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Crop Science 41:1130-1136 (2001)
© 2001 Crop Science Society of America

CROP PHYSIOLOGY & METABOLISM

Inhibition of Net Nitrate Uptake by Ammonium in Pima and Acala Cotton Roots

Muhammad Aslam, Robert L. Travis* and Donald W. Rains

Dep. of Agronomy and Range Science, Univ. of California, One Shields Avenue, Davis, CA 95616-8515

* Corresponding author (rltravis{at}ucdavis.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
This study was conducted to determine whether the inhibition of net NO-3 uptake by NH+4 in cotton roots is due to inhibition of NO-3 influx per se, and/or enhancement of efflux. Two cotton species, Pima (Gossypium barbadense L.) and Acala (G. hirsutum L.), which differ in NO-3 influx and efflux, were used. Seedlings were grown hydroponically for 10 to 11 d and then induced for NO-3 uptake with 0.01, 0.10, or 1.0 mM NO-3. Net uptake was determined by following NO-3 depletion from uptake solutions, containing 0.1 mM NO-3, in the presence or absence of 10 mM NH+4. Roots induced with 0.01 mM NO-3 contained only 5 to 6 µmol NO-3 g-1 fresh weight (FW) after the induction treatment. In these roots, net NO-3 uptake was equivalent to influx, and was not inhibited by NH+4. Roots induced with 0.1 mM NO-3 contained 36 to 38 µmol NO-3 g-1 FW, and NH+4 inhibited net NO-3 uptake albeit after a lag of {approx}12 min. In contrast, roots induced with 1.0 mM NO-3 contained 65 µmol NO-3 g-1 FW, and NH+4 immediately inhibited net uptake. Exposure to NH+4 increased NO-3 efflux by both species. At similar root NO-3 concentrations, NH+4 inhibited net NO-3 uptake and stimulated efflux more in Pima than in Acala cotton. These results indicate that the response of both cotton species to NH+4 is dependent upon root NO-3 concentration, and supports the argument that NH+4 has little effect on NO-3 influx, but inhibits net uptake by stimulating efflux.

Abbreviations: FW, fresh weight • HPLC, high-performance liquid chromatography


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
EXPOSURE OF PLANT ROOTS to NH+4 inhibits net NO-3 uptake (Aslam et al., 1994; Kronzucker et al., 1999). Inhibition of net NO-3 uptake by NH+4 may be rapid or slow. The slow effect of NH+4 and/or its assimilation products may be due to the inhibition of induction of NO-3 transporter(s) at the transcriptional and/or post transcriptional level (Glass and Siddiqi, 1995; Aslam et al., 1996b; Krapp et al., 1998; Zhuo et al., 1999; Vidmar et al., 2000b). However, rapid inhibition is assumed to result from the direct effect of NH+4 on the plasma membrane (Ullrich et al., 1984; Ayling, 1993). Since net NO-3 uptake is the difference between influx and efflux across the plasma membrane (Morgan et al., 1973; Jackson et al., 1976), the rapid inhibition of net uptake by NH+4 may be a result of either inhibition of influx and/or the enhancement of efflux.

Early investigators, using 15NO-3 or 36ClO-3, concluded that in a number of plant species the rapid inhibition of net NO-3 uptake by NH+4 is due to enhancement of NO-3 efflux (Jackson et al., 1976; Doddema and Telkamp, 1979; Deane-Drummond and Glass, 1983; Deane-Drummond, 1985). Later studies, in which 13NO-3 was the nitrate source, concluded that NH+4 inhibited influx, but had no effect on efflux (Glass et al., 1985; Ingemarsson et al., 1987; Oscarson et al., 1987; Lee and Drew, 1989; Ayling, 1993). If the former scenario were correct, then NH+4 should not inhibit net uptake by roots containing low NO-3 because efflux would be negligible (Aslam et al., 1994). However, inhibition would occur as the NO-3 concentration increased. We tested this hypothesis by comparing the effect of NH+4 on net NO-3 uptake by barley (Hordeum vulgare L.) roots containing variable NO-3 concentrations (Aslam et al., 1994). In that study, NH+4 had little effect in low-NO-3 roots, but inhibited uptake by roots containing more NO-3. Similarly, NH+4 had no effect on net NO-3 uptake by uninduced roots or roots in which the NO-3 transport system was induced with NO-2 (Aslam et al., 1994). Under these conditions, efflux was negligible and net uptake was equivalent to influx (Aslam et al., 1994; Kronzucker et al., 1999). These results supported the earlier literature, which suggested that NH+4 had no effect on NO-3 influx, but inhibited net uptake by stimulating efflux. Kronzucker et al. (1999) recently revitalized this controversy by reporting that NH+4 both inhibited 13NO-3 influx and enhanced efflux in barley roots. The increase in efflux was greater in uninduced (86%) and NO-2-induced (62%) plants, compared with plants induced with NO-3 (47%). Nevertheless, they concluded that inhibition of influx was the dominant factor in decreasing net NO-3 uptake.

