Ammonium Uptake by Rice Roots - Plant Physiology

Plant Physiol. (1993) 103: 1249-1258

Ammonium Uptake by Rice Roots' 1. Fluxes and Subcellular Distribution of 13NH4+ Miao Yuan Wang, M. Yaeesh Siddiqi, Thomas J. Ruth, and Anthony D. M. Glass*

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 V6J 2A3 (J.J.R.)

predominant form of N available to crop species. Relatively less is known about the uptake and subcellular partitioning of NH4+in higher plants. Nevertheless, in rice (Oryza sativa L.) cultivation (Sasakawa and Yamamoto, 1978), in forest ecosystems (Lavoie et al., 1992), in Arctic tundra (Chapin et al., 1988), and even in winter varieties of cereals growing in cold soils (Bloom and Chapin, 1981), NH4+ may represent the more important form of available N. In a previous report (Wang et al., 1991)it was demonstrated that net fluxes of NH4+into rice roots gradually acclimated between 0.1 and 1 m~ extemal [NH4+] so that net flux at steady state varied little among plants grown in these concentrations. Nevertheless, there is a lack of information about fluxes between subcompartments in relation to acclimation or to the mechanism(s) of NH4+ uptake. For example, Presland and McNaughton (1986)failed to observe 13NH4+efflux from maize roots. By contrast, a sizable net efflux of endogenous ''NH4+ was reported in wheat, oat, and barley upon transfer to 15NH4+solution, although there was no exact correlation between root ammonium concentration and net I4NH,+ efflux (Morgan and Jackson, 1988a, 1988b). The intemal NH4+ concentration of plant roots can be readily assayed after extraction by methods based on colorimetry or ion-specific electrodes (Fentemet al., 1983a; Morgan and Jackson, 1988a, 198813; Roberts and Pang, 1992). However, such analyses fail to provide information on the subcellular distribution of NH4+. On the basis of biochemical analysis, it was concluded that more than one intracellular pool of NH4+existed in roots of rice (Yoneyama and Kumazawa, 1974, 1975; Arima and Kumazawa, 1977). Two other methods have also been employed to determine subcellular NH4+ distribution, namely efflux analysis (Macklon et al.,

l h e time course of "NH4+ uptake and the distribution of 13NH.,+ among plant parts and subcellular compartments was determined for 3-week-old rice (Oryza safiva 1. cv M202)plants grown hydroponically in modified Johnson's nutrient solution containing 2,100, or 1000 PM NH4+(referred to hereafter as C2, C100, or ClOOO plants, respectively). At steady state, the influx of 13NH4+was determined to be 1.31, 5.78, and 10.11 pmol g-' fresh weight h-', respectively, for C2, C100, and ClOOO plants; efflux was 11, 20, and 29%, respectively, of influx. The NH4+flux to the vacuole was calculated to be between 1 and 1.4 pmol g-' fresh weight h-'. By means of 13NH4+efflux analysis, three kinetically distind phases (superficial, cell wall, and cytoplasm) were identified, with tllz for 13NH4+exchange of approximately 3 s and 1 and 8 min, respectively. Cytoplasmic [NH4+]was estimated to be 3.72, 20.55, and 38.08 mM for C2, G100, and GlOOO plants, respectively. These concentrations were higher than vacuolar [NH,'], yet 72 to 92% of total root NH4+ was located in the vacuole. Distributions of newly absorbed 13NH4+between plant parts and among the compartments were also examined. During a 30-min period GlOO plants metabolized 19% of the influxed 13NH4+.l h e remainder (81%)was partitioned among the vacuole (20%),cytoplasm(41%), and efflux (20%). O f the metabolized 13N, roughly one-half was translocated to the shoots.

The short-lived radioisotope 13N(tllz = 9.97 min) has been used as a tracer in studies of the fluxes of NOs- and NH4' into intact roots of com and barley plants (McNaughton and Presland, 1983; Glass et al., 1985; Lee and Clarkson, 1986; Hole et al., 1990; Siddiqi et al., 1991). It provides a methodology for the measurement of unidirectional fluxes (influx or efflux) across biological membranes over extremely short times and with great sensitivity (McNaughton and Presland, 1983).Because of its strong 7 emission, I3Ncan be determined rapidly and accurately, with little sample preparation, even in intact plants, by gamma counting techniques (McNaughton and Presland, 1983; Cooper et al., 1985; Meeks, 1992). The major emphasis in studies of N uptake has been upon Nos-, reflecting the widely held perception that NO3- is the

Abbreviations: CEC, cation-exchange column; DMRT, Duncan's multiple range test; G2, G100, and GlOOO plants, rice seedlinggrown i n MJNS containing 2, 100, or 1000 PM NH4+,respectively; MJNS, modified Johnson's nutrient solution; [NH4+],, [NH4+], [NH4+],, and [NHd+], ammonium concentrations ( p or~ m ~of)extemal medium, root, cytoplasm, and vacuole, respectively; @m, flux of NH4+across the tonoplast into vacuole; &, &, and &t, inward, outward, and net fluxes of NH,+ (pmol g-' fresh weight h-l) across the plasmalemma, respectively; Qi, Q,, Qv, ammonium contents (pmol g-' fresh weight) of root, cytoplasm, and vacuole, respectively; S, and S,, radioisotopic specific activities of extemal media and cytoplasmic compartments, resuectivelv;

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

1249

Wang et al.

