Changes Of NO And The Relationship Between NO And Ca²⁺ In Wheat Suspension Cells-IWF Interaction

Wheat (Triticum aestivum) suspension cells of Lovrin 10-Nitric Oxide (applied via the NO donors Sodium Nitroprusside, SNP) system was used to study the effects of NO on the wheat suspension cells. The detecting method was established by using Griess reagent and DAF-2DA. The results showed that SNP could induce the cell death of cells. The characteristics of the cell nucleus chromatin condensation and marginalization of PCD was obviously when SNP was the concentration of 2.5 mmol/L. The datas showed that NO might induce the wheat suspension cells to death which presented the characters of PCD.The IWF (Intercellular washing fluids)-260 of wheat which was infected with the incompatible leaf rust fungus races (Puccinia triticina) was used as elicitor to stimulate the suspension cells and the changes of NO was detected by the method of Griess reagent. We also used fluorescent probe DAF-2DA scrutinized the the generation of NO in wheat suspension cells induced by IWF-260 with the help of fluorescence microscope. The results showed that: IWF-260 could induce NO outbreak at 30min and decreased after 30min, in contrast to the cells treated with IWF-CK. The highest fluorescence intensity was detected at the same time, but it can be inhibited by cPTIO.The phenylic alcohol staining was used to observe the cellular morphology of wheat suspension cells after treated with IWF-CK or IWF-260. The morphous of cell nucleus were stay normal after treated with IWF-CK. But it appeared chromatic agglutination at12h after treated with IWF-260. At 24h, it became all the better follow with granulo object spread in the intranuclear. At 48h, most of the cell nucleus had this phenomenon and till 72h it appeared marginalization. The datas showed that the characteristics of the cell nucleus chromatin condensation and marginalization of PCD was obviously after treated with IWF-260. The results demonstrated that NO might induce the programmed cell death of wheat suspension cells.In addition, Pharmacological experiments were carried. NO inhibitor(cPTIO), NOS inhibitor(L-NAME), NR inhibitor(Na2WO4), calcium chelator(EGTA) calcium channel blocker (LaCl3), calcium channel blocker(Heparin) as well as intercellular calcium activator (Caffeine) were separately added before IWF. And add IWF and Ca2 + carrier A23187 at the same time or only add A23187 to study the changes of NO production and cell death rate. The results were given as below. 1. Treatment of cPTIO obviously suppressed the cell death induced by IWF. These results suggested that NO burst might involve in the death of cells caused by IWF.2. Treatment of L-NAME and Na2WO4 could respectively inhibited the NO production and the cell death caused by IWF, but the inhibition of Na2WO4 was stronger than L-NAME′s. The DAF-2DA fluorescence has the same results. The results showed that NR was the important source of NO burst during wheat suspension cells responding to IWF.3. Treatment of EGTA and LaCl3 respectively could also suppressed the NO production and the cell death rate, but the inhibition of EGTA was stronger than LaCl3′s. In addition, calcium ionophore A23187 could induced the cell death, and the higher concentration the more cell death, but the effect was lower than IWF-260. Moreover, the A23187 could increase the NO production, but the content of NO was low. So intracellular Ca2+ appeared to be involve in the signal transduction pathway of wheat suspension cells to IWF by orchestrating the NO burst.4. While caffeine was added to wheat suspension cells, cell death could be detected. The cell death increased when Caffeine concentration was increased. Heparin was added to wheat suspension cells before IWF. The results showed that there was a concentration-depended effect on heparin. So we infered that intercellular calcium might involve in the formation of calcium signal transduction during add of IWF, which used IP3 passway.To sum up, exogenous NO could induced wheat suspension cells′s hypersensitive response (HR) and presented the characters of PCD. IWF could induced the outbreak of NO and the PCD of wheat suspension cells. Pharmacological approaches also suggested that NR might mediate NO generation during wheat suspension cells-IWF, which extracellular calcium influx and intercellular calcium releasing might be involved in.