Ammonium Uptake by Rice Roots - NCBI

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Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 124 ... 13NH4+ influx into roots of intact rice plants (Wang et al.,.
Plant Physiol. (1993) 103: 1259-1267

Ammonium Uptake by Rice Roots' II. Kinetics of 13NH4+lnflux across the Plasmalemma Miao Yuan Wang, M. Yaeesh Siddiqi, Thomas J. Ruth, and Anthony D. M. Class" Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 124 (M.Y.W., M.Y.S., A.D.M.G.); and Tri-University Meson Facility, University of British Columbia Campus, Vancouver, British Columbia, Canada V6T 2A3 (T.J.R.) operating at low [NH4+], and a linear diffusive component at elevated [NH4+],(Fried et al., 1965; Ullrich et al., 1984). In N-starved Lemna both NH4+uptake by the saturable system and depolarization of plasmalemma potential were found to exhibit the same concentration dependence ( K , values for both processes were 17 PM).At higher [NH,'], the uptake by the linear system was not accompanied by further depolarization of membrane potential (Ullrich et al., 1984). The saturable component of NH4+uptake was sensitive to some metabolic inhibitors (Sasakawa and Yamamoto, 1978) and to changes of root temperature (Bloom and Chapin, 1981). In addition, NH4+uptake is subject to negative feedback, supposedly from N metabolites (Lee and Rudge, 1986; Morgan and Jackson, 1988; Clarkson and Lüttge, 1991). Youngdahl et al. (1982) demonstrated that NH4+uptake in rice decreased with plant age. However, despite these studies, the mechanism(s) of NH4+ uptake by roots of higher plants remain unclear. In particular, the high-concentration system represents virtually unexplored temtory. Ammonium is unique among inorganic cations because following absorption by plant roots, it is rapidly assimilated into organic pools. This has made the analysis of uptake and the subsequent fate of absorbed NH4+much more complicated than for cations such as K+ or ca'+. The availability of 13N to this laboratory has enabled us to measure short-term 13NH4+influx into roots of intact rice plants (Wang et al., 1991, 1993). This is critically important for two main reasons. First, this technique allows determination of the particular flux (in this case unidirectional plasmalemma influx) that is responding to the imposed conditions. By contrast, net uptake measurement, often obtained by means of long-term deple-

Short-term influxes of 13NH4+ were measured in intact roots of 3-week-old rice (Oryza safiva 1. cv M202) seedlings that were hydroponically grown at 2, 100, or 1000 HM NH4+.Below 1 m M external concentration ([NH4+],), influx was saturable and due to a high-affinity transport system (HATS). For the HATS, VmaX values were negatively correlated and K,,, values were positively correlated with NH4+provision during growth and root [NH4+]. Between 1 and 40 mM [NH4+],, 13NH,+ influx showed a linear response due to a low-affinity transport system (LATS). l h e 13NH4+influxes by the HATS, and to a lesser extent the LATS, are energy-dependent processes. Selected metabolic inhibitors reduced influx of the HATS by 50 to 80%, but of the LATS by only 31 to 51%. Estimated values for Qlo (the ratio of rates at temperatures differing by 1O'C) for HATS were greater than 2.4 at root temperatures from 5 to 10°C and were constant at approximately 1.5 between 5 and 30'C for the LATS. lnflux of 13NH4+ by the HATS was insensitive to external p H in the range from 4.5 to 9.0, but influx by the LATS declined significantly beyond p H 6.0. l h e data presented are discussed in the context of the kinetics, energy dependence, and the regulation of ammonium influx.

Despite the potential benefits of nitrate for the growth of rice (Oryza sativa L.) plants, especially under anaerobic conditions (Malavolta, 1954; Bertani et al., 1986), ammonium is the predominant and most readily bioavailable N form in paddy soil (Yu, 1985). It is the prefened N species taken up by rice (Fried et al., 1965; Sasakawa and Yamomoto, 1978), and in terms of the efficiency of fertilizer utilization, ammonium is superior to nitrate in paddy soil (Craswell and Vlek, 1979). Ammonium uptake systems have been well defined as concentrative, energy-dependent, and camer-mediated in algae (Smith and Walker, 1978), fungi (Kleiner, 1981), bacteria (Kleiner, 1985), and cyanobacteria (Boussiba and Gibson, 1991). However, compared with the extensive investigations of NOS- uptake, the kinetics and energetics of ammonium transport in higher plants have received relatively little attention. In both rice plants and Lemna, NH4+uptake followed a biphasic pattem, with a saturable camer-mediated system

Abbreviations: CCCP, carbonylcyanide m-chlorophenyl-hydrazone; CN-, (sodium) cyanide; DES, diethylstilbestrol; DMRT, Duncan's multiple range test; DNP, 2,4-dinitrophenol; G2, G100, and G1000, rice seedlings grown in MJNS containing 2, 100, or 1000 PM N&*, respectively; HATS or LATS, high-affinity or low-affinity transport systems, respectively; K, the extemal ion concentration giving half of the maximum rate (PM); MJNS, modified Johnson's nutrient solution; [NHI+], and [NH4+]i,extemal and root (intemal) ammonium concentration, respectively; pCMBS, p-chloromercuribenzene-sulfonate; (210, ratio of rates at temperatures differing by 10°C; RGR, relative growth rate; SHAM, salicylhydroxamic acid; V, the calculated maximum rate of ion influx (&mo1g-I fresh weight h-').