1250

1990) and NMR spectroscopy (Lee and Ratcliffe, 1991; Roberts and Pang, 11992).These studies recognized several NH4+ fractions of root!j, which correspond to those of the superficial, Water Free Space, Donnan Free Space, the cytoplasm, and the vacuole. The present work was undertaken to investigate the kinetics, metabolic requirements, and regulation of NH4+ flux in rice seedlings using 13NH4+.This communication reports the results of compartmental analyses, using 13NH4+efflux, to estimate the f112 values of NH4+ exchange and the size of major compartments, as well as NH4+fluxes between these compartments. Together with data obtained frqm chemical fractionation, it was possible to develop a detailed analysis of the initial fatr: of absorbed 13NH4+.In addition, the t1/2 values for 13NH4+exchange provide essential parameters for the design of appropriate protocols for influx measurement, particularly the tluration of 13NH4+loading and postwash treatments. To evaluate the methodology of the compartmental analyses, influx and net flux of NH4' were also measured by independent methods.

Plant Physiol. Vol. 103, 1993

pumps (Circulator model IC-2, Brinkmann Instruments 1:Canada] Ltd., Rexdale, Ontario) and aerated continuously. The pH of growth media was maintained at 6.0 f 0.5 by adding powdered CaC03 (1-3 g/tank), according to measured pH values, once or twice daily. Production of 13NH4+

The short-lived radioisotope I3N (t112 = 9.97 min) was produced, as described by Siddiqi et al. (1989), by 20-MV proton irradiation of H 2 0 on a CP42 cyclotron (Nordion Intemational, Inc., Vancouver, British Columbia). Coni aminants in the %o3-sample (mainly "F) were removed by passing the samples twice through a SEP-PAC Alumina-N cartridge (Waters Associates). Reduction of I3NO3- to ':'NH3 was achieved by using Devarda's alloy at 7OoC in a water bath (Vaalburg et al., 1975; Meeks et al., 1978); I3NH3was separated from remaining species by distillation and tra pped in acid solution as 13NH4+. Measurement of Fluxes

MATERIALS A N D METHODS

I

Rice (Oryza satiua L. cv M202) seeds were surface sterilized in 1% NaOCl for 30 min and rinsed several times with deionized distilled water. Seeds were allowed to imbibe overnight in aerated deionized distilled water at 38OC and were placed on plastic mesh mounted on Plexiglas discs. The discs were set in a Plexiglas tray filled with deionized distilled water just above the leve1 of the seeds, and seeds were allowed to germinate in a growth chamber in the dark (at 38OC) for 4 d. During the following 2 d, the temperature was stepped down to 2OoC (by 9OC/d). The discs containing 1week-old rice seedlings were transferred to 40-L Plexiglas tanks located in a walk-in growth room where growth conditions were main tained as follows: temperature, 20 k 2OC; RH, 75%; and irradiance, 300 pE m-' s-' under fluorescent Iight tubes (VITA LITE, Duro-Test) on a cycle of 16 h of light and 8 h of dark. Plants were 3 weeks old when used for flux studies.

Standard procedures for 13NH4+uptake were as follows. (a) Loading. Rice roots were loaded in l3NH4+-labeledMJNS (hereafter referred to as loading solution) for design ated periods. (b) Prewash and postwash. Prior to and after loading, roots were prewashed and postwashed in unlabeled MJNS (hereafter referred to as washing solution) for 5 and 3 min, respectively. The choice of these times is rationalized in "Discussion." Experiments were conducted at steady !state with respect to [NH4+],, i.e. the [NH4+],of washing solutions and loading solutions were the same as those provided during the growth period. Immediately after the postwash peiiod, plants were cut into shoots and roots and the surface liquid adhering to the roots was removed by a standard 30-s spin in a slow-speed table centrifuge (Intemational Chendcal Equipment, Boston, MA), Roots and shoots were introduced into separate scintillation vials and immediately counted in a y counter (Minaxi 7-5000, Packard, Downers Grove, IL). The fresh weights of roots and shoots were recorded immediately after counting.

