Eighty-five maize lines were evaluated in a primary screen for differences in uptake rate at 1 mM KNO 3 ; 15 were statistically separable into either "high" or "low" populations. Kinetic parameters were determined by iterative, least-squares regression using a model combining both a saturable and a linear component. Using the inbred B73 as an arbitrary standard, it was determined that the saturable component had a Km of 224 μM and a Vmax of 0.107 μmolNO 3 /mg root dry weight/hr. The ,linear component had a first order rate constant of 6.1 × 10 -2 μmol NO 3 /mg root dry weight-hr/mM. These values compared favorably with other published determinations of nitrate uptake kinetic parameters for saturable systems. No other constants have been reported for a linear component of nitrate uptake in higher plants.

MATERIALS AND METHODS ['4NJnitrate. Immediately after harvesting, the tissue was frozen with Dry Ice. The frozen tissue was then lyophilized and finally ground to a fine powder. Ten g ground shoot tissue were extracted with three successive 500-ml aliquots of MCW4 (13:4:3 by volume, see reference 12). The extract was separated into two phases, chloroform and methanol:water. The chloroform phase was discarded. The methanol:water phase was surface-aerated overnight at room temperature to remove methanol. The remaining liquid, which contained [14N]nitrate, organic nitrogen, and some dissolved methanol, was used for the subsequent procedural tests. Nitrate in the liquid was determined using the method of Cataldo et al. (2).Stem exudate was collected from 5-day-old, dark-grown com seedlings (DeKalb XL-45) which had been cultured on a minus-N nutrient solution (6) for 3 previous days. The resulting exudate was nitrate-free (verified by the method of Cataldo et al. (2), data not shown) and was used in the tests as collected.A salt solution of (NH4)2SO4 and KNO3 was also used in the various tests for comparison.Reduction of Nitrate during Digestion. The extent to which nitrate was reduced during Kjeldahl digestion was tested by adding selected quantities of KlNO3 to each of the three kinds of samples previously described. The final '5N enrichment of nitrate was approximately 50 atom %. Appearance of '6N in reduced N was used to indicate that reduction of nitrate had occurred during digestion of the sample. The atom per cent excess 15N observed in the reduced N fraction was used to calculate the total quantity of nitrate (14N + 15N) that had been reduced.The Kjeldahl procedure of McKenzie and Wallace (8) was followed, using 2.5 ml 36 N H2SO4, 1.5 g K2SO4, and 50 mg HgO as HgSO4. Samples were digested in a heating block for 1 h at 200°C to remove H20. The temperature was then increased and the samples were digested for an hour after the block reached 380°C (total time approximately 3 h). Ammonium was determined directly on the digestate (3).Nitrate Volatilization. The proposed procedure depends on the removal of nitrate by volatilization as nitric acid from concentrated H2SO4. The completeness ofnitrate volatilization was tested by heating selected quantities of umol)

The effects of C02-limited photosynthesis on 15N03-uptake and reduction by maize (Zea mays, DeKalb XL-45) seedlings were examined in relation to concurrent effects of CO2 stress on carbohydrate levels and in vitro nitrate reductase activities. During a 10-hour period in CO2-depleted air (30 microliters of C02/ per liter), cumulative 5N03-uptake and reduction were restricted 22 and 82%, respectively, relative to control seedlings exposed to ambient air containing 450 microliters of CO2 per liter. The comparable values for roots of decapitated maize seedlings, the shoots of which had previously been subjected to CO2 stress, were 30 and 42%. The results demonstrate that reduction of entering nitrate by roots as well as shoots was regulated by concurrent photosynthesis. Although in vitro nitrate reductase activity of both tissues declined by 60% during a 10-hour period of CO2 stress, the remaining activity was greatly in excess of that required to catalyze the measured rate of 15NO3-reduction. Root respiration and soluble carbohydrate levels in root tissue were also decreased by CO2 stress. Collectively, the results indicate that nitrate uptake and reduction were regulated by the supply of energy and carbon skeletons required to support these processes, rather than by the potential enzymatic capacity to catalyze nitrate reduction, as measured by in vitro nitrate reductase activity.Both the uptake and reduction of nitrate by higher plants can be restricted when concurrent photosynthesis is limited by subambient CO2 levels (CO2 stress) (2, 9). In one view, CO2 stress limits the energy available for one or more of the processes which regulate the utilization of exogenous nitrate: (a) nitrate uptake, (b) reduction of nitrate to ammonium, and (c) synthesis of amino acids and macromolecules from ammonium. The enhanced the rate at which nitrate was reduced, especially in leaftissue (1, 2, 25). In addition, exogenously supplied sucrose increased nitrate reduction not only in the dark but also in the light (1, 9, 12).The evidence which supports the alternative possibility of a limitation in the enzymatic capacity for nitrate reduction is less conclusive. Carbon dioxide stress severely restricted the induction of NR in nitrogen-depleted rice leaves (21), and it enhanced the decay of NR in nitrate-grown Perilla leaves (1 1). In contrast, CO2 stress stimulated the induction (by light and nitrate) of NR in ammonium-grown maize plants (18), but had no effect on induction in excised leaves (26).Although the evidence linking nitrate reduction to carbohydrate supply appears to be more conclusive than that linking it to NR activity, to our knowledge no direct comparisons have been made. Therefore, the present research was initiated to examine with maize seedlings the regulatory effects of C02-limited photosynthesis on '5NO3-uptake and reduction, and to compare the effects with those on in vitro NR activity. Both intact and decapitated seedlings were used in order to determine whether root as well as shoot processes were aff...

