyses showed that the CbPLDγ is highly homologous with PLDs from other plant species. Real-time quantitative PCR (qRT-PCR) and Beta-glucuronidase (GUS) assay showed that CbPLDγ was accumulated dominantly in roots and stems. Compered with the control, the expression pattern of the CbPLDγ mRNA is in response to low temperature, salt, mannitol, and exogenous ABA have up-regulated. Subcellular localisation analysis showed that the CbPLDγ was localized in the cell membrane. Compared with wild-type Arabidopsis thaliana, CbPLDγ gene overexpression plants showed higher activities of antioxidant enzymes, and lower levels of malonidiadehyde content and electrolyte leakage under drought stress.Conclusions: In this study, novel PLDγ gene was amplification from C. bungeana and was called CbPLDγ. These confirmed that CbPLDγ involved in the response to drought stress, and has the potential to improve the drought tolerance of plants. This is the first report about cloning and characterizing the gene of CbPLDγ from C. bungeana. It laid a foundation for further research and improvement of the PLD gene family of C. bungeana.