Mechanical Stimulation-induced Nitric Oxide Burst, Possible Pathways And Involvement Of Ca～(2+) And CaM In Tobacco Cultured Cells

Research on NO in plants has recieved considerable attention in recent years due to its function in plant growth and development and its role in plants response to various biotic and abiotic stresses as a signaling molecule. Mechanical stimulation (MS) is ubiquitous in environments where plants survive, such as wind, rain, touch, wound, press, infection of plant diseases, and so on. So it is valuable to study NO, about its production and synthesis pathways, its action mechanism, in plants exposed to MS. In this experiment, tobacco suspension cultured cells were used as material and increased rotational speed of a shaker incubator as MS factor. The study was aimed to investigate the formation of NO, its possible synthesis pathways and mechanism in tobacco suspension cultured cells under MS stress conditions.The results indicate that application of MS on the tobacco suspension cultured cells by raising rotational speed in the shaker incubator could rapidly induce NO formation within tens of minutes. The increase in NO release was positively and significantly correlated with the strength of the MS. The amount of NO in cells is up to the maximum at 30 minutes and this rapidly NO accumulation was reversed by the addition of the NO scavenger cPTIO. The investigation on the possible pathways of this MS-induced NO formation, by using Nitric oxide synthase (NOS) inhibitor N^G-Methyl-L-arginine (L-NMMA) and Nitrate reductase inhibitor NaN₃, indicated that NOS involved in the MS-induced NO production in the tobacco suspension cells. L-NMMA reduced the total MS-induced NO by almost 90 percent, but NaN₃ did not influence it. In addition, the results also showed that this MS-induced NO formation required the mediation of calcium and calmodulin (Ca²⁺-CaM). The NO formation decreased for some extent by pretreatment with the Ca²⁺ chelator EGTA, the plasma membrane Ca²⁺ channel blocker La³⁺, the intracellular Ca²⁺ channel antagonist ruthenium red (RR), the CaM inhibitors chlorpromazine (CPZ) and trifluoperazine (TFP). All of these results suggest that Ca²⁺-CaM involved in the MS-induced NO formation in the tobacco suspension cultured cells.