Provision of Nutrients

Compartmental Analysis

Rice plants were grown hydroponically in MJNS, in which ammonium (NH4C1)was the only source of N, and Si was added as NaZSiO3.5H20. MJNS was also the medium used to carry out a11 experiments. The composition of MJNS ( p ~ ) was 200 for Ca, K, and P, 100 for Mg, 300 for S, 16 for B, 5 for Si and Fe, 1 for Mn and Zn, 0.3 for Cu and Mo. [NH4+], was varied as indicated at the appropriate places. Rice plants were grown in MJNS containing 2, 100, or 1000 PM [NH4+], and are referred to hereafter as G2, G100, or GlOOO plants, respectively. The concentrationsof nutrients in growth media were maintained by infusion of appropriate stock solutions, through peristaltic pumps (Technicon Proportioning Pump 11, Technicon Instrument Corp., Tarrytown, NY).Generally, 2 L/d of stock solution was supplied and stock concentrations were detennined from daily chemical analyses of media samples. Solutions were mixed continuously by circulating

For better time control of the separation of washing sdutions from the I3NH4+-labeledroots during the efflux process and to reduce disturbance of roots, we devised a siniple apparatus in which to perform the efflux study. The spou t of a plastic funnel (100 mm diameter) was cut to fit into the barrel of a 25-cc plastic syringe, into which it was sealecl. A length of rubber tubing replaced the needle end of the syringe and a metal spring clip on the tubing functioned as drainage control. A small hole was drilled in the wall of the syringe barrel near the bottom and a needle was introduced throiigh this hole to provide aeration. This technique also resulted in good mixing of the washing solution. Roots of rice seedlings used for compartmental analysis were immersed for 30 min in the loading solution. These prelabeled roots were carefully introduced into the syringe barrel for elution. Samples of 20 mL of washing solution

Conditions for Plant Crowth

Fluxes and Distribution of 13NH4+in Rice Roots were poured into the 'efflux-funnel" and allowed to exchange with the 13N-labeled roots. After a prescribed interval, this solution was drained from the funnel directly into a 20-mL scintillation via1 by opening the drainage clip. Fresh washing solution was poured into the efflux-funnel from the top immediately after closing the drainage clip. The duration of successive washes were: 1 X 5 s, 1 X 10 s, 7 X 15 s, 2 X 30 s, 5 X 1 min, and 5 X 2 min. After the last wash, the plants were cut into shoots and roots and introduced into separate scintillation vials. The radioactivities of a11 samples were counted immediately. The radioactivities released from intact rice roots into efflux solutions during the 18-min efflux experiments were counted, converted to efflux rates, and plotted versus time in semilog plots (see Fig. 1).This method of analysis is required because NH4+is rapidly metabolized in rice roots (Yoneyamo and Kumazawa, 1974) and converted into amino acids and proteins. As a consequence, standard methods of compartmental analysis (Walker and Pitman, 1976), based on semilog plots of cpm remaining in the tissue plotted against time, are not appropriate. Hence, the values of log of rate 13NH4+released against time were plotted using the methods detailed by Lee and Clarkson (1986) in an automated computer analysis (Siddiqi et al., 1991). To be assured that the 13Nspecies that had effluxed from the roots was 13NH4+rather than any metabolic products, two other sets of 13NH4+-labeled roots were effluxed for 30 min in 750 mL of washing solution. Two 20-mL samples of the efflux solution from each beaker were taken and separated by the CEC procedure (see below) and counted for radioactivities. Checks of the Fluxes Derived from Efflux Analysis

After loading for 10, 20, and 30 min, respectively, at steady-state conditions, influx of 13NH4+was also determined by two independent methods: (a) the accumulation of 13Nby seedling roots and (b) the rate of depletion of 13NH4+from loading solution. In addition, the net flux of NH4+was also measured based on the rate of depletion of 14NH4+.The [NH4+], was determined colorimetrically (Solorzano, 1969). Time Course of 13NH4+Uptake In the time-course experiments, G2 or GlOO plants were exposed to 2 or 100 PM 13NH4+-labeledloading solutions, respectively, for times ranging from 10 s to 31 min. As described in the section on measurement of fluxes, roots were subjected to a standard prewash, loading, and postwash procedure. Spatial and Biochemical Allocation of Absorbed 13NH4+: CEC Separation

13NH4+was separated from its immediate metabolic products according to the methods of Fentem et al. (1983a) and Belton et al. (1985). After prewash, loading, and postwash, roots and shoots were separated, weighed, and frozen in liquid N. These frozen l3NH4+-1abeledtissues were first counted in the y counter and then ground with liquid N in a precooled porcelain mortar and extracted with 10 mL of 10 mM sodium acetate buffer (pH 6.2). The resulting slurry was