The authors wish to acknowledge continuing financia1 support for this research from the Potash and Phosphate Institute of Canada and from the Natural Sciences and Research Council of Canada. * Corresponding author; fax 1-604-822-6089. 1259

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tion experimenb, actually measures the difference between influx and efflux, which is especially relevant because N (either NH4+or NO3-) efflux has been reported to be significant, particularly at elevated concentrations of N (Breteler and Nissen, 1982; Morgan and Jackson, 1988; Wang et al., 1993). Second, by judicious choice of appropriate influx and desorption times, based upon the half-lives for exchange of the subcompartments of the root (Lee and Clarkson, 1986; Presland and McNaughton, 1986; Siddiqi et al, 1991; Wang et al., 1993), it is possible to measure the plasmalemma influx as opposed to other fluxes (to vacuole or to stele) that result from long-tem experiments (Cram, 1968). The objective of this study was to investigate the mechanisms and characteristics of ammonium uptake by rice plants. We have particularly emphasized short-tem responses of 13NH4+influxes to changes in [NH4+], of uptake solutions over a wide range of extemal concentrations in order to define the transport mechanisms responsible for influx across the plasma mernbrane. We have examined the influence of prior NH4+provision on the kinetic parameters for influx by both components of the biphasic system for NH4+transport. In addition, the sensitivities of these fluxes to metabolic inhibitors, short-tem variations in temperature, and pH were determined with a view toward clarifying the mechanisms of these fluxes. MATERIALS AND METHODS Crowth Conditions and Production of 13NH4+

Seed germination and growth conditions were as previously reported (Wang et al., 1993). In short, rice (Oryza sativa L. cv M202) seeds were germinated in distilled, deionized water and grown hydroponically in MJNS containing ammonium (NHKl) as the only source of N. MJNS was also the medium used to cany out a11 influx experiments. The composition of MJNS and the maintenance of the levels of nutrients and pH were as described in Wang et al. (1993). Growth conditions were maintained as follows: temperature, 20 f 2OC; RH, 75%; irradiance, 300 pE m-’ s-’ under fluorescent light tubes with spectral composition similar to sunlight on a cycle of 16 h of light and 8 h of dark. The short-lived radioisotope I3N (half-life = 9.97 min) was produced in the form of l3NO3-by the CP42 cyclotron (Nordion Intemational, Inc., Vacouver, British Columbia) and converted to 13NH4+by use of Devarda’s alloy and a distillation method, as outlined in Wang et al. (1993). RCR

Rice seedlings were grown in 2, 100, and 1000 p~ NH4+ (designated, hereafter, as G2, G100, and GlOOO plants, respectively) to represent inadequate, adequate, and excess N provision. Total fresh weights of plants were recorded for three treatments at ages of 14, 21, and 28 d. They were used to calculate RGR. influx Measurement

Three-week-old plants were used in a11 influx measurements. Plasmalemma fluxes of 13NH4+into root cells were measured by exposing intact roots to 13N-labeled NH4+so-

lutions. Prior to the 10-min loading in 13NH4+-labeled MJNS with the appropriate [NH4+],, rice roots were prewashed for 5 min in an identical but unlabeled MJNS with isame [NH4+],. Immediately following the 13NH4+loading, roots were postwashed for 3 min in solutions identical to those used for prewashing. The duration of the influx period!j and the subsequent postwash were reported in a previous paper (Wang et al., 1993). Plant roots were then excised anti immediately introduced into scintillation vials for counting in a y counter (MINAXI 7-5000, Packard). Roots were weiighed for flux calculation immediately after counting.

I

Kinetic Study

Influxes of G2, G100, or GlOOO plants were measured in 13N-labeledMJNS varying in [NH4+],from 2 FM to 40 I n M in perturbation experiments. Perturbation experiments are defined as those in which plants are grown at one pariicular [NH4+Io,and influxes are measured in a range of [NH4+],. Measured 13NH4+influxes at various [NH4+], were fitted to the Michaelis-Menten equation [V = (V,,, . [NH4+],)/(Km+ [NH4+],)]and a more comprehensive equation [ V = (V,,, . [NH4+],)/(Km [NH4+],) + b . [NH4+], a] by means of a nonlinear regression method using the computer program Systat (Wilkison, 1987). In the equation, V (pmol g-’ fresh weight h-’) stands for the influx measured at a particular [NH4+],. V,,, is the calculated maximum rate of influx, and K, ( p ~represents ) [NH4+],giving half of the maximum influx; b and a are constants characterizing the linear phase. P.t each concentration tested, influxes were determined in twa to six separate experiments each with three or four replicate:,. Each replicate consisted of about 20 rice seedlings. Based on the results of the kinetics studies (see ‘Rejults”), measured NH4+ influx from (1 m~ [NH,’], appeared to result from a saturable HATS. Since the influx by the HATS had saturated between 0.1 and 1.0 m~ [NH4+],, influx from 0.1 mM [NH4+],was selected as a concentration representative of the HATS in the following studies. Above 1 m~ [PJH4+], measured NH4+ influx appeared to result from the participation of both the HATS and a LATS. Therefore, the difference between measured influx at concentrations :>1 mM [NH4+],and the saturated values of the HATS were taken to represent fluxes due to the LATS.

+

+

Metabolic inhibitor Study

Influxes were measured in MJNS containing representative levels of either 0.1 m~ to estimate the activities of the HATS or 20 mM NH4Cl for the HATS plus LATS in the presence or absence of different metabolic inhibitors. The following inhibitors were used 10 p~ CCCP dissolved in ethanol; CNplus SHAM dissolved in water. The resulting alkaline pH was adjusted by titration with H2S04to pH 6. Fifty micro) in molar DES dissolved in ethanol; DNP (0.1 m ~ dissolved ) in water; pCMBS (1 m ~ ) ethanol; Mersalyl (50 p ~ dissolved dissolved in water. The acidic pH was adjusted by titration with Ca(OH)*to pH 5.8. Ethanolic solutions of CCCP, DES, and DNP were added to the nutrient solutions to give a final ethanolic concentration of 1%.Control solutions were treated with ethanol at the same concentration.