We have reexamined this issue in our analysis of nitrogen metabolism in Pima and Acala cotton. These species were selected because of their inherent differences in NO-3 influx and efflux rates (Aslam et al., 1997). Our results show that, in both species, short-term exposure to NH+4 had no effect on NO-3 influx, but inhibited its net uptake by stimulating efflux.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Seedling Growth
Acid-delinted seeds of Pima (cv. S-7) and Acala (cv. Maxxa) cotton were germinated in the dark at room temperature as previously described (Aslam et al., 1997). After 6 d, the seedlings were transferred to a stainless steel screen (mesh size 5 mm x 5 mm) suspended {approx}5 cm above the surface of 5 L aerated N-free, one-quarter-strength Hoagland solution (Hoagland and Arnon, 1950) contained in a plastic beaker. To avoid diurnal fluctuations in NO-3 uptake due to light/ dark transitions and temperature (Aslam et al., 2001), the seedlings were transferred into a growth chamber (Western Environmental, Napa, CA) set for continuous light and 28°C with 60 to 65% relative humidity. Light was supplied by metal halide and incandescent lamps. The photosynthetic photon flux density, measured at the top of plant canopy with a LI-COR quantum sensor (Lincoln, NE), was 600 µmol m-2 s-1. After 1 d, the seedlings were transplanted into 70 L plastic pots containing aerated one-quarter-strength Hoagland solution and 0.01, 0.02, 0.1, or 1.0 mM KNO3 or 0.1 mM KNO2, for 3 to 4 d. The solutions were analyzed for NO-3 or NO-2 using high-performance liquid chromatography (HPLC) (Thayer and Huffaker, 1980). Desired NO-3 or NO-2 concentrations in the nutrient solutions were maintained by daily addition of appropriate volumes of 1.0 M KNO3 or KNO2, respectively. Solution pH was maintained at 6.0 ± 0.1 by adding H2SO4 as needed. The seedlings were grown for a total of 10 to 11 d.

Measurement of Net NO-3 Uptake
Net uptake by intact seedlings was determined by following NO-3 depletion from the uptake solution. Measurements were made in a miniature controlled environment chamber as described before (Aslam et al., 1997). The environmental conditions of that chamber were the same as described above for the plant growth chamber. The miniature chamber was part of a fully automated system fabricated by Goyal and Huffaker (1986). Plants were maintained in nutrient solutions containing the same concentrations of NO-3 in which they were grown. One hour before measurement of NO-3 uptake rates, 12 to 13 seedlings (with roots weighing {approx}4 g FW) were placed in a flat bottom Pyrex glass tube (35 mm diam and 180 mm height) with 100 mL of aerated uptake solution containing 2.0 mM MES [(n-morpholino)ethanesulfonic acid] (pH 6.0), 0.2 mM CaSO4, 0.1 mM NO-3 and 0, 1 or 10 mM NH+4 as indicated in the figure and table legends. Ammonium was supplied as (NH4)2SO4. During this period NO-3 concentration in the uptake solutions was maintained by adding KNO3. In one experiment the seedlings were placed in nutrient solution containing 0.02 mM KNO3 for 6 h to deplete the metabolic NO-3 pool. Uptake solutions were aerated throughout the experiments to ensure thorough mixing. The samples (0.5 mL) were withdrawn by the automated sampling system at 1.5-min intervals for 30 min for NO-3 determination. Cumulative uptake was determined from the NO-3 depletion curves as described by Goyal and Huffaker (1986). Net uptake rates were then calculated by linear regression analysis of the cumulative uptake curves.

