Inhibitory effects of elevated endogenous cytokinins on nitrate reductase in ipt-expressing tobacco are eliminated by short-term exposure to benzyladenine.

Using a novel system for expressing ipt gene from Agrobacterium tumefaciens in tobacco (Nicotiana tabacum L., cv. Petit Havana SR1), we were able to grow seedlings and teratoma-like tissue with increased content of cytokinins. This material enabled us to investigate new regulatory aspects of nitrate reduction. We grew control plants and plants with elevated cytokinins on MS media, with or without nitrate and benzyladenine (BA). We determined in vitro nitrate reductase (EC 1.6.6.1) activity (NRA) in this plant material. Initially, we found that ipt-expressing plants always displayed lowered levels of NRA when compared to wild-type SR1 plants. We determined that long-term exposure of tobacco plants and tissue to cytokinins caused up to 60% decrease in NRA. Exposure to 40 mM nitrate was able to induce the activity in such plants 3-fold, increasing the activity in SR1 plants more than 5-fold. We were able to restore wild-type levels of NRA in ipt-expressing plants by simultaneous induction of NR with BA and nitrate. Our results suggest that regulation of NR by nitrate and cytokinin is a result of overlaying cytokinin-driven regulatory processes, with those acting in the short-term having a positive effect on NRA, and those acting over extended periods of time having inhibitory effects on NRA.