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The influence of the allelopathic compound ferulic acid (FA) on nitrogen uptake from solutions containing both NO(3) (-) and NH(4) (+) was examined in 8-day-old nitrogen-depleted corn (Zea mays L.) seedlings. Concurrent effects on uptake of Cl(-) and K(+) also were assessed. The presence of 250 micromolar FA inhibited the initial (0-1 hours) rate of NO(3) (-) uptake and also prevented development of the NO(3) (-)-inducible accelerated rate. The pattern of recovery when FA was removed was interpreted as indicating a rapid relief of FA-restricted NO(3) (-) uptake activity, followed by a reinitiation of the induction of that activity. No inhibition of NO(3) (-) reduction was detected. Ammonium uptake was less sensitive than NO(3) (-) uptake to inhibition by FA. An inhibition of Cl(-) uptake occurred as induction of the NO(3) (-) transport system developed in the absence of FA. Alterations of Cl(-) uptake in the presence of FA were, therefore, a result of a beneficial effect, because NO(3) (-) uptake was restricted, and a direct inhibitory effect. The presence of FA increased the initial net K(+) loss from the roots during exposure to the low K, ammonium nitrate uptake solution and delayed the recovery to positive net uptake, but it did not alter the general pattern of the response. The implications of the observations are discussed for growth of plants under natural conditions and cultural practices that foster periodic accumulation of allelopathic substances.

The effects of CO(2)-limited photosynthesis on (15)NO(3) (-) uptake and reduction by maize (Zea mays, DeKalb XL-45) seedlings were examined in relation to concurrent effects of CO(2) stress on carbohydrate levels and in vitro nitrate reductase activities. During a 10-hour period in CO(2)-depleted air (30 microliters of CO(2)/ per liter), cumulative (15)NO(3) (-) uptake and reduction were restricted 22 and 82%, respectively, relative to control seedlings exposed to ambient air containing 450 microliters of CO(2) per liter. The comparable values for roots of decapitated maize seedlings, the shoots of which had previously been subjected to CO(2) 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 CO(2) stress, the remaining activity was greatly in excess of that required to catalyze the measured rate of (15)NO(3) (-) reduction. Root respiration and soluble carbohydrate levels in root tissue were also decreased by CO(2) 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.

An experiment was conducted to determine the extent that NO(3) (-) taken up in the dark was assimilated and utilized differently by plants than NO(3) (-) taken up in the light. Vegetative, nonnodulated soybean plants (Glycine max L. Merrill, ;Ransom') were exposed to (15)NO(3) (-) throughout light (9 hours) or dark (15 hours) phases of the photoperiod and then returned to solutions containing (14)NO(3) (-), with plants sampled subsequently at each light/dark transition over 3 days. The rates of (15)NO(3) (-) absorption were nearly equal in the light and dark (8.42 and 7.93 micromoles per hour, respectively); however, the whole-plant rate of (15)NO(3) (-) reduction during the dark uptake period (2.58 micromoles per hour) was 46% of that in the light (5.63 micromoles per hour). The lower rate of reduction in the dark was associated with both substantial retention of absorbed (15)NO(3) (-) in roots and decreased efficiency of reduction of (15)NO(3) (-) in the shoot. The rate of incorporation of (15)N into the insoluble reduced-N fraction of roots in darkness (1.10 micromoles per hour) was somewhat greater than that in the light (0.92 micromoles per hour), despite the lower rate of whole-plant (15)NO(3) (-) reduction in darkness.A large portion of the (15)NO(3) (-) retained in the root in darkness was translocated and incorporated into insoluble reduced-N in the shoot in the following light period, at a rate which was similar to the rate of whole-plant reduction of (15)NO(3) (-) acquired during the light period. Taking into account reduction of NO(3) (-) from all endogenous pools, it was apparent that plant reduction in a given light period ( approximately 13.21 micromoles per hour) exceeded considerably the rate of acquisition of exogenous NO(3) (-) (8.42 micromoles per hour) during that period. The primary source of substrate for NO(3) (-) reduction in the dark was exogenous NO(3) (-) being concurrently absorbed. In general, these data support the view that a relatively small portion (<20%) of the whole-plant reduction of NO(3) (-) in the light occurred in the root system.