Measurement of NO-3 Efflux
Nitrate efflux was determined by following the release of the absorbed NO-3 from the intact roots in an efflux solution that contained 2.0 mM MES (pH 6.0), 0.5 mM CaSO4, 1.0 mM KNO2, and 0 or 10 mM NH+4, as previously described (Aslam et al., 1994). The experimental procedure for the measurement of efflux was the same as described above for the measurement of net NO-3 uptake. A set of 15 seedlings was rinsed for 10 s in N-free uptake solution and then transferred into the efflux solution. Continuous sampling (0.5-mL aliquots at 1.5-min intervals) was initiated immediately after the seedlings were transferred to the efflux solution. Accumulation of NO-3 in efflux solution was followed for 30 min. Cumulative efflux was calculated from NO-3 concentrations and solution volume data, as described by Goyal and Huffaker (1986). Efflux rates were calculated by linear regression analysis of the cumulative efflux data, as described by Aslam et al. (1996a).

NO-3 and NO-2Determination
Nitrate was extracted from 2 to 3 g FW of roots by homogenizing the tissue in a chilled mortar containing 4 mL distilled, deionized H2O per g of tissue and a small amount of acid-washed sand. The extracts were centrifuged at 30000 x g at 4°C for 15 min, and the supernatants were used to determine NO-3 concentrations. Nitrate concentrations of uptake solutions and tissue extracts were determined spectrophotometrically by measuring A210 after separation by HPLC on a partisil-10 SAX (Phenomenex, Torrance, CA) anion-exchange column (Thayer and Huffaker, 1980). The NO-2 concentration of the uptake solutions was also determined by measuring A210 after separation by HPLC (Thayer and Huffaker, 1980).

Data Analysis
The experiments were repeated three to four times with two to three replications. Although the absolute values for flux rates varied from experiment to experiment because of differential NO-3 accumulation and other biological variability, the trends of the treatment effects were similar. Therefore, the results of representative experiments are presented in the figures. Standard errors of the means were calculated for the data presented in tables. All results are reported on root FW basis.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Effect of NH+4 on Net NO-3 Uptake
Influence of Root NO-3 Concentrations
Figure 1 (A,B,C) shows the effect of NH+4 on cumulative NO-3 uptake across a 30-min period by Pima cotton roots varying in internal NO-3 concentrations. Roots of seedlings grown in 0.01 mM NO-3 contained relatively low NO-3 concentration (5.3 µmol g-1 FW) (Table 1), and exposure to 10 mM NH+4 had no effect on net NO-3 uptake (Fig. 1A). Uptake rates were similar whether or not NH+4 was present in the uptake solutions (Table 2). Seedlings grown in 0.1 mM NO-3 contained moderate NO-3 concentration (37.8 µmol g-1 FW) (Table 1). NH+4 inhibited net NO-3 uptake, but only after a 12-min lag period (Fig. 1B). The uptake rate then decreased by 45% between 18 and 30 min when exposed to NH+4 (Table 2). On the other hand, when seedlings were grown in 1.0 mM NO-3 and root NO-3 concentration was high (65.4 µmol g-1 FW), NO-3 uptake was immediately inhibited (Fig. 1C) with 25% of the inhibition occurring during the initial 12-min exposure (Table 2). Inhibition increased to 47% with longer exposure to NH+4 (Table 2). Inhibition of net uptake was similar in roots of seedlings grown in 0.1 or 1.0 mM NO-3 during the 18- to 30-min treatment period (Table 2). Exposure to NH+4 also had no effect on net NO-3 uptake by roots of Acala cotton seedlings grown in 0.01 mM NO-3 (Fig. 1D). While exposure to NH+4 inhibited NO-3 uptake by roots of Acala cotton grown in 0.1 or 1.0 mM NO-3 (Fig. 1E,F), the inhibition was less severe than that in Pima (Fig. 1, Table 2), even though root NO-3 concentrations were similar in both species (Table 1). Exposure to NH+4 also had no effect on net NO-3 uptake by Pima roots induced with NO-2 (Fig. 2). These results indicate that the response to NH+4 is dependent upon root NO-3 concentration.



