Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Abstract

Abstract.Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO 3-content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wildtype controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO 3-content was observed in plants grown in nitrogenreplete conditions at low irradiance (170~tmol photons.m -2 .s-l). This relationship disappeared at higher irradiance (450~tmol photons.m-2.s -1) because the maximal extractable NR activity in the leaves of the wildtype plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO 3-accumulated in the leaves of plants grown at 450~tmol photons.m-2.s -~ than in those grown at 170 ~tmol photons.m-e.s -~ in N-replete conditions. The foliar NO 3-level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO 3-was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO 3-in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of

Interactions Between Carbon and Nitrogen Metabolism

“…Either conditions are less favourable for NR activity in vivo than in vitro or other factors are more important in controlling flux from nitrate into amino acids. Constitutive expression ofNR in tobacco gave extractable activities that were between two-and fivefold higher than wild-type tobacco but had no marked effect on chlorophyll contents, protein levels, photosynthesis or biomass, although glutamine levels were increased twofold (Foyer et al 1994b;Ferrario et al 1995). The last observation does suggest some effect of overexpression on flux from nitrate to glutamine: metabolism downstream of glutamine synthesis is obviously able to absorb this perturbation.…”

“…Constitutive expression should theoretically remove the requirement for nitrate-induced activa-tion of transcription and allow NR protein to remain high even at limiting nitrate supply. However, although nitrate does not influence the phosphorylation state of the enzyme, it appears to be necessary to prevent decreases in NR capacity due to protein turnover: the imposition ofN deficiency caused extractable NR activities to decrease almost to zero in transformants and wild-type plants alike (Ferrario et al 1995(Ferrario et al , 1996. Conversely, compensatory modifications at several levels (including transcription, protein turnover and phosphorylation status) dampened the impact offewer nia genes on nitrate assimilation in tobacco (Scheible et al 1997b).…”

“…One explanation may be effects of nitrate at the level ofNR protein stability (Ferrario et al 1995). A second could be the influence of phosphorylation on enzyme activity (Kaiser and Huber 1994).…”

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

“…Nitrate reductase protein and NR activity have been shown to decrease more quickly than the NR mRNA when plants are deprived of NOf (Galangau et al 1988) Nicotiana plumbaginifolia expressing a constitutive NR. The shaded areas on the figures represent the dark period activity in both the 35S-NR expressors and in untransformed controls (Ferrario et al 1995). The apparent insensitivity of the NR activation state to exogenous NO~-in the presence of microcystin-LR, a phosphatase inhibitor, suggests that the NR-protein kinase is not regulated by NO3.…”

“…Only mature leaves were chosen for disks; these were harvested after 2 h of illumination. The remaining plants were then watered with a complete nutrient solution containing 0.05 mM NO;-for 5 d (see Ferrario et al 1995 for details), the time necessary for the plants to develop two new leaves depleted of nitrate. These are therefore designated as low-N plants, as opposed to the high-N plants described above.…”