Nitrate Uptake and Assimilation by Wheat Seedlings during Initial Exposure to Nitrate

Abstract

Nitrate uptake, reduction, and translocation were examined in intact, 14-day-old, nitrogen-depleted wheat (Triticum vulgare var. Knox) seedlings during a 9-hour exposure to 0.2 mM Ca(NO3)2. The nitrate uptake rate was low during the initial 3-hour period, increased during the 3-to 6-hour period, and then declined. By the 3rd hour, 14% of the absorbed nitrate had been reduced, and this increased to 36% by the 9th hour. Shoots accumulated reduced '5N more rapidly than roots and the ratio of reduced "N to '5N-nitrate was higher in the shoots. A significant proportion of the total reduction occurred in the root system under these experimental conditions. Accumulation of "N in ethanol-insoluble forms was evident in both roots and shoots by the 3rd hour and, after 4.5 hours, increased more rapidly in shoots than in roots. An experiment in which a 3-hour exposure to 0.2 mM Ca('NO3)2 was followed by a 12-hour exposure to 0.2 mM Ca ("NO3)2 revealed a half-time of depletion of root nitrate of about 2.5 hours. A large proportion of this depletion, however , was due to loss of '5N-nitrate to the ambient "N-nitrate solution. The remaining pool of '5N-nitrate was only slowly available for reduction. Total "N translocation to the shoot was relatively efficient during the first 3 hours after transfer to Ca ("NO3)2 but it essentially ceased after that time in spite of significant pools of "N-nitrate and a-amino-"N remaining in the root tissue. Many higher plant species (3, 9, 10, 14, 15), as well as tobacco cell cultures (6) and Penicilliumii chrysogenum (5), exhibit an apparent induction pattern of nitrate uptake. In each instance the rate of uptake increased substantially after a period of exposure to nitrate. The induction of nitrate reductase in corn roots (18, 23) parallels the increase in rate of nitrate uptake (9), and nitrate translocation to the xylem, as well as nitrate uptake and reduction, was restricted by protein-synthesis inhibitors (9, cf. 12). It follows that, upon exposure to nitrate, plants should develop enhanced capabilities for nitrate uptake, reduction in roots, translocation, and reduction in shoots. To examine the developmental patterns of these processes, two experiments were conducted. The first involved exposure of nitrogen-depleted plants to nitrate solutions highly enriched in "N; the results show the effectiveness with which absorbed nitrate (a) accumulated in roots and shoots, (b) was reduced, and (c) was incorporated into protein during a 9-hr period. In the second experiment, a 3-hr exposure to highly enriched "N-nitrate was followed by an additional 12 hr in "N-nitrate, permitting an assessment of the fate of the initially absorbed "N-nitrate during the early phases of recovery from the nitrogen depleted state. MATERIALS AND METHODS Wheat seed (Triticum vulgare var. Knox) were kept moist with 10-' M CaSO4, germinated 3 days in darkness, and transferred to small plastic cups containing holes in the bottom through which the roots were threaded. Each cup contained six seedlings and henceforth will be referred to as a culture. Roots of the required numbers of cultures were placed in 13-liter plastic tanks containing nutrients at one-fifth the concentrations given by Hoagland and Arnon (7) for minus nitrogen solutions. The plants were grown in these solutions for 11 days in a controlled environment chamber. Sixteen hr of light and 8 hr of darkness were used. The light intensity was 194 hectolux at plant height, and temperature was 24 +2 C and 17 + 2 C during the light and dark periods, respectively. These growth conditions produced seedlings low in nitrogen and high in carbohydrate content (16). Thus the seedlings would be expected to absorb and assimilate nitrate nitrogen at a rapid rate, provided the required transport, reduction, and assimilatory systems were present. Both experiments were conducted in the growth chamber at 25 C and 194 hectolux. For the first experiment, cultures were exposed to aerated 0.2 mm Ca("NO,), containing 95 atom % "N and were harvested at 0, 3, 4.5, 6, and 9 hr. Each harvest consisted of 32 cultures. For the second experiment all cultures were exposed to 0.2 mm Ca("NO,)2 with 97.5 atom % "N for 3 hr. Then 28 cultures were harvested while the remainder were placed in 0.2 mm Ca("NO,),. Subsequently, harvests of 28 cultures each were made at 4.5, 6, 9, 12, and 15 hr after initiation of the experiment. All solutions were adjusted initially to pH 6.5 with Ca(OH),, and adjustments to pH 6.5 were made with Ca(OH)2 and H.SO, every 15 to 30 min. At the designated times, cultures were removed from the treatment solution, rinsed thoroughly with deionized H20, separated at the root-shoot junction, blotted with cheesecloth to remove excess water, weighed, quickly frozen, and lyophilized. At each harvest, plants were divided at random into two groups, permitting duplicate analyses of all components. The lyophilized tissue was extracted with 70% ethyl alcohol and the insoluble residue was analyzed for total N and "N. The ethyl alcohol-soluble material was evaporated to dryness, and 1102