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  		Fig. 1. Cumulative net NO-3 uptake by roots of Pima (left panel) and Acala (right panel) cotton grown in 0.01 (A, D), 0.10 (B, E) or 1.00 (C, F) mM NO-3 for 3 d, and then exposed to uptake solutions. Cumulative Net NO-3 uptake was measured by following the depletion of NO-3 from uptake solutions initially containing 0.1 mM NO-3 ± 1 or 10 mM NH+4. See Table 1 for root NO-3 concentrations.

 

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  		Table 1. NO-3 concentrations in roots of Pima and Acala Cotton grown in 0.01, 0.10, or 1.0 mM NO-3 for 3 d. FW = fresh weight.

 

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  		Table 2. Net NO-3 uptake rates by roots of Pima and Acala cotton grown in 0.01, 0.01, or 1.00 mM NO-3 for 3 d and then placed in uptake solutions. Net NO-3 uptake rates were measured by following the depletion of NO-3 from the uptake solutions initially containing 0.1 mM NO-3 ± 10 mM NH+4. See Fig. 1 for the time-course kinetics and Table 1 for root NO-3 concentrations. FW = fresh weight.

 


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  		Fig. 2. Net NO-3 uptake by roots of Pima cotton grown in 0.10 mM NO-2 for 2 d, then exposed to uptake solutions containing 0.1 mM NO-3 ± 10 mM NH+4. Uptake rates were determined after 0, 15, and 60 min treatment with NH+4. Root NO-3 concentration was 1.8 ± 0.3 µmol g-1 FW. Vertical lines above the bars represent the respective +SE of the means of four replicates.

 
Reversal of NH+4 Inhibition
Figure 3 shows the effect of NH+4 on net NO-3 uptake rates by roots grown in different NO-3 concentrations across a longer period. NH+4 had no effect on net uptake by roots grown in 0.01 mM NO-3 (4.7 µmol NO-3 g-1 root FW), even when the exposure period was 5 h. When roots were grown in 0.02 mM NO-3 (13.8 µmol NO-3 g-1 root FW), net uptake was inhibited after 15 min of exposure to NH+4; however, this inhibition gradually diminished within 2 h (Fig. 3). In contrast, when roots were grown in 0.10 or 1.0 mM NO-3 (35.5 and 67.8 µmol g-1 root FW, respectively) NH+4 inhibited net NO-3 uptake for a longer period (Fig. 3).



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  		Fig. 3. Net NO-3 uptake rates (expressed as % of the control) by roots of Pima cotton seedlings grown in 0.01, 0.02, 0.10, or 1.0 mM NO-3 for 3 d, then exposed to uptake solutions. Net NO-3 uptake was measured in the presence of 0.1 mM NO-3 ± 10 mM NH+4. Tissue NO-3 concentrations for roots grown in 0.01, 0.02, 0.10, and 1.0 mM NO-3 were 4.7, 13.8, 35.5, and 67.8 µmol g-1 FW, respectively. Vertical lines through data points reflect ± SE of means of three replicates. Net NO-3 uptake rates at 100% varied from 4 to 5 µmol g-1 FW h-1.

 
Effect of Pretreatment with Low NO-3
Figure 4 shows the effect of NH+4 on net NO-3 uptake by Pima roots grown in 0.1 mM NO-3 (control) for 4 d, then pretreated with 0.02 mM NO-3 for 6 h. Nitrate concentrations of control and pretreated roots were 52.6 ± 1.5 and 47.2 ± 1.5 µmol g-1 FW, respectively. Net NO-3 uptake by control roots was inhibited by {approx}55% between 18 to 30 min after exposure to NH+4 (Fig. 4A, Table 3). In contrast, inhibition of NO-3 uptake by pretreated roots did not occur until after 20 min of exposure to NH+4, and was only 17% (Fig. 4B, Table 3). Likewise, NH+4 had little effect on net NO-3 uptake by similarly pretreated Acala roots (2000, unpublished data).



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  		Fig. 4. Cumulative net NO-3 uptake by roots of Pima cotton grown in 0.1 mM NO-3 for 4 d and pretreated with 0.02 mM NO-3 for 6 h. Control seedlings were left in 0.1 mM NO-3 (A), while pretreated seedlings (B) were placed in 0.02 mM NO-3 for 6 h. Net NO-3 uptake was measured in the presence of 0.1 mM NO-3 ± 10 mM NH+4. Initial root NO-3 concentrations were 52.6±1.5 (control) and 47.2 ± 1.5 (pretreated) µmol g-1 FW. Results are from a representative experiment.

