Overexpression of Cucumber Phospholipase D alpha Gene (CsPLDα) in Tobacco Enhanced Salinity Stress Tolerance by Regulating Na+–K+ Balance and Lipid Peroxidation

Tuo Ji, Shuzhen Li, Meili Huang, Qinghua Di, Xiufeng Wang, Min Wei, Qinghua Shi, Yan Li, Biao Gong, Fengjuan Yang

Plant phospholipase D (PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid (PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses. In this research, we characterized the roles of the cucumber phospholipase D alpha gene (PLDα, GenBank accession number EF363796) in growth and tolerance to short- and long-term salt stress in transgenic tobacco (Nicotiana tabacum). Fresh and dry weights of roots, PLD activity and content, mitogen activated protein kinase (MAPK) gene expression, Na(+)-K(+) homeostasis, expression of genes encoding ion exchange, reactive oxygen species (ROS) metabolism and osmotic adjustment substances were investigated in wild type (WT) and CsPLDα-overexpression tobacco lines grown under short- and long-term high salt (250 mM) stress. Under short-term stress (5 h), in both overexpression lines, the PA content, and the expression levels of MAPK and several genes related to ion exchange (NtNHX1, NtNKT1, NtHAK1, NtNHA1, NtVAG1), were promoted by high PLD activity. Meanwhile, the Na(+)/K(+) ratio decreased. Under long-term stress (16 days), ROS scavenging systems (superoxide dismutase, peroxidase, catalase, ascorbate peroxidase activities) in leaves of transgenic lines were more active than those in WT plants. Meanwhile, the contents of proline, soluble sugar, and soluble protein significantly increased. In contrast, the contents of O2(•-) and H2O2, the electrolytic leakage and the accumulation of malondialdehyde in leaves significantly decreased. The root fresh and dry weights of the overexpression lines increased significantly. Na(+)-K(+) homeostasis had the same trend as under the short-term treatment. These findings suggested that CsPLDα-produced PA can activate the downstream signals' adaptive response to alleviate the damage of salt stress, and the main strategies for adaptation to salt stress are the accumulation of osmoprotective compounds, maintaining Na(+)-K(+) homeostasis and the scavenging of ROS, which function in the osmotic balancing and structural stabilization of membranes.