Kinetics of NH4+ lnflux across the Plasmalemma of Rice Roots

In this study, 3-week-old G2 and GlOO plants were used. Before labeling with radioisotope, rice roots were treated with unlabeled MJNS containing the same concentrations of CNplus SHAM for 30 min. There were no pretreatments for the other inhibitors. Measurements of influx were undertaken as in the kinetic study. Each inhibitor experiment was repeated twice with three replicates for each treatment. Each replicate consisted of about 20 seedlings. Therefore, the means for influxes and SE values were calculated from six replicates and represented the mean for approximately 120 seedlings. Temperature Study

Rice plants were grown under the same conditions as described previously, so that they were adapted to 20 f 2OC. Influxes were subsequently measured in MJNS with either 0.1 or 20 mM NH4Cl at solution temperatures of 5, 10, 20, and 3OOC. During the prewash, uptake, and postwash, solutions were maintained at the designated temperatures. The measurements of influx were undertaken as in the kinetic study. Solution pH Profile Study

Rice plants were grown in MJNS containing 2 PM NH4+ under the conditions described in "Materials and Methods" and adapted to growth media at pH 6. Uptake solutions were adjusted to pH values of 3.0, 4.5, 6.0, 7.5, and 9.0 by additions of HCl or NaOH. To examine the effects of solution pH on 13NH4+influx, roots were exposed to the designated pH levels during the 5-min prewash, the 10-min influx, and the 3-min postwash. Influxes of 13NH4+were measured in either 0.1 or 10 m NH4+ solution. The choice of 10 m NH4+ rather than 20 m~ was dictated by the desire to minimize additions of HCl or NaOH in adjusting pH levels in the uptake solutions.

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up to 6.85 pmol g-' fresh weight, respectively. As shown in Figure 2, increasing [NH4+liwas associated with decreasing V,,, values, from 12.8 through 8.2 down to 3.4 pmol g-' fresh weight h-I, and increasing K, values, from 32 through 90 up to 188 PM, for G2, G100, and GlOOO plants, respectively. LA TS

In the higher range from 1 to 40 m, the relationship between [NH4+],and 13NH4+influx was linear (Fig. 3A). The y intercepts of these lines (13.21, 10.14, and 4.59 for G2, G100, and G1000, respectively) decreased according to the ammonium provision during the growth and agreed well with the corresponding V,,, for the HATS (Table I). Thus, we concluded that the measured fluxes at elevated [NH4+], result from the combined activities of the HATS and the LATS. To evaluate the effect of prior NH4+provision on the LATS for 13NH4+influx without the influence of the HATS, the V,,, values for HATS were subtracted from the measured influxes at elevated [NH4+],values. The derived LATS values were replotted accordingly (Fig. 3B). As shown in Figure 3B, 13NH4+influx by LATS is higher for GlOOO than for GlOO or G2. Slopes of the lines increased according to the NH4+leve1 during the growth period (0.67 for G2, 0.79 for G100, and 1.30 for GlOOO in Table I). These linear relationships at high [NH4+],were confirmed by means of F tests for linearity (Zar, 1974). Statistical analyses revealed that the slope of the GlOOO line was significantly different from the slopes of the G2 and GlOO lines (data not shown). Effect of Metabolic lnhibitors on the lnflux of 13NH4+

In most cases I3NH4+influxes of G2 plants were reduced by the presence of metabolic inhibitors in the uptake solutions as shown in Figure 4. Net reductions of influxes, listed in Table 11, were calculated by using the influx of the control as

RESULTS Kinetics of 13NH4+lnflux

Influxes of 13NH4+in response to externa1 concentrations in the range from 0.002 to 40 m [NH4+],were resolved into two distinct phases, presumably mediated by two separate transport systems: at low [NH4+],(4m),a saturable HATS, and at high [NH4+], (>1 m ~ ) the , combined activities of a saturated HATS and a linear LATS.

-m

"

E

7

-i

10

M

1 s HA TS

In the low concentration range (4 mM [NH4+],),the values of 13NH4+influx into roots of G2, G100, or GlOOO rice plants conformed to Michaelis-Menten kinetics (Fig. 1). The kinetic parameters of V,,, and K, were estimated using nonlinear regression analysis (Table I) to fit the Michaelis-Menten equation. Analysis by means of a more comprehensive equation (see 'Materials and Methods") gave similar trends although actual values of V,,, and K, were slightly different (data not shown). With increasing provision of NH4+from 2 through 100 to 1000 PM in the period of 2 weeks prior to uptake measurements, root [NH4+Iiincreased from 2.37 through 4.31

-

'G1000'

P

X

-

z

CI

O O

0.1

0.2

0.3

Externa1

0.4

O5

ammonium

0.6

0.7

0.8

concentration

0.9

1.0

(mM)

Figure 1. lnflux of 13NH4+ into rice roots at low concentrations in

perturbation experiments. Rice seedlings (3 weeks old) were grown at 2, 100, or 1000 p~ NH4+(G2 [AI, G100 [O], or GlOOO [O], respectively). Each data point is the mean of 16 replicates with SE values shown as vertical bars. The solid lines are estimated from V,, and K, values (Table I ) of G2, (2100,and GlOOO plants, respectively.

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70

Table 1. Kinetic parameters for saturable and linear 13NH4+ influx of 3-week-old rice roots of C2, CIOO, or ClOOO as functions of fNH4+lo

70 > '61000'

I :."I

'

-I

T'

Y

'6100'

The relationships between 13NH4+influx and [NH4+], of uptake solution were estimated from Michaelis-Menten kinetics for influx measured between 2 and 1000 PM [NH4+], and for linearity in the ranRe of 1 to 40 ITIM, where "a" is the interceDt and "b" is the sloDe.