1251

passed through Whatman filter paper and then washed three times each with 5 mL of the same buffer solution. Radioactivity remaining on the filter was referred to as root debris. The filtrate was passed through the CEC filled with 3 mL of resin (Dowex-50, 200-400 mesh, Na+ form), resulting in an eluate (Off-CEC) and a CEC-bound fraction (On-CEC). Radioactivities of the various fractions were determined by y counting. Two sets of GlOO plants, containing 100 to 120 plants each, were subjected to prewash and loading in 100 PM 13NH4+for 30 min, then postwashed for 3 min and immediately frozen in liquid N for y counting and CEC separation. Chemical Assay of Ammonium Concentration of Root Tissue

Qi of G2, G100, and GlOOO seedlings were determined as follows. After desorbing in NH4+-free MJNS for 3 min to remove 14NH4+located in the cell wall, the roots were cut, weighed, and frozen for further CEC separation as described above. To assay Qi, the NH4+adsorbed on the CEC column was eluted using 250 ITU~KC1 and determined by the indophenol blue colorimetric method (Solorzano, 1969). Calculation of

&v

The results of CEC separation quantified the unmetabolized I3NH4+fraction in roots following 30 min of 13NH4+ loading. This amount (Q*c+V) represented the combined values of cytoplasmic and vacuolar (Q*") radioactivities that can be converted to a chemical quantity (Qc+V) after dividing by the specific activity of 13NH4+in the extemal solution (SJ: Qc+v

=

Q*c+v/So

(1)

The specific activity of 13NH4+within the cytoplasm (S,) during loading will increase to its steady-state value according to the rate constant for tracer exchange of the cytoplasm (kc = 0.693/t1,2) as given in the following equation (Walker and Pitman, 1976): S, = S, (1

- e+')

(2)

Thus, if S, and tIl2 are known, S, can be determined for any particular time ( t ) . By 30 min of loading (equivalents to 4 cytoplasmic half-lives, see Table 11), the specific activity of cytoplasmic 13NH4+(S,) is brought to approximately 94% of S, and 13NH4+accumulated within the cytoplasm also reaches about 94% of Qc (in Table IV). Therefore, the proportion of Qc+, transferred to the vacuole is given by: Qv

= Qc+,

- 0.94 . Qc

(3)

and from Equation 3, the $ J can ~ be roughly estimated (method I). The portion of Q*c+V that is transferred to the vacuole (Q*v) is given by: Q*v

=

(Qv/Qc+v)

. Q*c+v

(4)

Wang et al.

1252


The accumulation of tracer in the root vacuole is related to 4, and the spetific activity of the cytoplasm at each interval: 7-1

Q * v (t)

= 4-

. s c (t)

(5)

Q * v (t)

= 4-

*

2

(6)

5b&

and Z

(R'

11

S c (t)

c 0.99)

4-

(R'

111

The sum of tracer accumulation within the vacuole Q*" (= Z (tJ is given by Equation 4, and Z S, (t) can be calculated for each minute from Equation 2. Therefore, as method 11, it is possible to estimate & more rigorously from Equation 6 . RESULTS

0 4 . .

.

.

O

I

.

.

.

5

I5

10

Compartmental Analysis

Efilux time

Analysis of the 13Nreleased into washing solutions during compartmental malysis revealed that 99.5% of the radioactivity was retained on the CEC (Table I). Since positively charged amino acids (Arg, His, and Lys) represented only 5% of total amino acids in 3-week-old rice roots (Yoneyama and Kumazawa, 1974), we interpreted this result to indicate that 13NH4+was the predominant N species released from roots and adsorbed ort the cation-exchange resins. The influence of [NH4+],on compartmental analyses was investigated by using G2, G100, or GlOOO plants to represent inadequate, adequate, and excess N supply, respectively, prior to efflux measurements. A representative sample of such data (18-min efflux) for GlOOO plants is shown in Figure 1. Three distinct phases, having different slopes with high r z values, were fouind for each of the three types of plants tested (G2, G100, and G1000). These compartments were tentatively defined as corresponding to (a) the superficial solution adhering to roots, (b) the cell wall, and (c) the cytoplasm, respectively. The flIP values for exchange of these compartments were calculated to be approximately 3 s, 0.5 to 1 min, and 7 to 8.5 min, respectively (Table 11). According to DMRT, there were no sigpificant differences among these values for plants grown under different concentrations of NH4+,except for the cell-wall fraction of G2 plants. One important part of the compartmental analysis was to calculate the fluxes of NH4+across the plasmalemma of root cell. These calculated fluxes are in good agreement with the values obtained by more direct methods using the same root material (Table 111). 4K varied with the NH4+level provided during the growth period. Average NH4+influx values for G2, G100, and GlOOO plants were estimated to be 1.32 f 0.07, 6.08 f 0.61, and 10.16 f 0.31 pmol g-' fresh weight h-', respectively. @net was estimated by subtracting the esti-

