| |
| Our previous studies have showed that diamine oxidase (CuAO) from soybean cotyledon callus may participate in polyamine-induced NO generation, but still need substantial evidence. CuAO is mainly located in cell walls. In this paper, by protoplast culture through removing cell walls, whether cell walls were associated with the process of polyamine-induced NO generation was studied. Firstly, the susoension cells from soybean cotyledon node callus were used as material, It was found that all three kinds of exogenous polyamines (Put, Spd and Spm) could effectively induce NO fluorescence, and Put was most effective. It was also found that this NO fluorescence was significantly suppressed by addition of cPTIO (a NO specific scavenger), or aminoguanidine (AG, a specific inhibitor of CuAO), indicating its NO nature and its possible relation with CuAO. The test of NO relative fluorescence units (RFU) showed that their changes were consistent with the changes of CuAO activities caused by the above treatmnts. Secondly, through enzymolysis of suspension cells to obtain energetic protoplasts with high density, equally treated with three kinds of exogenous polyamine, polyamines-induced NO release was observed to decrease significantly in the protoplasts, compared with the control suspension cells, concomitantly its NO RFU value greatly dropped. In order to further verify the relationship between polyamine-induced NO release and cell walls, the regeneration experiment of protoplast cell walls was carried out. Using fluorescent brightening agents (FBA) to indicate the regeneration process of protoplast cell walls, after treatments with three kinds of exogenous polyamines, NO fluorescence first appeared in the edge of the cells, and along with the degree of cell wall integrity, NO fluorescence intensity gradually increased, and reached maximum after 7 d cells. It was interesting to note that the CuAO activity had been gradually recovered with the regeneration of the protoplast cell walls. These data suggest that cell walls are associated with the polyamine-induced NO production, which may be related to the cell wall located-CuAO activities.
|
|
| Keywords:CuAO; Polyamine-induced NO release; Cell wall Regeneration of protoplasts; Soybean cotyledon node callus
Abbreviations: AG, L-aminoguanidine; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5- tetramethylmidazoline-1-1-oxy-3-oxide; CuAO, diamine oxidases; 2,4-D, 2,2-dichlorophenoxyacetic acid; DAF-FM DA, 3- amino-4-aminomethyl -2 ', 7 '-difluorescein-diacetate; FDA, fluorescein diacetate; KM8P, protoplast medium; KT, Kinetin; MS, Murashige and Skoog medium; MSB, callus growth medium; PAs, polyamines; Put, putrescine, PWS, protoplast wash solution; RFU, relative fluorescence units; SNP, sodium nitroprusside; Spd, spermidine; Spm,spermine; FBA: fluorescent brightening agents. |
|