G2 HATS"

12.8 f 0.2b 8.2 f 0.7 3.4 f 0.2 32.2 f 2.1 90.2 23.2 188.1 f 34.5 13.21 10.14 4.59 0.67 0.79 1.30 0.97 0.97 0.99 0.41 1.94 1.19 0.67 0.79 1.30 0.98 0.96 0.98

V,,

*

)K,,, HATS+ LATS

83

b ir2

LATS

G 1O00

ClOO

#a

lb 1 2.

HATS represents the high-affinity transport system, measured below 1 mM [NH4+],. lnflux measured at concentrations above 1 mM [NH4+], is considered to be the combined contributions of both high- and low-affinity transport systems (HATS LATS). LATS represents the low-affinity transport system and is estimated by V,, and K, were estisubtracting HATS from HATS LATS. mated by nonlinear regression with k SE. a

+

+

zero reduction (0%). The HATS f o r NH4+ i n f l u x was reduced by 81 to 87% by the protonophore (CCCP) o r the uncoupler of electron transport chain (CN- plus SHAM) a n d inhibitors of A T P synthesis (DNP). These three treatments reduced the LATS by only 31 t o 51% . The ATPase inhibitor DES reduced 13NH4+ i n f l u x due t o the HATS by 51% but h a d negligible effects o n LATS. Extemal protein modifiers of the membrane surface, pCMBS a n d Mersalyl, reduced 13NH4+ influx of H A T S by about 40%, with slightly less or similar reductions o f LATS (22-46%). These pattems of inhibition were also observed f o r GlOO plants (data n o t shown).

Effect of Root Temperature on 13NH4+lnflux

o

o o

'GIOOO'

A

'GZ'

'GIW'

. 5

15

10

Externa1

20

25

Ammonium

35

30

Concentration

, I 40

45

(mM)

Figure 3. lnflux of "NH4+ into rice roots at high concentrations in perturbation experiments. A, lnfluxes of "NH4+ into rice rciots of

G2 (A), ClOO (O), or ClOOO (O), respectively, were plotted against [NH4+],. Each data point is the mean of more than six replicates with SE values shown as vertical bars. 6,The estimated LATS fluxes after subtracting the V,, of the HATS of C2, C100, or G1000, respectively, from the corresponding measured influxes I:in A). These plotted lines of LATS have the same slopes as their corresponding lines in A but with slightly different values of the intercept: 0.53, 1.96, and 0.99 for C2, G100, and G1000 plants, respectively.

to the g r o w t h temperature o f 2OoC (data n o t shown). Table

111 shows the calculated Qlo values for G 2 a n d GlOO lplants in the temperature range f r o m 5 to 30OC. In this temperature range the Qlo values f o r HATS fel1 from >2.4 between 5 a n d 10°C t o 1.25 between 20 a n d 3 O O C . The results o f F tests in conjunction with D M R T demonstrated that Qlo values for the different temperature ranges were significantly different for the H A T S (P < 0.01). In contrast, there were n o significant differences between the Qlo values f o r L A T S in the same three temperature ranges for b o t h G2 a n d GlOO plantlj (P >

Short-term perturbations of root temperature significantly affected the i n f l u x o f 13NH4+ into rice roots that were adapted

'5

r 250

I

o 0.0

4 '

"

I

'

2.0

Root

J.

'GZ' ~

~

.

J.

'G100'

'

4.0

ammonium

"

I

~

'

'GLOOO' '

6.0

concentration

I

.

8.0

.

.

O 10.0

Control

CCCP

CN+SHAM

Inhibitors

DES

in

DNP

uptake

Marselyl

pCMBS

solution

(mM)

Figure 2. Relatioiiship between kinetic parameters of NH4+uptake and root ammonium concentrations ([NH4+]i) of rice seedlings. The values of V,, (O) and K, (A) from Table I were plotted against [NH4+]i for (22, (2100, or C1000 plants, indicated by downward arrows on the x axis.

Figure 4. Effect of metabolic inhibitors on 13NH4+influx. Rice plants were grown in MJNS containing 2 @M NH4CI. lnfluxes of "NH4+ were measured in MJNS with either 0.1 or 20 mM NH4+ in the presence or absence of a specific metabolic inhibitor. Each data point is the average of more than six replicates with SE values shown as vertical bars.

Kinetics of NH4+ lnflux across the Plasmalemma of Rice Roots

Table 11. Reduction of 13NH4+influx into roots of C2 plants by

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Table IV. Effect of uptake solution pH on 13NH4+influx into rice roots of 3-week-old C2 plants grown at p H 6.0 in M/N5

various metabolic inhibitors ~

Treatment

None

CCCP CN + SHAM DES DNP

10 mM

KJCMBS

of

+

Leve1

Control

Mersalyl

lnflux of "NH4+ was measured in MJNS at various pH levels (3.0, 4.5, 6.0, 7.5, and 9.0) with [NH4+], at either 0.1 mM for the HATS or 10 m M for the HATS LATS. The value of LATS was obtained by subtracting the values of HATS from HATS LATS of each treatment.

Percent Reduction

lnhibitor

1

mM

50 mM 0.1 mM 50 m M 0.5 mM

HATS"

LATS'

O 84.58 80.84 53.96 86.72 41.97 41.33

O 30.72 43.20 4.00 50.55 22.40 46.1 1

HATS

"The influxes of HATS were measured in the representative [NH4+],(0.1 mM). Reduction of HATS (%) was calculated by setting the control value, the influx value measured in 0.1 mM NH4CI uptake solutions without the inhibitor, as 0%. ' Reduction of LATS (%) was calculated by first determining the influx due to LATS by subtracting the influx values measured at 0.1 mM NH4+ from that at 20 m M NH4+ for control and for each inhibitor treatment, respectively. The reduction of influx value due to LATS under control conditions was then set at 0%.

0.05). Nevertheless, Qlo values for the LATS were significantly greater than 1.