= 0.97)

3-

(min)

+-

I

O

I

Figure 1. The rate of 13NH4+ released (log[cpm] g-' fresh weight

min-') during 18 min of efflux from intact rice roots of GlOOO plants

(see text for details).

mated values of 13NH4+efflux (derived from efflux analysis) from the influx of l3NH4+,or by measuring net deplebion of I4NH4+from the uptake solution. Both methods gave similar results with average values of 1.09 f 0.03, 4.56 & 0.24, and 6.75 f 0.67 pmol NH4+g-' fresh weight h-'for G2, (3100, and GlOOO plants, respectively. The & and &.t values of GlOO plants were 4-fold higher than those of G2 plants (Table 111). Fluxes of GlOOO plants were about 1.5 times the values of GlOO plants. Efflux values, expressed as percentages of influx, were 11, 20, and 29% for G2, G100, and GlOOO plants, respectively (Fig. 2). Since the volumes of subcellular compartments are very different (Steer, 1981; Patel et al., 1990), it is necessalry to distinguish between NH4+ content (Q), expressed as moles per unit weight of roots (pmol g-'), and [NH4+],expressed as moles per unit volume of a compartment ( p or~ mM). The results of estimated cytoplasmic NH4+ concentration ([NH4+],)and chemically assayed total root NH4+ coritents (Q,) of G2, G100, and GlOOO plants, as well as calculated values of [NH4+],,[NH4+IV, Qc, and Qv are presented together in Table IV. Values of [NH4+],and [NH4+],were higher with

~~

~

Table II. Estimated means for t~ values of 13NH4+exchange for. three compartments (superficial, cell wall, and cytoplasm) from the

efflux analysis Rice roots of 3-week-old G 2 , (2100, and C1000 plants were loaded in 13N-labeledMJNS for 30 min and effluxed in unlal2eled identical MJNS for 18 min at steady-state conditions with regard to [NH4+],. Each mean is t h e average of four individual efflux tests

*

~

Table 1. Separation of "N-labeled compounds in the loading solution, efflux solution, and shoot extract, by CEC Each mean is t h e average of two replicates & SE.

In loading solution In efflux solution In shoot extract

SE.

Compartments

G2

GlOO

G iooa

Radioactivity Adsorbed on CEC (Percent of Total cpm in Sample)

I. Superficial ( s ) ~ 3.42 f 1 .OOa 3.83 f 0.24a 3.38 f 0.37a 11. Cell wall (min) 1.06 f 0.10b 0.57 f 0.09a 0.43 f 0.D6a III. Cvtoolasm (min) 6.95 +- 1.14a 7.36 +- 0.12a 8.33 f O.bOa

99.7 f 0.1 (2) 99.5 f 0.5 (2) 0.7 f 0.2 (2) '

a DMRT was used to compare the means of each compartrnent. Only means followed by a different letter are significantly different at t h e 5% level of significance.

Fluxes and Distribution of 13NH4+ in Rice Roots

1253

Table 111. Comparison of the mean I3NH4+ fluxes across the plasmalemma of root cells Each mean is the average of three or four replicates f SE. G2

Methods

C 1O00

ClOO pmol g-' fresh weight h-'

lnflux (6.d (1) 13NH4+ efflux analysis" (2) "NH4+ accumulated in rootsb (3) 13NH4+depletion of mediumb (4) 13NH4+ depletion of mediumb

Net flux MneJ (5) "NH4+ efflux analysis" (6) I4NH4+ depletion of medium" Efflux (7) "NH4+ efflux analysis" (8) Subtracted (6) from (2) (9) Subtracted (6) from (4) a

Based on 30-min uptake.

1.20 1.39 1.37 1.33

f 0.02 f 0.02 f 0.01

5.97 f 0.41 5.27 f 0.20 6.99 f 0.51 6.1 1 f 0.32

10.51 10.16 10.29 9.66

f 2.04 f 0.23 f 0.29 f 0.63

1.O6 f 0.07 1.11 f 0.04

4.80 f 0.39 4.32 f 0.15

7.41 f 1.55 6.08 f 0.27

0.13 f 0.02 0.27 f 0.02 0.22 f 0.1 7

1.17 f 0.14 0.94 f 0.05 1.79 f 0.47

3.09 f 0.56 4.09 f 0.04 3.58 f 0.36

Based on 10-min uptake.

higher levels of NH4+ provision. The values of [NH4+],,were about 5- to 6-fold higher in G100, and about 10-fold higher in GlOOO plants than in G2 plants. The values for the vacuolar pool were based on the differences between the total in the roots (Qi) and the cytoplasmic pool (QJ. Of the total NH4+ of the roots, 92% was localized within the vacuole in G2 plants and about 72 to 76% was localized in GlOO and GlOOO plants. Chemical and radioisotopic quantities for various compartments used in calculating &., are presented in Table V. The specific activity of cytoplasm (S, (t)) was calculated for each minute from t = 1 to 30 min. Both Z Q*v(t)and Z Sc (t) were used for estimating &.,. The & estimated by methods I and I1 are given in Table V. Metabolism and Translocation of 13N