 

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  		Table 3. Net NO-3 uptake rates by roots of Pima cotton grown in 0.1 mM NO-3 for 4 d and pretreated with 0.02 mM NO-3 for 6 h. One set of seedlings was left in 0.1 mM NO-3 (control) and the other set was placed in 0.02 mM NO-3 for 6 h (pretreated). Net NO-3 uptake was measured in the presence of 0.1 mM NO-3 ± 10 mM NH+4. Root NO-3 concentrations were 52.6 ± 1.5 (control) and 47.2 ± 1.5 (pretreated) µmol g-1 fresh weight (FW). See Figure 4 for the time-course kinetics.

 
Comparative Effect of NH+4 on NO-3 Efflux and Net Uptake
Figure 5 shows the comparative effect of NH+4 on net NO-3 uptake and efflux from roots of Pima and Acala cotton. After an initial lag of {approx}12 min, NH+4 inhibited net uptake in both species, while efflux was enhanced in the presence of NH+4. The time courses of the two responses indicate that stimulation of efflux occurred about the same time that net uptake was inhibited. During the initial 12 min of exposure to NH+4, there was little effect on net uptake or efflux rates (Table 4). Between 18 to 30 min, net uptake was inhibited in Pima and Acala by 35 and 20%, respectively, while efflux was stimulated by 49 and 28% (Table 4).



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  		Fig. 5. Cumulative net NO-3 uptake and efflux from roots of Pima (A) and Acala (B) cotton grown in 0.1 mM NO-3 for 3 d, then exposed to the uptake or efflux solutions. Net NO-3 uptake and efflux were measured in the presence or absence of 10 mM NH+4. Root tissue NO-3 concentrations for Pima and Acala were, respectively, 44.8 ± 2.3 and 48.9 ± 2.7 µmol g-1 FW.

 

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  		Table 4. Net NO-3 uptake and efflux rates by roots of Pima and Acala cotton grown in 0.1 mM NO-3 for 3 d. Net NO-3 uptake and efflux were measured in the presence or absence of 10 mM NH+4. NO-3 concentrations in Pima and Acala roots were 44.8 ± 2.3 and 48.9 ± 2.7 µmol g-1 fresh weight (FW), respectively. See Figure 5 for the time-course kinetics.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
In an earlier communication, we reported that Pima and Acala cotton roots differ considerably in NO-3 influx and efflux rates (Aslam et al., 1997). In this report, we examine the effect of NH+4 on these processes. Since net NO-3 uptake is modulated by influx and efflux (Morgan et al., 1973; Jackson et al., 1976), of particular interest was whether NH+4 inhibits net NO-3 uptake by decreasing influx and/or stimulating efflux.

We previously reported that NH+4 did not inhibit net NO-3 uptake or stimulate efflux from barley roots unless their internal NO-3 concentration was above a certain threshold (Aslam et al., 1994). Likewise, in this study, 10 mM NH+4 did not inhibit net NO-3 uptake by cotton roots (Fig. 1A,D) when the internal NO-3 concentration was 5 to 6 µmol g-1 FW (Table 1). In fact, exposure to even 50 mM NH+4 did not reduce net uptake in these low NO-3 roots (2000, unpublished data). Similarly, when the seedlings were pretreated with NO-2, and root NO-3 concentration was only 1.8 ± 0.3 µmol g-1 FW, NH+4 had little effect on net uptake (Fig. 2). If NH+4 inhibited net uptake by reducing NO-3 influx, then that process should have been inhibited under these conditions. Addition of NH+4 did inhibit net uptake by roots grown in 0.1 mM NO-3, but only after a lag of {approx}12 min (Fig. 1B,E). On the other hand, when the seedlings were grown in 1.0 mM NO-3, and root NO-3 concentration was high (Table 1), net uptake was inhibited immediately in Pima roots (Fig. 1C). At similar internal NO-3 concentrations, the inhibition of net uptake was greater in Pima than in Acala cotton (Fig. 1). The time courses of net NO-3 uptake and efflux indicate that when net uptake was inhibited, efflux was stimulated (Fig. 5). These results suggest that inhibition of net uptake by NH+4 may be due to stimulation of efflux when sufficient NO-3 is available.