LATS

+

PH

Influx'

3.0 4.5 6.0 7.5 9.0 3.0 4.5 6.0 7.5 9.0

6.91 f 1.43 12.02 f 0.46 13.22 f 0.27 14.51 f 0.39 12.94 f 0.30 15.75 f 0.45 18.63 f 2.80 18.07 f 0.49 11.44 f 1.37 9.29 f 1.54

LSD~

*

Percent of

Control'

ns

53 87

Control ns

1O0 1o9

ns * ns

95 87 103

Control

1O0

** *

63 51

a Each value (?SE) (pmol g-' fresh weight h-') is the average of four means of duplicate experiments. Each mean is derived from three replicates. 'LSD test made pairwise comparisons between the control at pH 6.0 and other treatments. *, Significant at 1% level; *, significant at 5% level; ns, not significant. 'The percentages of control were calculated using the NH4+ influx measured

at pH 6.0 as 100%.

Effect of Solution pH on 13NH4+lnflux DISCUSSION

The effect of uptake solution pH on 13NH4+influx was also investigated. The percentage of the control was computed on the basis of the influx value at pH 6.0 for either HATS or LATS (Table IV). In this case, an LSD test was used for making painvise comparisons between the control and other treatments. In the range from 4.5 to 9.0, solution pH had only a small effect on I3NH4+influx from 0.1 m [NH4+],, whereas I3NH4+influx by LATS decreased very significantly with increasing ambient pH beyond pH 6.0. By contrast, reduction of solution pH down to 3.0 drastically reduced 13NH4+influx by HATS as well as by LATS.

Table 111. Calculated Qlo values for 13NH4+influx by the HATS or LATS of 3-week-old rice plants grown at 20°C with 2 or 100 p~

NH4Cl IC2 and GlOO dants)

HATS LATS'

Temperature Range '

G2 Plants"

DMRTb

GlOO Plants

DMRT

5-10°C 10-20°C 20-30°C 5-10°C 10-20°C 20-30°C

2.48 f 0.04 1.79 f 0.08 1.25 f 0 . 1 6 1.41 f 0 . 2 1 1.49 f 0.06 1.56 f 0.06

A

2.59 f 0.21 1.68 f 0.22 1.44 f 0 . 1 6 1.54 f 0.27 1.90 f 0.46 1.33 f 0.12

A B

B

C

B

a Each value (&SE) is the average of three means from duplicate experiments; each mean is derived from three repliDMRT compared all possible pairs of treatment means. cates. Means having a common letter were not significantly different at the 1% significance level. Both F tests and DMRT indicated that means for the LATS were not significantly different at the 1% or even 5% level.

Kinetics of Ammonium Uptake

In an earlier report in this series it was demonstrated that the half-lives for 13NH4+exchange of the cell wall and cytoplasmic phases of rice roots (G2, G100, or GlOOO plants) were approximately 1 and 8 min, respectively (Wang et al., 1993). By using 10-min exposures to 13NH4+and 3-min postwashes, estimates of plasmalemma influxes rather than net flux or quasi-steady fluxes to vacuole were obtained (see Cram, 1968). The results of the present study revealed that NH4+influx across the plasmalemma into rice roots exhibits a biphasic pattern: in the low range (below 1 m [NH4+],), influx occurred via a saturable HATS, whereas from 1 to 40 m [NH4+],, a second, nonsaturable LATS became apparent. This biphasic pattern of uptake has been reported for NH4+ uptake by L e m a (Ullrich et al., 1984), for K+ uptake by com roots (Kochian and Lucas, 1982), and for NO3- uptake by barley roots (Siddiqi et al., 1990). Plasmalemma 13NH4+influx at low [NH4+],conformed to Michaelis-Menten kinetics (Table I) in accord with earlier studies of net NH4+uptake by rice (Youngdahl et al., 1982; Wang et al., 1991). This has also been found to be the case for roots of other species, including com (Becking, 1956), ryegrass (Lycklama, 1963), and barley (Bloom and Chapin, 1981), where net NH4+ uptake rates saturated in the range from 100 to 1000 ~ L M[NH4+],. The significance of this HATS for NH4+ in rice roots is that it allows plants to absorb sufficient N (NH4+)from very low levels in the rhizosphere to meet the minimum requirement for plant growth. In the present experiments, for example, by 3 weeks the RGRs were independent of [NH4+], from 100 to 1000 p~ NH4+. The

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RGRs calculatecl from total fresh weights of both GlOO and GlOOO plants vvere at approximately 0.16 d-' for the 3rd week of growth, whereas for G2 the value was approximately 0.06 d-'. By the 4th week the differences in RGR had diminished to 0.05, 0.06, and 0.06 d-', respectively, for G2, G100, and GlOOO plants. The reduced growth rates of G2 plants were accompaniied by increased root:shoot ratios, and leaves were slightly paler than those of plants grown at higher [NH4+],. At the higher range of [NH4+],(1-40 m ~ ) a, linear LATS also participatecl in NH4+uptake by rice roots, as is the case for other ions and plant species (Kochian and Lucas, 1982; Ullrich et al., 1984; Face and McClure, 1986; Siddiqi et al., 1990). The y intercepts for lines of measured influx (due to both transport systems) against [NH4+],were in good agreement with the corresponding V,,, values for the HATS (Table I), which suggests that the HATS and LATS are additive. Despite the irnportance of NH4+as principal source of N for many plant species and the increasing availability of techniques for the measurement of short-term I3NH4+and 15NH4+influxes, few detailed influx isotherms (as distinct from net uptake isotherms) have been reported for NH4+ influx into roots of higher plants. Nevertheless, Ullrich et al. (1984) were able to demonstrate linear kinetics of NH4+ uptake by Lemna between 0.1 and 1.0 m~ [NH,'], using a depletion method. The question of the saturation of this apparently linear system at higher concentrations remained unresolved. Cle,arly, it is difficult to measure net fluxes by employing concentration depletion methods at high extemal concentrations vvithout extending the uptake experiment for long periods of time. By using short-lived radioisotopes, such as I3N, it has been possible to measure unidirectional fluxes of NOs- and NH4+at the plasmalemma of intact plant roots (Glass et al., 1985; Lee and Clarkson, 1986; Presland and McNaughton, 1'386; Ingemarson, 1987; Siddiqi et al., 1990; Wang et al., 1993). Even at concentrations as high as 40 m ~ , there was no evidence of saturation Òf the LATS system (Fig. 3). Energetics of Ammonium Uptake

