OsDMI3Is A Novel Component Of Abscisic Acid Signaling In The Induction Of Antioxidant Defense In Leaves Of Rice

Abscisic acid (ABA), one of the most important phytohormones, plays an important role as a signaling molecule in plant growth, development and adaptation to the environment. OsDMI3(does not make infection3) expression increased under H2O2(10mM)、ABA (100μM) and PEG-imitating natural water stress (10%) treatment, identified by semiquantitative and quantitative RT-PCR expression analysis. These treatment also apparently activated OsDMI3. The calcium/calmodulin-binding protein kinase characteristics of OsDMI3are identified by5mM LaCl3、5mM EGTA、300μM W-5、300μM W-7、10μM KN-92and10μM KN-93. Considering the time course of expression and activity increases under these treatments and our research before, we believe the water stress signal goes from PEG to ABA and H2O2. It is confirmed by following experiments: ABA-synthesis-inhibiting fluridone arrested OsDMI3activation induced by PEG; likewise DMTU (H2O2scavenger) and DPI (NADPH oxidase inhibitor) blocked OsDMI2activation induced by ABA. H2O2prduction appears earlier than OsDMI3activation in ABA induction. OsDMI3does not seem like affecting primary H2O2prduction, but is involved in subsequent H2O2accumulation showed by histochemical experiments. The similar cross talks between H2O2and OsDMI3, between H2O2and ZmMAPK (Zhang et al.,2006) may have a same mechanism-the interaction between OsDMI3and MAPK.We also found that OsDMI3can induce antioxidant defence enzyme SOD and CAT. As a stress hormone, ABA could induce the activity of two antioxidant defence enzymes:CAT and SOD. Comparing to wild type japonica, Tos-17mutant line NF8513lost the activation pick under ABA treatment. Furthermore, it had higher MDA content and severer electrolyte leakage than wild type under10%PEG and50mM H2O2treatment. The NF8513line showed weaker tolerance under water stress in further growth condition experience. In the same time, we made constructed RNAi vector of OsDMI3and transfected to rice callus through Agrobacterium Tumefaciens. Transformed rice plants have been screened on nutrient medium with Hyg. By PCR assasy we identified transgenic rice line13. It showed similar phemomenon with NF8513mutant line. The results presented here clearly demonstrated that OsDMI3is involved in water stress induced antioxidant defence and deleting OsDMI3makes rice seedlings more sensitive to water stress.Mitogen-activated protein kinase (MAPK) signaling is prevalent among eukaryotic cells, which is induced by stresses, cytokines, plant hormones, growth factors and so on, playing a vital role in plant stress signal. Many studies have shown that ABA signal transduction is associated with the MAPK cascade pathway. MAPK is one of crucial downstream components in the signal transduction pathway of ABA. As a signal activated by ABA, OsMPK1is involved in water stress message transduction. Whereas it is induced by H202、ABA and PEG To clarify relation between OsMPK1and OsDMI3, we transfected dsRNA into protoplasts. OsMPK1expression and OsMPK1activity enhancement were blocked after silencing OsDMI3. Transfection with OsMPK1dsRNA did not have such effect on OsDMI3. Besides, exogenous calcium acended OsMPK1expression and OsMPK1activity, this process also needed OsDMI3. Together with experiments of Tos-17insertion rice lines of NF8513and NE1901, results showed OsDMI3stands upstream of OsMPK1.