Experiments in which roots containing low NO-3 concentration recovered from the NH+4 treatment provide further evidence that NH+4 stimulates efflux rather than inhibiting influx (Fig. 3). If NH+4 inhibited net NO-3 uptake by preventing influx, then there should be no reversal of the effect upon prolonged exposure to NH+4, irrespective of root NO-3 concentration. However, that was not the case in our experiments. When roots grown in 0.02 mM NO-3 (13.8 µmol NO-3 g-1 FW) were exposed to NH+4, net uptake was inhibited, but that inhibition was relieved after {approx}2 h (Fig. 3). This varied response to NH+4 can only be explained in the context of initial stimulation of efflux by NH+4. When roots were grown in 0.01 mM NO-3 and their internal NO-3 content was only 4.7 µmol g-1 FW, presumably there was insufficient NO-3 to support efflux, hence there was no effect on net uptake. Conversely, when the roots contained 13.8 µmol NO-3 g-1 FW, enough was present to stimulate efflux after 15 min of exposure to NH+4. This effect was transient, however, presumably because efflux was not stimulated when root NO-3 concentration decreased. In contrast, because of the abundance of NO-3, efflux continued for a longer period from roots grown in 0.1 or 1.0 mM NO-3, and recovery of net uptake did not occur (Fig. 3).

The delayed time-dependent effect of NH+4 on net uptake and/or efflux (Fig. 5) suggests that the initial effect was on the cytoplasmic NO-3 (metabolic) pool. Apparently, after {approx}12 min of exposure, the storage pool (vacuole) was also affected. Consequently, net NO-3 uptake was inhibited, and efflux was stimulated by NH+4 (Table 4). That the metabolic pool is initially affected by NH+4 is evident from the results presented in Fig. 4. When the roots were pretreated with 0.02 mM NO-3 for 6 h, the response to NH+4 was both delayed and reduced (Table 3). Presumably, pretreatment with 0.02 mM NO-3 decreased the concentration of NO-3 in the metabolic pool, even though total root NO-3 decreased only 10%. In this regard, the response to NH+4 in cotton differs from that in barley where the inhibition of net NO-3 uptake and/or stimulation of efflux was immediate even in roots grown in 0.1 mM NO-3 and containing only 25 to 30 µmol NO-3 g-1 FW (Aslam et al., 1994).

These results contradict earlier 13NO-3 studies which concluded that NH+4 inhibited influx but had no effect on efflux (Glass et al., 1985; Ingemarsson et al., 1987; Oscarson et al., 1987; Lee and Drew, 1989; Ayling, 1993). Recently, Kronzucker et al. (1999) reported that NH+4 inhibited 13NO-3 influx and stimulated efflux in barley roots. Nevertheless, the NH+4 effect on influx was still considered the dominant factor in the inhibition of net uptake. They also observed that NH+4 inhibited both influx and net uptake of NO-3, and stimulated efflux in NO-2-induced roots; however, inhibition of net uptake and influx was about one-third of that by NO-3-induced roots (Kronzucker et al. 1999). In our studies, NH+4 had little effect on net NO-3 uptake by NO-2-induced roots of either cotton (Fig. 2) or barley (Aslam et al., 1994). It is not known as to why this discrepancy occurred. If NH+4 inhibits NO-3 influx, then the percent inhibition should be about the same in both NO-3- and NO-2-induced roots. Although Kronzucker et al. (1999) did not provide data on root NO-3 concentration for NO-2-induced roots, the cytoplasmic NO-3 concentration, as determined by 13NO-3 exchange, was even higher in roots induced with 0.1 mM NO-2 than in roots induced with 0.1 mM NO3. Presumably enough NO-3 was present in NO-2-induced roots to allow NH+4 to stimulate efflux. Kronzucker et al. (1999) argued that NO-2 be an appropriate analog for NO-3, and that the transport system induced by NO-2 may be different than that induced by NO-3. Vidmar et al. (2000a) recently reported, however, that induction with NO-2 increased the HvNRT2 transcript (that which encodes for high-affinity NO-3 transporter) level. This suggests that both ions induce the same transport system. Induction with NO-2 also increased the transcript level for the putative high affinity NO-3 transporter in cotton (F. Fritschi, R.L. Travis, D.W. Rains, 2000, unpublished data).

