Nitrate Uptake into Barley ( Hordeum vulgare ) Plants ' A NEW APPROACH USING 36 C 103-AS AN ANALOG FOR N 03 -

Evidence is presented that chlorate is an extremely good analog for nitrate during nitrate uptake by intact barley (Hordeum vulgare cv. Fergus) roots. The depletion of C103or NO3from uptake media over 2 to 6 hours by seedlings was found to be dependent on combined N03plus C103concentrations, and total anion uptake was equivalent at different N03-/CI03ratios. After loading barley seedlings with 3'CI03for 6 hours, kinetic parameters were derived from the analysis of efflux of 13"CI chlorate into unlabeled solution. On the basis of this analysis, the half times for exchange for the cytoplasmic and vacuolar phases were 17 minutes and 20 hours, respectively. Data pooled from a number of different experiments were used to calculate kinetic constants (Km and V,,) for 36CI03influx into barley roots at different external C103-/NO3ratios, using short (10 minutes) influx times. There appeared to be no discrimination by the root cells between C103and N03-. Lineweaver-Burk analysis of the interaction between nitrate and chlorate were characteristic of competitive inhibition at low nitrate concentrations (0-0.5 mM). At higher concentrations, in the range of >1 mm, similar interactions between these ions were evident. There is relatively little information available on the detailed kinetics of nitrate transport into barley plants. Even the more recent investigations (2, 7, 21) have described net nitrate flux into barley plants over periods of greater than 2 h duration. The use of the heavy isotope `5N03 as a tracer is not without difficulties, and again for whole plant studies uptake periods of 9 h or greater are no exception (1, 14). In part, this is due to the large internal pools of nitrogen (6). As far as the authors are aware N03 has so far only been used for the study of nitrate assimilation in tobacco culture cells (24) and soybean roots and nodules (23). Moreover, this isotope is not very convenient for routine experiments because of its extremely short half-life (10 min). Doddema and Telkamp (9) have shown that nitrate competes with chlorate for uptake into Arabidopsis thaliana. Other workers have obtained similar Km and Vmax values for nitrate reductase activity (NR) using chlorate or nitrate as substrates for the enzymes from Chlorella (25) or tomato (13). In higher plants, the chlorite which results from the action of NR is not reduced by nitrite reductase and apparently accounts for many of the toxic symptoms caused by chlorate treatment (15). Indeed, chlorate resistance has been used to screen for nitrate reductase mutants in Aspergillus (3) and Pisum (12). Tromballa and Broda (27) have studied the uptake and reduction of 36C103and 'CIO4in Chlorella fusca. They found that increasing external nitrate concentrations depressed accumulation 'Financial support from NSERC Canada is gratefully acknowledged. of tracer in the cells, and the authors assumed that competition between C103 and N03 took place at both the uptake and reduction steps. Cram (4) and Smith (26) have suggested that accumulated nitrate and chloride exert a common negative feedback on the subsequent uptake of these ions. The demonstration, therefore, that Claccumulation by carrot discs led to a reduction of 'C103 uptake (5) is consistent with the belief that this ion may serve as a tracer for nitrate. To date these are the only examples in the literature which give some indication that 36C103could be used as a tracer for NO3-. There are other known examples where two ions are so similar in chemical properties that the cellular transport mechanisms fail to discriminate between them. Such examples are sulfate-selenate, chloride-bromide, calcium-strontium, and rubidium-potassium (10, 11, 18). The convenience associated with the considerably longer half-life of 86Rb+ (18.7 d) than 42K' (12.4 h) has led to the widespread use (11) of the former as a tracer for K+. Provided that certain limitations of this method are taken into account (22), the technique has greatly enhanced current understanding of K+ transport in higher plants. From the foregoing discussion, the available evidence in the literature suggests that the use of 36C103(half-life 3.07 x 105 years) as a tracer for NO3is a reasonable proposition. The aim of the present inquiry was to explore this possibility during NO3 uptake by barley plants. MATERIALS AND METHODS Growth of Plants. Barley plants (Hordeum vulgare cv. Fergus) were grown as described previously (7) in modified Johnson's solution at one-tenth concentration with calcium sulfate and potassium sulfate replacing nitrates. Nitrate was provided at 10 AM. Previous work has shown that at this concentration there is no net nitrate flux into the plants, but that the nitrate uptake system is fully 'induced' (7). In all experiments, barley plants were 7 to 9 d old. Net Flux Measurements. These were obtained by measuring depletion of the uptake solution containing 0.5 mm CaSO4 + 0.25 mM K2SO4 + (0-0.5 mM) nitrate and/or chlorate. Disappearance of chlorate and nitrate was monitored using an HNU (HNU Systems Inc., MA) nitrate electrode. We have found that nitrate and chlorate are virtually indistinguishable by this method of analysis (Fig. 1, insert). 36C03Synthesis. 36C103was prepared by the electrolysis of 36Cland the products were separated using TLC. 36C103was counted in a Triton X-based scintillation fluid by means of a Searle Isocap scintillation counter (8). Efflux Analysis. Barley plants were loaded for 6 h in 0.1 mM C103+ 0.25 mm K2SO4 + 0.5 mm CaSO4 labeled with 36C103(final specific activity, 0.013 ,uCi/,mol). The unlabeled medium contained 0.1 mm KNO3 + 0.25 mm K2SO4 + 0.5 mm CaSO4. After loading, the roots were briefly rinsed in distilled H20 to prevent carry-over of label from 36C103loading solution and then 50 www.plantphysiol. org on July 23, 2018 Published by Downloaded from Copyright © 1982 American Society of Plant Biologists. All rights reserved. NO3UPTAKE IN BARLEY USING 36C103-