Roots of decapitated maize seedlings (Zea mays L.) were exposed for 12 hours to 1.0 millimolar KNO(3) (98.5 atom per cent (15)N) in the presence and absence (control) of 0.1 millimolar p-fluorophenylalanine (FPA), an analog of the amino acid phenylalanine. FPA decreased nitrate uptake but had little effect on potassium uptake. In contrast, accumulation of both ions in the xylem exudate was greatly restricted. The proportion of reduced (15)N-nitrogen that was translocated at each time was also restricted by FPA. These observations are interpreted as indicating that synthesis of functional protein(s) is required for nitrate uptake and for transport of potassium, nitrate, and reduced-(15)N from xylem parenchyma cells into xylem elements. The effect of FPA on nitrate reduction is less clear. Initially, FPA limited nitrate reduction more than nitrate uptake, but by 8 hours the cumulative reduction of entering nitrate was similar ( approximately 35%) in both control and FPA-treated roots. A relationship between nitrate uptake and nitrate reduction is implied. It is suggested that nitrate influx regulates the proportion of nitrate reductase in the active state, and thereby regulates concurrent nitrate reduction in decapitated maize seedlings.

The influence of nitrogen stress on net nitrate uptake resulting from concomitant (15)NO(3) (-) influx and (14)NO(3) (-) efflux was examined in two 12-day-old inbred lines of maize. Plants grown on (14)NO(3) (-) were deprived of nitrogen for up to 72 hours prior to the 12th day and then exposed for 0.5 hour to 0.15 millimolar nitrate containing 98.7 atom% (15)N. The nitrate concentration of the roots declined from approximately 100 to 5 micromolar per gram fresh weight during deprivation, and (14)NO(3) (-) efflux was linearly related to root nitrate concentration. Influx of (15)NO(3) (-) was suppressed in nitrogen-replete plants and increased with nitrogen deprivation up to 24 hours, indicating a dissipation of factors suppressing influx. Longer periods of nitrogen-deprivation resulted in a decline in (15)NO(3) (-) influx from its maximal rate. The two inbreds differed significantly in the onset and extent of this decline, although their patterns during initial release from influx suppression were similar. Except for plants of high endogenous nitrogen status, net nitrate uptake was largely attributable to influx, and genetic variation in the regulation of this process is implied.

It is unclear if the relative content of NO(3) (-) and reduced N in xylem exudate provides an accurate estimate of the percentage reduction of concurrently absorbed NO(3) (-) in the root. Experiments were conducted to determine whether NO(3) (-) and reduced N in xylem exudate of vegetative, nonnodulated soybean plants (Glycine max [L.] Merr., ;Ransom') originated from exogenous recently absorbed (15)NO(3) (-) or from endogenous (14)N pools. Plants either were decapitated and exposed to (15)NO(3) (-) solutions for 2 hours or were decapitated for the final 20 minutes of a 50-minute exposure to (15)NO(3) (-) in the dark and in the light. Considerable amounts of (14)NO(3) (-) and reduced (14)N were transported into the xylem, but almost all of the (15)N was present as (15)NO(3) (-). Dissimilar changes in transport of (14)NO(3) (-), reduced (14)N and (15)NO(3) (-) during the 2 hours of sap collection resulted in large variability over time in the percentage of total N in the exudate which was reduced N. Over a 20-minute period the rate of (15)N transport into the xylem of decapitated plants was only 21 to 36% of the (15)N delivered to the shoot of intact plants. Based on the proportion of total (15)N which was found as reduced (15)N in exudate and in intact plants in the dark, it was estimated that 5 to 17% of concurrently absorbed (15)NO(3) (-) was reduced in the root. This was much less than the 38 to 59% which would have been predicted from the relative content of total NO(3) (-) and total reduced N in the xylem exudate.

