Figure 7. Effect of cold stress and L-NNA on activities of NOS-like enzyme reaction in pollen tubes.

Pollen grains from Camellia sinensis were incubated under control and cold conditions for 2 h with or without 300 μM L-NNA, then the pollen tubes were collected and grounded for crude enzyme preparation according to Zhao et al. [15] with slight modification. For the detection of NOS-like activity, samples were added to a reaction mixture containing the substrate and all cofactors of the NOS reaction, and were incubated for 10 min at 37°C. NO production from NOS-like activity was detected by EPR at 0, 5 and 10 min, respectively. A, NO production from crude extracts of pollen tubes incubated at 25°C. B, NO production from crude extracts of pollen tubes treated by 300 μM L-NNA at 25°C. C, NO production from crude extracts of pollen tubes incubated at 4°C. D, NO production from crude extracts of pollen tubes treated by 300 μM L-NNA at 4°C. E showed the mean relative EPR signal height of NO from NOS-like activity, and data are the means of three replicates ± SD. Means with different letters are significantly different at P<0.05 (E). All the representative spectra are from three independent measurements and were obtained by the accumulation of five recordings.