The influx of ammonium by HATS is clearly dependent on metabolic energy. In the present study metabolicinhibitors CCCP, DNP, or CN- plus SHAM diminished l3WH4+influxes of HATS by more than 80% (Table 11). The effects of these inhibitors on the LATS were much smaller (31-51% inhibition). Further evidence from the Qlo values (Table 111) supported the notion of energy dependence. A QIO value greater than 2 is considered to indicate the metabolic dependence of physiological processes such as ion transport. Short-term perturbations of temperature between 5 and 10°C significantly increased the Qlovalues for HATS up to 2.5 compared 1.5 between 20 and 30OC. In a 7-h perturbation of root temperature, Sasakawa and Yamamoto (1978)concluded that the uptake of ammonium by 9-d-old rice seedlings was closely associatedwith metabolism.However, such long-term studies probably measure the Qlofor NH4+assimilation rather than the transpart process. The values of Qlo estimated from Ta and Ohira's data (1982) provided values larger than 2.5 for I5NH4+absorption by rice roots between 9 and 24OC.

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Lower Qlo values (1.0-1.6) were reported for net ammcinium uptake of low temperature-adapted ryegrass (Clarkson and Wamer, 1979), barley (Bloom and Chapin, 1981), and oilseed rape (Macduff et al., 1987), indicating that NH4+ trarksport had acclimated to the low-temperature growth condjtions. Consistent with the results of the metabolic inhibitor studies, the present Qlo study indicated that LATS was less serisitive to changes of root temperature than the HATS (Table 111). The apparent energy dependence of the HATS may not necessarily mean that NH4+ uptake is an active transport process, although active transport systems for ammonium have been proposed in bacteria, fungi, and algae (Kleiner, 1981; Schlee and Komor, 1986, Singh et al., 1987). The accumulation of NH4+ against its concentration gradient could be achieved by active or passive uptake mechanisms: the former could be achieved by direct use of metabolic energy to carry a solute across a membrane toward a region of higher electrochemical potential, whereas the latter could be done by solute flux across a membrane along the electrochemical potential gradient, in response to a favorabk electrical gradient. According to compartmental analysis (Wang et al., 1993), the cytoplasmic concentration of NH4+ in G2 roots was estimated to be 3.7 m. Using this value and -116 rnV as the measured plasmalemma membrane electrical pot ential difference for G2 plants (Wang et al., 1992), predictions derived from the Nernst equation indicated that net ammonium uptake would be active only when [NH4+],falls l~elow 42 ~ L MThis . is rather similar to the value of 67 ~ L Mcalciilated for Lemna (Ullrich et al., 1984). However, this calculation serves only to predict the feasibility of the process occiirring under the prescribed conditions. The precise relationship between the calculated electrochemical potential difference for an ion and the putative transport systems, predicted on the basis of concentration-dependent influx curves, are difficult to realize. In the present case, for example, there iire no discontinuities in the uptake curve corresponding to the predicted concentration at which the switch between active and passive transport (approximately 42 ~ L M[NH 1' o) occurs. This issue is raised as a waming against a too-literal interpretation of the thermodynamic predictions. Although on thermodynamic grounds influx is uphill below 42 ~ L Mand downhill beyond 42 PM, the kinetic data reveal no apparent change of transport mechanism. The characteristics of the two transport systems for NH4+ influx have significant features in common with those described for K+ uptake, in which (incidentally) there is yet no clear consensus regarding the mechanisms of influz into higher plant roots. Likewise, the mechanism of the apparently active transport of ammonium below 42 ~ L Mis unknown. It might occur by means of a specific ATPase or a secondary transport system such as an NH4+:H+symport that is dlriven by the proton motive force. As proposed for K+ uptake by Neurospora for each K+ entering, one H+ is co-transported and 2H+ are extruded by the proton pump (RodriquezNavarro et al., 1986). The net result is a 1:l K+:H+exchange. 1s it possible that NH4+influx is mediated by an analogous system? It has long been documented that NH4+ uptiike is associated with strong acidification of the extemal media (e.g. Becking, 1956). Likewise in the present study, when pH was

Kinetics of NH4+lnflux across the Plasmalemma of Rice Roots not adjusted daily in the initial growth experiments, externa1 pH dropped so low that plants failed to grow normally. Regarding the passive uptake of ammonium at higher concentrations, several authors have proposed that NH4+ influx may occur by an electrogenic uniport in response to the electrical gradient (Kleiner, 1981; Ullrich et al., 1984). When ambient concentration is beyond the predicted threshold for active uptake, the concentrative NH4+uptake may be due to a facilitated transport system driven by the electrochemical potential difference for NH4+.This has two components: the difference in chemical potential of NH4+ ( A ~ N H ~between +) cytoplasm and outside and the electrical potential difference (A*) generated in part by proton efflux across the transducing membrane. The actual mechanistic link, if one exists, between NH4+influx and the proton motive force across the plasma membrane is unclear at present. Certainly, the results of the treatments with the protonophore a (CCCP) or the uncouplers of ATP formation (DNP and CNplus SHAM), which caused greater than 81% reduction of influx due to HATS, are consistent with a dependence of NH4+influx on transmembrane proton motive force. Further support for this hypothesis is provided by the effect of the ATPase inhibitor DES, which reduced 13NH4+influx due to HATS by 54% but had negligible effects on LATS. Regulation of Ammonium Uptake