The effect of ambient ammonium (0.5 millimolar [(14)NH(4)](2)SO(4)) added to a nutrient solution containing 1.0 millimolar K(15)NO(3), 99 atom per cent (15)N, upon [(15)N]nitrate assimilation and utilization of previously accumulated [(14)N]nitrate was investigated. Corn seedlings, 5-day-old dark-grown decapitated (experiment I) and 10-day-old light-grown intact (experiment II), which had previously been grown on K(14)NO(3) nutrient solution, were used. In both experiments, the presence of ambient ammonium decreased [(15)N]nitrate influx (20% after 6 hours) without significantly affecting the efflux of previously accumulated [(14)N]nitrate. In experiment I, relative reduction of [(15)N]nitrate (reduction as a percentage of influx) was inhibited more than was [(15)N]nitrate influx. Nevertheless, in experiment I, where all reduction could be assigned to the root system, the absolute inhibition of reduction during the 12 hours (13 micromoles/root) was less than the absolute inhibition in influx (24 micromoles/root). The data suggest that the influence of ammonium on [(15)N]nitrate influx could not be totally accounted for by the decrease in the potential driving force which resulted from restricted reduction; an additional impact on the influx process is indicated. Reduction of [(15)N]nitrate in experiment II after 6 hours accounted for 30 and 18% of the tissue excess (15)N in the control and ammonium treatments, respectively. Relative distribution of (15)N between roots and exudate (experiment I), or between roots and shoots (experiment II) was not affected by ammonium. On the other hand, the accumulation of [(15)N]nitrate in roots, shoots, and xylem exudate was enhanced by ammonium treatment compared to the control, whereas the accumulation of reduced (15)N was inhibited.

A series of experiments was conducted to characterize alterations in carbohydrate utilization in leaves of nitrogen stressed plants. Two-week-old, nonnodulated soybean plants (Glycine max [L.] Merrill, ;Ransom'), grown previously on complete nutrient solutions with 1.0 millimolar NO(3) (-), were transferred to solutions without a nitrogen source at the beginning of a dark period. Daily changes in starch and sucrose levels of leaves were monitored over the following 5 to 8 days in three experiments. Starch accumulation increased relative to controls throughout the leaf canopy during the initial two light periods after plant exposure to N-free solutions, but not after that time as photosynthesis declined. The additional increments of carbon incorporated into starch appeared to be quantitatively similar to the amounts of carbon diverted from amino acid synthesis in the same tissues. Since additional accumulated starch was not degraded in darkness, starch levels at the beginning of light periods also were elevated. In contrast to the starch effects, leaf sucrose concentration was markedly higher than controls at the beginning of the first light period after the N-limitation was imposed. In the days which followed, diurnal turnover patterns were similar to controls. In source leaves, the activity of sucrose-P synthase did not decrease until after day 3 of the N-limitation treatment, whereas the concentration of fructose-2,6-bisphosphate was decreased on day 2. Restricted growth of sink leaves was evident with N-limited plants within 2 days, having been preceeded by a sharp decline in levels of fructose-2,6 bisphosphate on the first day of treatment. The results suggest that changes in photosynthate partitioning in source leaves of N-stressed plants resulted largely from a stable but limited capacity for sucrose formation, and that decreased sucrose utilization in sink leaves contributed to the whole-plant diversion of carbohydrate from the shoot to the root.

The use of the relative ureide content of xylem sap [(ureide-N/total N) x 100] as an indicator of N(2) fixation in soybeans (Merr.) was examined under greenhouse conditions. Acetylene treatments to inhibit N(2) fixation were imposed upon the root systems of plants totally dependent upon N(2) fixation as their source of N and of plants dependent upon both N(2) fixation and uptake of exogenous nitrate. Significant decreases in the total N concentration of xylem sap from plants of the former type were observed, but no significant decrease was observed in the total N concentration of sap from the latter type of plants. In both types of plants, acetylene treatment caused significant decreases in the relative ureide content of xylem sap. The results provided further support for a link between the presence of ureides in the xylem and the occurrence of N(2) fixation in soybeans. The relative ureide content of xylem sap from plants totally dependent upon N(2) fixation was shown to be insensitive to changes in the exudation rate and total N concentration of xylem sap brought about by diurnal changes in environmental factors. There was little evidence of soybean cultivars or nodulating strains affecting the relative ureide content of xylem sap. ;Ransom' soybeans nodulated with Rhizobium japonicum strain USDA 110 were grown under conditions to obtain plants exhibiting a wide range of dependency upon N(2) fixation. The relative ureide content of xylem sap was shown to indicate reliably the N(2) fixation of these plants during vegetative growth using a (15)N method to measure N(2) fixation activity. The use of the relative ureide content of xylem sap for quantification of N(2) fixation in soybeans should be evaluated further.