When 13NO-3 is used to measure NO-3 influx, it is assumed that there is no simultaneous efflux of 13NO-3. However, NO-3 influx measured by using radioactive tracer is underestimated, because of a concurrent efflux of the tracer (Siddiqi et al., 1989). Lee and Drew (1986) reported that NO-3 influx, as measured by 13NO-3 across a period of 15 min from 0.15 mM NO-3 solution, would be underestimated by as much as 26 to 29% because of this efflux. Siddiqi et al. (1989) pointed out that the magnitude of this error depends on the rate of increase of cytoplasmic specific activity of 13NO-3 and the rate of efflux. Previously, we (Aslam et al., 1994) argued that perhaps the presence of NH+4 stimulates such an efflux. This would account for the observed stimulation of NO-3 efflux reported by Kronzucker et al. (1999) in uninduced and NO-2-induced barley roots. Since the measurement of 13NO-3 influx is based on the net accumulation of the tracer, any concurrent efflux of 13NO-3 would be reflected by decreased NO-3 influx.

In interpreting the results presented here, one must consider whether the depolarizing effect of NH+4 on the plasma membrane might explain the inhibition of NO-3 uptake by NH+4. Nitrate transport across the plasma membrane of root cells occurs via the NO-3/H+ symport mechanism (Ullrich and Novacky, 1981; Ullrich, 1987; McClure et al., 1990). Exposure of roots to NH+4 causes transient depolarization of membrane potential which, through its effect on proton motive force (Ullrich et al., 1984; Ayling, 1993), might inhibit the NO-3/H+ cotransport system. However, K+, which also transiently depolarizes the plasma membrane (Newman et al., 1987), did not inhibit NO-3 uptake (Glass and Siddiqi, 1995; Wang et al., 1996). Thus, the decrease in electrical potential does not seem to be a reasonable explanation for the rapid inhibition of NO-3 influx by NH+4 (Crawford and Glass, 1998). Furthermore, any changes in the proton motive force due to membrane depolarization would also be expected to inhibit the absorption of other anions facilitated by proton transport (Lee and Drew, 1989). In this respect, the results have been variable. While Lee and Drew (1989) observed no consistent decrease in phosphate influx on exposure of barley roots to NH+4, Ayling (1993) reported a moderate inhibition. In contrast, Ullrich et al. (1984) and Ayling (1993) reported inhibition of phosphate influx similar to that of NO-3 in fronds of Lemna gibba L. and tomato [Lycopersicon esculeutum Mill.] roots, respectively. The differing responses of phosphate uptake to NH+4, when considered with the dependence of the NH+4 response on root NO-3 concentration, and the reversal of inhibition by NH+4 in low NO-3 roots, provide further evidence that NH+4 may have a more specific effect on net uptake. That effect is likely on efflux rather than influx.


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Our results show that the inhibitory effect of NH+4 on net NO-3 uptake by cotton is exerted predominantly through the stimulation of efflux with little or no effect on influx. In both species, the response to NH+4 was dependent upon root NO-3 concentration. When root NO-3 concentration was very low, NH+4 had no effect on net uptake (influx), even up to 5 h. When roots contained low to medium NO-3, however, net uptake was inhibited after {approx}12 min of exposure to NH+4. In contrast, net uptake by roots containing high NO-3 was inhibited immediately upon exposure to NH+4. Inhibition of net uptake by NH+4 in low NO-3 roots was alleviated after {approx}2 h, whereas inhibition continued for longer duration in medium to high NO-3 roots. The inhibition of net uptake by NH+4 was dependent upon NO-3 concentration in the metabolic pool. Exposure to NH+4 increased NO-3 efflux from both species. At similar root NO-3 concentrations, NH+4 inhibited net uptake and stimulated efflux more in Pima than in Acala cotton. Since both NO-3 influx and efflux are energy consuming processes (Aslam et al., 1996c; Glass et al., 1990), the stimulation of efflux by NH+4 places an extra burden on the plant in terms of energy cost, and may decrease NO-3 use efficiency.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
This research was supported in part by grants from Cotton Incorporated and the California Crop Improvement Association.

Received for publication September 19, 2000.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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Crop Science 2001 41: 991. [Full Text]  




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