Virtually none of the 13NH4+absorbed by rice roots was translocated to the shoots (Table I). It is improper to express the translocation of 13N (to the shoot) as pmol NH4+per g

1001

f 0.07

fresh weight of roots because (a) 13Nis transported from the root in the form of amino acids, and (b) the specific activities of these amino acid pools were unknown. Therefore, the translocation was expressed as a percentage of the total radioactivity (cpm accumulated in roots plus shoots during the loading period). This total radioactivity is equivalent to net absorption of 13NH4+.Further fractionation of root tissues of GlOO plants by the CEC separation revealed that about 8.6% of the radioactivity provided by influx during 30 min of 13NH4+loading was retained in a metabolized form (Table VI). By combining the I3N translocated to shoots (10%) with root debris (4%)and the Off CEC fraction (5%),an estimation of the proportion (19%)of absorbed 13NH4+that was metabolized during the 30 min was obtained. The partitioning of radioactivity was also calculated based on the total cpm remaining in roots (Table VI). Time Course

of 13NH4+lnflux in Rice Roots

The results of steady-state 13NH4+uptake by G2 and GlOO plants, establishing the pattern of 13NH4+accumulation in rice roots, are shown in Figure 3. The accumulation of 13NH4+ appeared to be linear for the duration of the 30-min uptake experiments; the coefficient of determination of these lines (0.87 and 0.99 for G2 and GlOO plants, respectively) were high. In a11 cases, the intercept on the ordinate differed significantly from zero (at 5% significancelevel). GlOO plants had a higher accumulation rate than G2 plants. The data for 13 N accumulation were used to calculate the rate of 13N accumulation (influx) as a function of time (Fig. 4). Based upon very short exposures (less than 2.5 min) to 13NH4+,the influx of GlOO plants appeared to be about 20 to 30% higher than the steady value of influx. Beyond 5 to 10 min, influx in both G2 and GlOO plants remained essentially unchanged; approximately 1 and 7.5 pmol g-' fresh weight h-' for G2 and GlOO plants, respectively.

-F l I / Efflux 29%

=

x E

I

'62'

'6100'

'61000'

Plants

as percentage of bOcfor G 2 , G100, and G1O00 plants at steady state based on the data of compartmental analysis in Table II. Figure 2. &, and

DISCUSSION The flI2 of 13NH4+ Exchange

Three kinetically distinct phases (I, 11, and 111) with t1I2 for 13NH4+exchange of approximately 3 s, 1 min, and 8 min,


Wang et al.

1254

Table IV. Ammonium pools in root cells of 3-week-old C2, ClOO, and ClOOO rice plants at steady state The contents of unmetabolized NH4+ in root tissues (Q,)and cytoplasm (QJ and vacuole (QJ and their corresponding NH4+ concentrations ([NH4+], and [NH4+],), as well as that the cell wall pool ([NH4+],), are presented. In parentheses, Qc or Qv, respectively, are presented as percentages of Q,. NH,+ Content

NH4+Concentration"

Plant

Q,b

Q',

Q/

[NH4+lw

pmol g-' fresh weight root

C2

ClOO ClOOO

2.38 4.31 6.85

0.19 (8%) 1 .O3 (24%) 1.94'(28%)

[NH,+l,

INHa+Iv

mM

2.19 (92%) 3.28 (76%) 4.91 (72%)

0.56 2.27 14.41

3.72 20.55 38.08

2.58 3.86 5.78

a The values of [NH4+], and [NH4+], were estimated from compartment analysis with four replicates each and [NH4+IVwas estimated from Qv. The values of Q, were obtained from chemical NH4+ assay with three replicates each and are the same as the values of [NH4+],. ' The values of Qc were calculated from [NH4+], based on the assumption that the cytoplasm had only 5% of total cell volume. The values for Qv are based on the difference between Q,and Qc and the assumption that the vacuole occupies 85% of cell volume.

