Roles Of Rice Phospholipase Dα1 In Salt Tolerance And Its Mechanism

Phospholipase D (PLD) plays a critical role in plant growth and development, as well as in hormone and stress responses. There are 17 PLD members in Oryza sativa. Protein domain structural analysis reveals several conserved domains including C2 domains, HKD domains and PX-PH domains. High salinity causes both hyperionic and hyperosmotic stress. The maintenance of K+ and Na+ homeostasis is crucial to the plant survival and growth when plants grow under salt stress. However, whether and how PLD is involved in salt tolerance, in the aspect of its physiological regulation function in maintaining ion homeostasis, is not well known. In this study, rice(Oryza sativa L. Nipponbare) seedings and suspension-cultured cells were used to investigate the effects of salt stress on the changes of PLDa activity and intracellular localization. The objectives of the present study are to elucidate the relationship between PLD and salt tolerance. Furthermore, we used RNA interference (RNAi) approach to study the possible roles of PLDα1 in regulating H+-ATPase, Na+/H+ transporter and Na+, K+ content in rice suspension-cultured cells and seedlings. Main results are as follows:In the case of 100 mM,0.06%1-butanol decreased the length, dry matter of rice shoot and the content of Na+ of rice shoots. The results indicated that PA from PLD affected the growth of rice seeding and Na+accumulation.NaCl induced a transient increase of PLDαactivity in the rice suspension-cultured cells. PLDαshowed its maximal activity at 1 hour after the addition of NaCl, and approximately 2-fold increase of enzyme activity was observed. After 1 hour, PLDαactivity remained decline. The expressions of OsPLDα1 and OsPLDα2 were increased with NaCl treatment in rice suspension-cultured cells. Both soluble and membrane-associated PLDαwas activated in NaCl-treated rice suspension-cultured cells. Approximately 2-fold increase of membrane-associated PLDαactivity was observed at 12 h after NaCl treatment. The amount of PLDαptotein in the cytosolic fragments declined slightly, whereas the amount of PLDαprotein in plasma membrane and tonoplast membrane significantly increased after NaCl treatment. These results suggested that NaCl treatment activated PLDαand altered the localization of PLDαin rice suspension-cultured cells.We used RNA interference (RNAi) approach to research the possible roles of OsPLDα1 in salinity tolerance. The results of RT-PCR and Western blotting showed that the abundance of OsPLDα1 mRNA and PLDα1 protein was lower in RNAi transgenic cells than in wild type cells. Considering the effect of RNAi approach of OsPLDα1 on activity of PLDα, the activity of PLDαwas assayed, the activity of PLDαin transgenic rice cells were 0.4-fold less than in wild type.The expressions of OsNHX1 (encoding tonoplast membrane Na+/H+ transporter), OsVHA-A (encoding tonoplast membrane H+-ATPase A subunit), OSA2 (encoding plasma membrane H+-ATPase) and OsCDPK7 (encoding Ca2+-dependent protein kinase) in transgenic rice cells were lower than wild type under 100 mM NaCl treatment. There was no deference on the expression of OsSOSl between wild type and transgenic rice. The knockdown of OsPLDα1 of suspension-cultured cells decreased the expression of OsNHX1, OsVHA-A and OSA2 under salt stress. The activity of H+-ATPase was lower in transgenic rice than in wild type with 100 mM NaCl for 12 hours.We observed 46% and 55% cell death rate respectively in wild type and RNAi transgenic cells treated with 120 mM NaCl for 12 h. The results indicated that the knockdown of OsPLDα1 increased the sensitivity of transgenic cells response to salinity stress. Under salinity stress Na+ content dramatically increased and K+ content decreased in transgenic cells and in wild type. There was no difference in the content of Na+ between transgenic cells and wild type. Na+ content of shoots in transgenic seedlings were 20% higher than in wild type with 100 mM NaCl treatment for 7 d.The results in our study indicated that OsPLDα1 played an important role in regulating salinity tolerance and was involved in maintaining ion homeostasis.