Potassium influx into roots of dark-grown decapitated maize seedling (Zea mays L., cv Pioneer 3369A) was examined in presence and absence of ambient ammonium and at various root potassium concentrations. Six-day old seedlings which were dependent on the endosperm reserves for their energy source were exposed to KCl (labeled with (86)Rb) ranging from 5 to 200 micromolar. At both low (13 micromoles per gram fresh weight) and high (100 micromoles per gram fresh weight) root potassium concentration, isotherms indicated two potassium influx systems, one approaching saturation at 50 to 100 micromolar potassium and an additional one tentatively considered to be linear. A mixed-type inhibition by ammonium for the low-concentration saturable system was indicated by a concomitant decrease in V(max) and increase in K(m). High root potassium concentration decreased V(max) but had little effect on K(m) of this system. The rate constant for the second quasilinear system was decreased by ambient ammonium and by high root potassium status. Transfer of high potassium roots to potassium-free solutions resulted in an increase in influx within 2 hours; by 24 hours influx significantly exceeded that of roots not previously exposed to potassium. In roots of both low and high root potassium concentrations, potassium influx was restricted progressively as ambient ammonium increased to about 100 micromolar, but there was little further inhibition as ammonium concentrations increased beyond that to 500 micromolar. The data imply that potassium influx has two components, one subject to inhibition by ambient ammonium and one relatively resistant.

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 (NO(3))(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 (15)N more rapidly than roots and the ratio of reduced (15)N to (15)N-nitrate was higher in the shoots. A significant proportion of the total reduction occurred in the root system under these experimental conditions. Accumulation of (15)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 ((15)NO(3))(2) was followed by a 12-hour exposure to 0.2 mm Ca ((14)NO(3))(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 (15)N-nitrate to the ambient (14)N-nitrate solution. The remaining pool of (15)N-nitrate was only slowly available for reduction. Total (15)N translocation to the shoot was relatively efficient during the first 3 hours after transfer to Ca ((14)NO(3))(2) but it essentially ceased after that time in spite of significant pools of (15)N-nitrate and alpha-amino-(15)N remaining in the root tissue.

The effects of accumulated [(14)N]nitrate and its utilization in decapitated, 5-day-old dark-grown corn roots on influx, accumulation, xylem deposition, and reduction of concurrently absorbed nitrate during an 18-hour exposure to 0.5 millimolar K(15)NO(3) nutrient solution were examined. A 20-hour pretreatment in 15.0 millimolar K(14)NO(3) high nitrate (HN) resulted in a 2-fold greater tissue nitrate level than pretreatment in 0.5 millimolar K(14)NO(3) low nitrate (LN). Upon transfer to the 0.5 millimolar K(15)NO(3) solution, the net nitrate uptake rate in HN roots after 2 hours was 52% of the LN rate, but increased to 93% at the end of the uptake period. Despite an enhanced [(14)N]nitrate efflux from HN roots to the uptake solution, the efflux differences between the two pretreatments did not compensate for the decrease in net nitrate uptake. The [(15)N]nitrate influx rate was initially restricted by 33% in the HN roots compared to LN roots, but it had decreased to 7% by the end of the 18-hour uptake period. At this time, the total tissue nitrate levels were similar for both pretreatments. The rate of accumulation of [(15)N]nitrate in the tissue was relatively constant for both pretreatments, but was 25% less in HN roots. Of the previously accumulated [(14)N]nitrate, 52 and 46% remained after 18 hours in the LN and HN roots, respectively. The [(14)N]nitrate decline for HN roots was initially more rapid than in the LN roots which was linear over time. Xylem transport and efflux more than accounted for the decline in [(14)N]nitrate of LN roots and all but 4% in the HN roots which was attributed to reduction. Compartmentation of the previously accumulated nitrate was evident from the higher atom per cent (15)N of xylem nitrate compared to that of the tissue nitrate of both LN and HN roots. During the first 2 hours, xylem transport of [(14)N]nitrate by the HN roots was 49% greater than for LN roots, while [(15)N]nitrate transport was 9% less in HN roots compared to LN roots. Even though the reduction of [(15)N]nitrate in HN roots was 31% less than LN roots during the first 2 hours, [(15)N]nitrate was reduced more rapidly than the previously accumulated [(14)N]nitrate. After the first 4 hours, the relative partitioning of absorbed [(15)N]nitrate between accumulation, reduction, and translocation was similar regardless of pretreatment.