Although the biphasic pattem of NH4+ influx was independent of the prior NH4+exposure, the individual systems, particularly the HATS, were extremely sensitive to prior NH4+ exposure (Figs. 2 and 3). Evidently NH,+ influx by the HATS was subject to regulation by negative feedback; with increasing [NH4+], in the growth medium, root [NH4+Iiincreased and NH4+influx decreased (Fig. 2). It is noteworthy that in the present case, negative feedback regulation appeared to affect both V,,, and K, values (Table I, Figs. 1 and 2). It has commonly been observed that V,,, is strongly and unequivocally influenced by the level of nutrient supplied during growth. By contrast, an effect on K, has rarely been observed (Lee, 1982). Only in the case of K+ (Glass, 1976) was the K, strongly influenced by K status, although other ions such as C1- do show small changes (Lee, 1982). In the present study, the values of K, were strongly influenced by the prior level of NH4+supply and are positively correlated with [NH4+],. Contrary to expectation, 13NH4+influxes due to the LATS were higher in plants previously maintained at 1000 PM NH4+ than in those maintained at 2 p~ NH4+. The reverse was found to be the case for 13N03- influx in barley (Siddiqi et al., 1990). This positive correlation between provision of NH4+ and 13NH4+influxes at high [NH4+],may indicate that the LATS may not be subject to regulation by negative feedback. Another possible explanation is that better N nutrition may provide more building materials (protein?) for constructing transporters. However, exposures to high [NH4+], (>1 mM) were brief, and in longer exposures NH4+ influx may be down-regulated in accord with expectation. The present study has demonstrated the strong negative down-regulation of influx by the HATS in response to elevated NH4+ supply during growth. At present the mechanism(s) and signals responsible for this down-regulation of

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uptake are unclear. Feedback signals may result from unmetabolized ammonium of root cells or reduced N (Lee, 1982; Morgan and Jackson, 1988). Lee and Rudge (1986) have suggested that in barley the uptake of NH4+ and Nos- are under common negative feedback control from a product of NH,' assimilation rather than NH4+ and/or NO3- accumulation per se. Reduced N pools that cycle in xylem and phloem from root to shoot have been implicated in the whole plant regulation of N uptake by plant roots (Cooper and Clarkson, 1989). However, Siddiqi et al. (1990) have suggested that in the case of NO3- influx, vacuolar accumulation of Nos- per se may also, at least indirectly, participate in flux regulation. Further support for this proposal has come from studies of nitrate reductase mutants of barley that are capable of normal induction of NO3- uptake and appear to show diminished 13N03-influxas NO3- accumulates (King et al., 1993). In the present study, also, there was a close negative correlation between NH4+ influx and [NH4+Iiin root tissues (Fig. 2). However, the altered NH4+status in G2, G100, and GlOOO plants was probably also associated with changes in organic N fractions. Since efflux was estimated to be 10 to 30% of influx for G2, G100, and GlOOO plants (Wang et al., 1993), negative feedback acts very strongly on the influx step of the HATS, but since efflux also increased with increasing [NH4+],, this flux will exert significant effects upon net uptake. Effect of pH Profile on Ammonium Uptake

In the present study, influx by the HATS was strongly reduced below pH 4.5. By contrast, in the range from pH 4.5 to 9.0, 13NH4+influx by the HATS appeared to be relatively insensitive to pH. I3NH4+influx by the LATS actually decreased with increasing ambient pH beyond pH 6.0. It has been reported for several species that the specific uptake rate of NH4+can be reduced by short-tem decreases in pH below 6.0 (Munn and Jackson, 1978; Marcus-Wyner, 1983; Vessey et al., 1990) and even tenninated entirely at pH 4.0 (TollyHenry and Raper, 1986). Tanaka (1959) suggested that rice is very sensitive to pH below 4. This probably reflects a general detrimental effect of such acidic conditions on the transport systems. In addition, it has been observed that when plants were grown at such low pH values over extended periods of time, the roots became stunted and discolored. It has been suggested that both high pH and/or high ammonium concentration of solution may result in high rates of NH3 uptake due to increased NH3 concentration and the higher permeability of cell membranes to NH3 than NH4+ (see Macfarlane and Smith, 1982). However, in many studies this expectation has not been observed, and uptake failed to increase at elevated pH (MacFarlane and Smith, 1982; Deane-Drummond, 1984; Schlee and Komor, 1986). Likewise, in the present study influxes of I3NH4+due to the LATS were reduced by 25 to 35% at higher pH values (7.5-9.0), despite a predicted increase of [NH3]from less than 0.1% of total [NH4+plus NH,] at pH 6.0, to 36% at pH 9.0 according to the pK, for NH4+ (9.25). Furthermore, membrane electrical potentials of rice roots have been shown to be depolarized by elevated am-

Wang et al.

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monium concentrations (Wang et al., 1992). These observations indicate t h e entry of cation (NH4+) rather than neutra1 ammonium (NH3). The evidence from our electrophysiological study of rice roots indicated a linear relationship between depolarization of membrane potential a n d influx of NH4+ from 1 to 40 m~ (data not shown). Therefore, a t elevated concentration a n d pH it is unlikely that simple diffusion of NH3 could be considered a s a major component of the influx of LATS. Nevertheless, i n their study using Lemna, Ullrich e t al. (1984) reported that depolarization of membrane potential w a s saturated a t 0.1 mkl, even though net uptake continued to 1 mM i n a linear pattem. This observation is consistent with NH3 entry by the LATS i n Lemna.

