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

Abstract

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.

Ammonium formation and assimilation in PSARK∷IPT tobacco transgenic plants under low N

“…It has been shown that high endogenous CK levels reduced the inducibility of NR by NO 3 − (Lexa and Genkov, 2002). This observation is in agreement with our results showing that the P SARK ::IPT (100) plants displayed a low response of NR activity to N deficiency (Table 2).…”

Extent of ipt gene expression and resulting amount of cytokinins affect activities of carboxylation enzymes in transgenic plants

“…The other transformant types had three (tobacco) to seven (potato) times smaller leaf area even if no increase of CK contents was detected. Other studies on transgenic plants bearing the ipt gene in various expression constructs generally confirmed the basic traits of such plants (Ondřej et al 1990, Šiffel et al 1992, Čatský et al 1993a,b, Pospíšilová et al 1997/98, Synková et al 1999, Jordi et al 2000, Synková and Valcke 2001, Lexa et al 2002. Leaf fresh masses of the NOT and MOD were very similar compared to the respective controls.…”

“…On the other hand, transgenic plants raised from seeds or tubers had just little elevated or unelevated CK contents. Such morphological abnormalities are inhibition of root formation, teratoma-like structures of multiple shoot meristems with small leaves, and short internodes in in vitro grown transformants (Thomas et al 1995, Lexa et al 2002, and reduced growth of stems, reduced apical dominance, reduced size of leaves, and increased number of short internodes in grafted or rooted plants (Synková et al 1999). Rate of gene expression depends among others on a site in the plant genome where the gene is incorporated and it can be time-dependent.…”

Plant growth response and nitrate reductase activities of roots of Chromolaena odorata in a model spent lubricating oil-polluted soil

“…The first step in the conversion of nitrate is catalyzed by nitrate reductase (NR). Lexa et al (2002) described this substrate-inducible plant enzyme as essential in nitrogen assimilation. Activity of this enzyme is considered to be a limiting factor for growth and protein production in plants (Jackson et al, 2008).…”