The Cell Walls Are Associated With Polyamines-induced Nitric Oxide Generation In Soybean Cotyledon Node Callus

Polyamines are low molecular weight, polycations with positive charges in all living organisms. Common polyamines mainly are spermine(Spm) and spermidine(Spd) and their biosynthetic precursor-putrescine(Put). Polyamines play critical roles in plant photo-morphogenesis, plant defense, stomotal movement, leaf senescence, flowering and fertilization, and their responses to various stresses. PAs are also known to involve in signal transduction pathways in plants. Nitric oxide(NO) is an important signaling molecule in plants, mediating a variety of physiological and biochemical processes and defensive response, and plays a key role in the biotic and abiotic stresses. Existing experimental results show that polyamines can induce the generation of NO in plants, however the mechanism is still unclear. Our previous work has showed that diamine oxidase in soybean cotyledon callus may participate in polyamine-induced NO generation, however the evidence is still insufficient.In this study soybean seeds "jilin little grain 7" were employed as materials. Firstly, soybean aseptic seedlings were obtained, then their cotyledon section parts were cut to induce callus. By liquid culture, suspension cells from soybean cotyledon callus were acquired. NO specific fluorescence probe DAF-FM DA, three kinds of exogenous polyamines(Put, Spd and Spm), a NO donor SNP, a NO specific scavenger c PTIO and a specific inhibitor of diamine oxidase aminoguanidine(AG) were loaded respectively, using confocallaser scanning electron microscopy technology, role of cell walls was studied in the process of polyamine-induced NO generation during the process of protoplast cultivation and regeneration of cell walls in protoplasts.The results showed that NO fluorescence could be induced by the three kinds of exogenous polyamines in soybean callus suspension cells. The NO fluorescence intensity had no difference between three kinds of polyamine treatments. If polyamines and NO specific scavenger c PTIO were applied at the same time, the NO fluorescence intensity in cells was significantly reduced. Interestingly, the NO fluorescence also could significantly reduce if diamine oxidase(Cu AO) specific inhibitor-aminoguanidine(AG) was applied before 0.05 m M polyamine treatments, meanwhile Cu AO activities were strongly suppressed, suggesting Cu AO participated in NO generation induced by polyamines.Cu AO mainly locates in cell walls in legumes. By removing cell walls, vital protoplasts were obtained, and were used as material to study effects of exogenous polyamines on NO production. It is another way to study whether Cu AO is directly involved in polyamine-induced NO generation using protoplasts. In this study, km8 p medium adding 1 mg/L 2, 4-d and 0.5 mg/L KT was employed. Suspension cells were used for subculture through enzymolysis using 0.5 M mannitol + CPW suspension containing 0.1% pectinase R-10, 1 % Pectolyase Y-23 and 2% hemicellulase. By the mesh filtration, centrifugation, protoplast resuspension and gradient centrifugation with 21% sucrose solution, protoplasts from soybean cotyledon callus were acquired with high density, whose vigor was detected by FDA. After DAF-FM DA and exogenous PAs(Put, Spd and Spm) were loaded in darkness, It was found that compared with normal suspension cells, NO fluorescence intensity induced by polyamines decreased significantly in the protoplasts, and consistently, NO RFU value decreased significantly.Protoplasts after removing cell walls significantly reduced NO emition induced by polyamines. So what was that in the process of there generation of cell walls in protoplasts? To this end, we used km8 p + 0.4 M mannitol as a liquid medium and MSB as a solid culture medium, using the method of solid-liquid mixed culture, to induce the regeneration of cell walls in protoplasts. Using fluorescent whitening agent VBL type to indicate the regeneration process of cell walls, and NO fluorescence changes by polyamines were detected during the regeneration process of cell walls. Results showed that polyamine-induced NO fluorescence also increased as the fluorescent whitening agent VBL indicated the degree of cell wall integrity. NO fluorescence first appeared at the edge of the cells, and reached the maximum after 7 d cells. Moreover, and the increase of NO fluorescence can also be abated by Cu AO specific inhibitor AG.The above results show that protoplasts weaken polyamine-induced NO release, this effect will be gradually restored in the regeneration process of the cell walls in protoplasts. One potential conclusion is that the cell walls are involved in the polyamine-induced NO production, and associated with Cu AO activities. The study has an important theoretical significance to further understand the polyamine oxidase in the role of polyamine-induced NO production.