CONCLUSION

The work described provides t h e first detailed characterization of NH4+influx across t h e plasmalemma of rice roots. Ammonium influx is biphasic a n d mediated by t w o discrete transport systems. Metabolic inhibitor studies and Qlo determinations indicated that both systems were energy dependent, although thc! HATS consistently showed greater sensitivity t o metabolic interference t h a n t h e LATS. Nevertheless, thermodynamic evaluations indicate that only a t quite low [NH4+],is there a need to invoke active transport of NH,+ against the elechochemical gradient. It is highly unlikely that t h e LATS is active. The HATS w a s found to be extremely sensitive to prior exposure to ammonium, a s indicated by the altered values of K , a n d V,,,. General insensitivity of influx to pH in the rarige from 4.5 to 9.0 argues strongly against significant entry of NH3 across the plasmalemma even a t high [NH4+],. ACKNOWLEDCMENTS

We would like to express our sincere appreciation to Bryan J. King, Jamail Mehroke, Michael Adams, Tamara Hurtado, Salma Jivan, and Xiaoge Chen for assistance and discussions; Dr. J.E. Hill (University of Califomia, Davis) for rice seeds; and TRIUMF (Vancouver, British Columbia, Canada:)for I3N. Received January 15, 1993; accepted May 10, 1993. Copyright Clearance Center: 0032-0889/93/103/1259/09. LITERATURE ClTED

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perennial ryegrass Lolium multiflorum and Lolium perenne. Plant Physiol64 557-561 Cooper HD, Clarkson DT (1989) Cycling of amino-nitrogrm and other nutrients between shoots and roots in cereals-a p3ssible mechanism integrating shoot and root in the regulation of nutrient uptake. J Exp Bot 40: 753-762 Cram WJ (1968) Compartmentation and exchange of chloiide in carrot root tissue. Biochim Biophys Acta 1 6 3 339-353 Craswell ET, Vlek PLG (1979) Fate of fertilizer nitrogen applied to wetland rice. In Nitrogen and Rice. Intemational Rice Research Institute, Los Bafios, Philippines, pp 174-192 Deane-Drummond CE (1984) Mechanism of nitrate uptak.e into Chara corallinn cells: lack of evidence for obligatory coupling to proton pump and a new N03-/N03- exchange model. Plant Cell Environ 7: 317-323 Fried MF, Zsoldos F, Vose PB, Shatokhin IL (1965) Characterizing the NO,- and N K + uptake process of rice roots by use of I5N labelled NH4N03. Physiol Plant 1 8 313-320 Glass ADM (1976) Regulation of potassium absorption in ,barely roots. An allosteric model. Plant Physiol 5 8 33-37 Glass ADM, Thompson RG, Bordeleau L (1985) Regulation of Nos- influx in barley. Studies using 13N03-. Plant Physiol 77: 379-381 Ingemarsson B (1987) Nitrogen utilization in Lemna. 11. Stutiies of nitrate uptake using I3NO3-. Plant Physiol 8 5 860-864 King BJ, Siddiqi MY, Ruth TJ, Warner RL, Glass ADM '(1993) Feedback regulation of nitrate influx in barley roots by nitrate, , nitrite, and ammonium. Plant PhysiollO2: 1279-1286 Kleiner D (1981) The transport of NH3 and NH4+ across biological membranes. Biochim Biophys Acta 639 41-52 Kleiner D (1985) Bacterial ammonium transport. FEMS Microbiol Rev 3 2 87-100 Kochian LV, Lucas WJ (1982) Potassium transport in com roots. I. Resolution of kinetics into a saturable and linear component'.Plant Physiol70: 1723-1 731 , Lee RB (1982) Selectivity and kinetics of ion uptake by barley 'plants following nutrient deficiency. Ann Bot 5 0 429-449 Lee RB, Clarkson DT (1986) Nitrogen-13 studies of nitrate fluxes in barley roots. I. Compartmental analysis from measurements of I3N efflux. J Exp Bot 185 1753-1767 Lee RB, Rudge KA (1986) Effects of nitrogen deficiency amn the absorption of nitrate and ammonium by barley plants. An.n Bot 57: 471-486 Lycklama JC (1963) The absorption of ammonium and nitrate by perennial ryegrass. Acta Bot Neerl 12: 361-423 Macduff JH, Hopper MJ, Wild A (1987) The effect of root temperature on growth and uptake of ammonium and nitrate by Elrassia napus L. in flowing solution culture. 11. Uptake from soliitions containing NH4N03. J Exp Bot 3 8 53-66 Macfarlane JJ, Smith FA (1982) Uptake of methylamine by Ulva rigida: transport of cations and diffusion of free base. J Exp Esot 3 3 195-207 Malavolta E (1954) Study on the nitrogenous nutrition of rice. Plant Physiol29 98-99 Marcus-Wyner L (1983) Influence of ambient acidity on the ahsorption of NO3- and NH,+ by tomato plants. J Plant Nutr 6: 657-666 Morgan MA, Jackson WA (1988) Reciproca1 ammonium trarisport into and out of plant roots: modifications by plant nitrogen ;;tatus and elevated root ammonium. J Exp Bot 4 0 207-214 Munn DA, Jackson WA (1978) Nitrate and ammonium upta'ke by rooted cutting of sweet potato. Agron J 70: 312-316 Pace GM, McClure PR (1986) Comparison of nitrate uptake dinetic parameters across maize inbred lines. J Plant Nutr 9 1095-1 111 Presland MR, McNaughton GS (1986) Whole plant studies using radioactive 13-nitrogen. IV. A compartmental model for the uptake and transport of ammonium ions by Zea mays. J Exp Bot 37: 1619-1 632 Rodriquez-Navarro A, Blatt MA, Slayman CL (1986) A potassiumproton symport in Neurospora crassa. J Gen Physiol87: 649-674 Sasakawa H, Yamamoto Y (1978) Comparison of the upta:ke of nitrate and ammonium by rice seedlings. Influences of light, temperature, oxygen concentration, exogenous sucrose, and metabolic inhibitors. Plant Physiol 62: 665-669

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