Nitric Oxide Has a Concentration-Dependent Effect on the Cell Cycle Acting EIN2 in Cultured Cells.

Ethylene is known to influence the cell cycle (CC) via poorly characterized roles whilst nitric oxide (NO) has well-established roles in the animal CC but analogous role(s) have not been reported for plants. As NO and ethylene signaling events often interact we examined their role in CC in cultured cells derived from wild-type (Col-0) plants and from ethylene-insensitive mutant plants. Both NO and ethylene were produced mainly during the first 5 days of the sub-cultivation period corresponding to the period of active cell division. However, in cells, ethylene generation was significantly reduced while NO levels were increased. With application of a range of concentrations of the NO donor, sodium nitroprusside (SNP) (between 20 and 500 μM) ethylene production was significantly diminished in Col-0 but unchanged in cells. Flow cytometry assays showed that in Col-0 cells treatments with 5 and 10 μM SNP concentrations led to an increase in S-phase cell number indicating the stimulation of G1/S transition. However, at ≥20 μM SNP CC progression was restrained at G1/S transition. In the mutant strain, the index of S-phase cells was not altered at 5-10 μM SNP but decreased dramatically at higher SNP concentrations. Concomitantly, 5 μM SNP induced transcription of genes encoding and in Col-0 cells whereas transcription of s and s were not significantly altered in cells at any SNP concentrations examined. Hence, it is appears that EIN2 is required for full responses at each SNP concentration. In cells, greater amounts of NO, reactive oxygen species, and the tyrosine-nitrating peroxynitrite radical were detected, possibly indicating NO-dependent post-translational protein modifications which could stop CC. Thus, we suggest that in cultured cells NO affects CC progression as a concentration-dependent modulator with a dependency on EIN2 for both ethylene production and a NO/ethylene regulatory function.