In our experiments, a strong competitive inhibitor of all three isoforms of animal NO synthase (NOS), L-NMMA, inhibited the accumulation of GUS activity in transgenic Arabidopsis harboring the GUS gene driven by a cytokinin-sensitive ARR5 promoter. In accordance with earlier publication , this inhibitor also suppressed cytokinin-dependent betacyanin accumulation in Amaranthus. In the Arabidopsis assay system, the effectiveness of L-NMMA was higher than in Amaranthus one: 1–2 mM L-NMMA inhibited cytokinin-induced effect in Arabidopsis at about 80–90%. In Amaranthus, similar inhibition was observed with 5 mM L-NMMA.
In addition to L-NMMA we have used its D-isomer (D-NMMA) which does not affect animal NOS. Experiments with L- and D-isomers have shown that both isomers were able to inhibit cytokinin action with similar effectiveness. Thus no functional difference between the active (in animals) L-form and its inactive D-analog was revealed in the Arabidopsis assay system.
To obtain more direct evidence, we treated Arabidopsis seedlings with NO. Arabidopsis seedlings were incubated in the presence of the NO donors NOR3 or SNAP. In our experiments neither SNAP nor NOR3, both tested in a wide concentration range, caused a cytokinin-like effect, namely the enhancement of GUS activity. This result argues against a role for NO as a direct messenger of the cytokinin signal. However, it does not exclude the possibility that NO has a role in a some parallel transduction pathway, which could be indispensable to effective cytokinin signaling. This possibility was tested experimentally by adding NO donors to Arabidopsis plants which were treated with BA and L-NMMA. However experiments showed that NO did not alleviate the L-NMMA inhibition of cytokinin-induced GUS activity. This result also does not support the participation of NO in cytokinin signal transduction to primary response genes, at least in Arabidopsis.
Next we explored whether L-NMMA inhibits cytokinin signaling at an early stage (before gene activation) or acts posttranscriptionally. To this end we analysed the steady state levels of GUS transcripts 35 min after cytokinin treatment of Arabidopsis seedlings. Results demonstrated that with no cytokinin treatment GUS gene expression was very low, the radioactive signal being hardly detectable. 35 min after cytokinin treatment a more than 30-fold increase of the GUS transcript was detected. 5 mM L-NMMA, which strongly inhibits the cytokinin-induced accumulation of GUS activity, had no influence on the cytokinin-induced accumulation of GUS transcripts. This result shows that L-NMMA acts posttranscriptionally.