This study was carried out to investigate the effect of the intracellular signaling molecule nitric oxide (NO) on osmoregulation of tobacco cells under osmotic stress caused by phenylethanoid glycosides 6000 (PEG 6000). The results show that the PEG stress induced a specific pattern of endogenous NO production with two peaks in tobacco cells in vivo. Treatments with the NO donor sodium nitroprusside (SNP) significantly improved vitality and re-growth capacity, lowered cell death rate and alleviated the damage of tobacco cells caused by PEG 6000 stress. Further study indicated that SNP treatments led to relatively lower cell solute potential and higher water potential, which was beneficial for maintaining cell pressure potential under PEG stress. These results indicate that NO could improve the tolerance of tobacco cells to osmotic stress by enhancing their osmoregulation capacity. In addition, SNP treatments increased the accumulation of proline, one of the important organic osmoregulators in the tobacco cells under normal culture condition as well as PEG stress. The investigation on proline metabolism pathways demonstrated that the SNP-induced proline accumulation might be a combined result of sequential activation of several key enzymes of proline biosynthesis, including glutamate dehydrogenase and Δ1-pyrroline-5-carboxylate synthetase of glutamate pathway, and arginase and ornithine aminotransferase of ornithine pathway, and the inhibition of proline dehydrogenase of proline degradation pathway. All of these results suggest that NO takes part in the response and adaptation of tobacco cells to osmotic stress by enhancing their osmoregulation capacity and proline accumulation.

Key words: Nitric oxide, osmoregulation, proline, tobacco suspension cells, osmotic tolerance.