respectively, were identified by means of compartmental analysis (Table 11). Phase I is probably due to the surface solution on roots camed over from the loading solution (Fig. 1).The second phase is attributed to the cell wall fraction, or the apparent free space, which is the sum of the Water Free Space and the Donnan Free Space (McNaughton and Presland, 1983, and refs. therein). The f1I2 of this phase (0.5-1 min) was shorter than the equivalent phase reported for corn roots (2.5 min) by Presland and McNaughton (1986), but similar to the f1I2 for Nos- exchange (0.5 min) in barley roots (Siddiqiet al., 1991).By using the efflux-funnel, shorter efflux intervals were achieved, which allowed for resolution of these two rapid phases (I and 11) and more accurate estimation of the cell wall tl/2. The third phase is believed to be the cytoplasm. The t1/2 of cytoplasmic exchange for G2, G100, and GlOOO plants ranged from 6.9 to 8.3 min, but the differences were statistically insignificant, although the cytoplasmic pool sizes varied according to the provision of NH4+ during growth (Table 11). Siddiqi et al. (1991) showed that barley roots treated with SDS or pretreatetl by immersion in water at 7OoC for 30 min accumulated antl released significantly less 13N03- from

Table V. Data used for calculation of the flux (&J from cytoplasm

vacuole The data were taken from the results of the compartmental analysis (Table IV) and root partitioning experiment (Table VI). The calculation procedure is in "Materials and Methods." I into

Parameter SO

Z S,(,, ( t = 30 min) Z Q*vca ( t = 30 min) Q*C+" QC+" QC QV

& (method I ) &v

(method 11:)

Value 164,214 3,361,875 79,666 238,982 1.46 0.97 0.49 0.97 1.42

Unit

cpm pmol-'

cpm pmol-' cpm g-'

cpm g-' pmol g-' pmol g-' pmol g-' pmol g-' h-' pmol g-' h-'

phase 111, but phase I1 appeared unaffected. These rc?sults were consistent with phase I11 being the cytoplasm. In studies of 13NH4+efflux from spruce roots, H. Kronzucker (personal communication) has found that elevated [Ca2+l0in the loading and washing solutions reduced the extent of phase,II for 13NH4+exchange in spruce roots (which had t1/2 simi!lar to those observed in rice), as would be expected if this phase corresponds to the cell wall compartment. The short t l l 2 of 13N decay and long tlI2 of exchange of the vacuole (Lei: and Clarkson, 1986; Macklon et al., 1990) preclude the estimation of vacuolar parameters by efflux analysis using 13N. lJsing 15NH4+,Macklon et al. (1990) estimated the half-lives for cytoplasmic and vacuolar exchange to be 44 min and 8.2 to 22.8 h, respectively, for excised onion roots. Cooper and Ford (cited in Macklon et al., 1990) observed much shorter t 1 / 2 values for cytoplasmic 13NH4+exchange, ranging from 4 to 10 min in roots of wheat. The latter values are much closer to those obtained in the present studies, i.e. 6.9 to 8 min

Table VI. Fractionation of radioactivity in shoot and root tissues of 3-week-old CIO0 plants After 30-min loading in 13N-labeled MJNS containing 100 p~

NH4+, plants were prepared for counting and separation according to "Materials and Methods." Radioactivities are expressed a'; percentages of total cpm in plants. Each analysis used 1O 0 to 120 Iplants and data eiven are means of two redicate exaeriments. Percent c p m in Plant

(A) In shoots

9.7 f 0.9

'

(B) In roots

Total Percent recovery after CEC (a) On-CEC (b) Root debris (c) Off-CEC (C) Metabolizeda

90.3 f 0.9 81.7 f 2.5 5.1 +. 1.0 3.5 f 0.6 18.3 +. 2.5

a Metabolized is the sum of lines A, (b),and (c) based on t h e total cpm in whole plants or the sum of lines (b) and (c) based on the total cpm in root.


I

n

. . . O

5

10

U 1

.

.

15

Uptake

U

cr

-

. . . . . . . . . . . . , . . . . 20

time

25

30

35

(min)

Time-course study of 13NH4+ uptake by C2 and GlOO roots at steady state. Three-week-old rice plants were grown and loaded in 13N-labeled MJNS containing 2 ~ L M(O) or 100 PM (A) [NH4+],. Uptake is expressed as the accumulation of 13NH4+ (pnol g-' fresh weight). Each data point is t h e average of three replicates with SE values shown as vertical bars. Figure 3.