An experiment was conducted to investigate the relative changes in NO(3) (-) assimilatory processes which occurred in response to decreasing carbohydrate availability. Young tobacco plants (Nicotiana tabacum [L.], cv NC 2326) growing in solution culture were exposed to 1.0 millimolar (15)NO(3) (-) for 6 hour intervals during a normal 12 hour light period and a subsequent period of darkness lasting 42 hours. Uptake of (15)NO(3) (-) decreased to 71 to 83% of the uptake rate in the light during the initial 18 hours of darkness; uptake then decreased sharply over the next 12 hours of darkness to 11 to 17% of the light rate, coincident with depletion of tissue carbohydrate reserves and a marked decline in root respiration. Changes also occurred in endogenous (15)NO(3) (-) assimilation processes, which were distinctly different than those in (15)NO(3) (-) uptake. During the extended dark period, translocation of absorbed (15)N out of the root to the shoot varied rhythmically. The adjustments were independent of (15)NO(3) (-) uptake rate and carbohydrate status, but were reciprocally related to rhythmic adjustments in stomatal resistance and, presumably, water movement through the root system. Whole plant reduction of (15)NO(3) (-) always was limited more than uptake. The assimilation of (15)N into insoluble reduced-N in roots remained a constant proportion of uptake throughout, while assimilation in the shoot declined markedly in the first 18 hours of darkness before stabilizing at a low level. The plants clearly retained a capacity for (15)NO(3) (-) reduction and synthesis of insoluble reduced-(15)N even when (15)NO(3) (-) uptake was severely restricted and minimal carbohydrate reserves remained in the tissue.

An experiment was conducted to investigate the reduction of endogenous NO(3) (-), which had been taken up by plants in darkness, during the course of the subsequent light period. Vegetative, nonnodulated soybean plants (Glycine max [L]. Merrill, ;Ransom') were exposed to 1.0 millimolar (15)NO(3) (-) for 12 hours in darkness and then returned to a solution containing 1.0 millimolar (14)NO(3) (-) for the 12 hours ;chase' period in the light. Another set of plants was exposed to (15)NO(3) (-) during the light period to allow a direct comparison of contributions of substrate from the endogenous and exogenous sources. At the end of the (15)NO(3) (-) exposure in the dark, 70% of the absorbed (15)NO(3) (-) remained unreduced, and 83% of this unreduced NO(3) (-) was retained in roots. The pool of endogenous (15)NO(3) (-) in roots was depleted at a steady rate during the initial 9 hours of light and was utilized almost exclusively in the formation of insoluble reduced-N in leaves. Unlabeled endogenous NO(3) (-), which had accumulated in the root prior to the previous dark period, also was depleted in the light. When exogenous (15)NO(3) (-) was supplied during the light period, the rate of assimilation progressively increased, reflecting an increased rate of uptake and decreased accumulation of NO(3) (-) in the root tissue. The dark-absorbed endogenous NO(3) (-) in the root was the primary source of substrate for whole-plant NO(3) (-) reduction in the first 6 hours of the light period, and exogenous NO(3) (-) was the primary source of substrate thereafter. It is concluded that retention of NO(3) (-) in roots in darkness and its release in the following light period is an important whole-plant regulatory mechanism which serves to coordinate delivery of substrate with the maximal potential for NO(3) (-) assimilation in photosynthetic tissues.

From 10 to 60% of the nitrate present in plant tissue extracts and stem exudates of corn (Zea mays L.) was found to be reduced during Kjeldahl digestion, even in the absence of added reducing agents. This reduction is of particular concern in [(15)N]nitrate assimilation studies, because it results in an overestimate of nitrate reduction. To overcome this problem, a method was developed for removing nitrate prior to Kjeldahl digestion, thereby preventing nitrate reduction. The procedure utilizes hydrogen peroxide for partial oxidation of organic matter in order to minimize the nitration of organic compounds. The free nitrates are then volatilized as nitric acid from concentrated sulfuric acid at 95 degrees C. When the proposed method was used as a pretreatment to Kjeldahl digestion, less than 0.5% of the applied nitrate was recovered in the reduced nitrogen fraction of plant tissue extracts and stem exudates.

Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in vitro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar NO(3) (-) for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO(3) (-) likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO(3) (-), the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO(3) (-) occurs during uptake and reduction of exogenous NO(3) (-). Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO(3) (-). It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO(3) (-), (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO(3) (-) is taken up by cells of the cortex and stele, and/or (c) NO(3) (-) is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO(3) (-) reduction.