McClure, P. R., Omholt, T. E. and Pace, G. M. 1986. Anion uptake in maize roots; Interactions between chlorate and nitrate [107][108][109][110][111][112] Effects of nitrate, chloride and chlorate ions upon nitrate and chlorate uptake by roots of maize {Zea mays L., cv. B73) seedlings were examined. Net nitrate uptake, ''ClOj' influx and *C1" influx (the latter two in a background of 0.5 mM K.NO3) displayed similar pH profiles with optima at pH 5.5 and below. External, non-labeled chloride had little effect on the accumulation of ' "CIO3" (both m 5 h and 20 min uptake assays), while nitrate and chlorate had almost identical, marked inhibitory effects. Nitrate pretreatment caused an apparent induction of both *ClOj and "NO, uptake activities. After 5 h of treatment in nitrate, the uptake activities of chloride-and chlorate-pretreated plants increased to that of nitrate-pretreated ptants. During 6 h exposure to chlorate, "^lOj uptake activity of nitrate-pretreated plants decreased to that of chlorate-and chloride-pretreated plants. The results support the existence of a shared nitrate/chlorate transport system in maize roots which is not inhibited by external chloride, and which is induced by nitrate, but not by chlorate or chloride. The suggestion is made that selection of chlorate-resistant mutants of maize can identify nitrate uptake as well as nitrate reductase mutants.Additional key words -Chloride uptake, nitrate reduction, nitrate uptake mutants, Zea mays. P. R. McClure (reprint requests) and T. E. Omholt,

Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [5Sjmethionine and in vitro translation. Nitrate enhanced the synthesis ofa 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with In the present study, we have searched for nitrate-inducible polypeptides in maize roots, in an attempt to identify nitrate membrane transport proteins. We have examined the profiles of polypeptides synthesized in maize roots incubated with or without nitrate. The spectra of in vitro translation products of nitrateinduced and noninduced total root RNA were also compared.MATERIALS AND METHODS Plant Material. Maize seeds (Zea mays L. var B73) were germinated in darkness at 29'C between germination paper saturated with 0.5 mM CaSO4. After 3 d, the roots ofthe seedlings were transferred to aerated solutions containing 0.5 mM CaSO4, 5 mM K2HPO4, 1 mM MgSO4, 1.25 mM K2SO4, micronutrients (13) and FeEDTA at 1 mM Fe. The seedlings were grown in an incubator at 29°C under fluorescent lights (about 100-200 ,uEm-2 -s) with a light/dark cycle of 16/8 h. During induction periods, typically during the first part of the light period on the 7th d after the beginning ofgermination, the roots ofthe seedlings were transferred to aerated solutions containing, in addition to 10 mm Mes (pH 5.5), either 5 mM Ca(N03)2 (induced) or 5 mM CaCl2 (noninduced). To label root proteins, the roots of 5 to 8 seedlings were bathed in either control or induction solutions (45 ml)