(Table 11). The longer f112 values reported by Macklon et al. (1990) may have arisen from species differences and/or differences of methodology. To select appropriate durations for the loading and washing periods employed in influx studies, it is important to estimate the fl12 values for 13NH4+exchange between different compartments (Cram, 1968). The choice of a 10-min loading time, used in the present study and in subsequent 13NH4+influx studies, was arrived at from considering the following. (a) The t112 of I3N decay is short (t1/2 = 9.97 min) and therefore the influx period should be as short as possible. As the isotope decays, the statistical uncertainty in the measurement of 13N retained by the plant roots or transported to the stem becomes as high as k15% after about 40 min (McNaughton and Presland, 1983). (b) If the loading time is long, compared with the f1/2 for cytoplasmic exchange for 13NH4+,the specific activity of the cytoplasmic pool may will be approach saturation and the 13NH4+efflux t e m maximized. The measured 13NH4+influx under these conditions would approximate the net flux (&et = &,c - 4 4 . (c) Although the overestimation of influx (see below) was minimized by 20 min, 10 min of loading reduced that overestimation to less than 10% (Fig. 4). The duration of the loading period and the postwash period is a compromise (Lee and Clarkson, 1986). Since the goal is to measure the unidirectional flux across the plasmalemma (&), I3N present in the cell wall should be removed during the postwash period. Based on the estimated t112 of the cell wall fraction, a short postwash period of 3 min (corresponding to three to six halflives, Table I) was adopted in a11 influx experiments. Therefore, to equilibrate the cell wall fraction to any changes of [NH4+IO,rice roots were always pretreated for 5 min in identical unlabeled MJNS before loading in I3N-labeled MJNS.

1255

Fluxes of 13NH,+ into Root Cells The results of the present study showed that 13NH4+appeared to be accumulated at a constant rate ( r 2 = 0.874 and 0.997, respectively) during 30 min of loading of G2 and GlOO plants under steady-state conditions (Fig. 3). Moreover, 13NH4+accumulation increased with increasing [NH4+], of the loading solution. This observation is similar to previous reports indicating that the accumulation of 13N (either as I3NO3- or I3NH4+)by plant roots increases in linear fashion during short (usually 4 5 min) loading periods (Presland and McNaughton, 1984; Lee and Drew, 1986). The data for 13NH4+accumulation by G2 and GlOO plants are also presented as plots of influx versus time (Fig. 4). Influx values based on very short exposures to 13NH4+were accompanied by large errors probably associated with the lower counts accumulated and a large multiplicative factor involved in calculating influx on a per hour basis. Nevertheless, the data indicated that initial influx values were 20 to 30% higher than those recorded after 2 to 5 min. After loading for more than 5 min, the influxes were 1 and 7.5 pmol g-' fresh weight h-I for G2 and GlOO plants, respectively, and notwithstanding some variation, remained reasonably constant for the next 25 min. Presland and McNaughton (1984) noted a higher rate of I3NH4+accumulation in maize roots during the first 2 min that they attributed to apoplasmic filling. In the present study, although the roots were subjected to a 3-min postwash, any tracer uptake by rice roots during the postwash period would represent an overestimate. The impact of these additional counts would be to overestimate the calculated influx values at shorter loading intervals due to the multiplicative effect in calculating fluxes on a per hour basis. This effect, which decreased as the duration of the influx period increased, was minimized at about 20 min (Fig. 4). This interpretation is in contrast to Lee and Ayling (1993), who argue that the lower counts recorded after 2 to 5 min represent an underestimate of influx due to release of absorbed I3N or I5N as cytoplasmic specific activity reaches steady state. We question this interpretation because the tl12

o i

. ..., ... .,.. .., . ... ,. .. . ,. . .. O

5

10

15

Uptake

time

20

, . 25

... ,

, , ,

,

30

35

(min)

Figure 4. lnfluxes of 13NH4+ into C2 and GlOO roots in the timecourse study at steady state. O, G2 plants; A,G100 plants. lnflux is expressed as pmol g-' fresh weight h-'. Each data point is the

average of three replicates with

SE values

shown as vertical bars.

1256

Plant Physiol. Vol. 1038,1993

W a n g et al.

for exchange of 13NH4+from the cytoplasmic phase was about 8 min for rice roots grown at various nitrogen conditions (Table 11) and the absolute value of the efflux from cytoplasm to outside (&) varied from 10 to 30% of influx (do,)according to compartmental analyses (Table 111). Therefore, we consider it unlikely that a significant reduction of measured influx would result from efflux of tracer during the short duration of these exposures. Values for d,,, dto, and dnetof I3NH4+determined by efflux analyses corresponded very well with those obtained by other, more direct methods (Table 111). This close correspondente allows us to accept the parameters derived from 13NH4+ compartmental analysis with some degree of confidence. Influxes of 13NH4+into rice roots under steady-state conditions increasecl according to the levels of [NH4+], in the growth media (Table 111). A similar trend was shown for net fluxes determined either by efflux analysis or by depletion methods. 9,,t tended to show only a small increase as [NH4+], increased from 104 to 1000 p~ (Table 111). This confirms our previous report that net uptake of NH4+ was acclimated to [WH4+],in growth media, although the acclimation was not achieved by G2 plants (Wang et al., 1991). These results demonstrated that NH4+ fluxes are closely related to the N status of plants, which is determined by plant growth conditions. Estimated effluxes of NH4+from rice roots were about 10, 20, and 29% of' the influx values for G2, G100, and GlOOO plants, respectively (Table 111, Fig. 2). In addition, efflux was positively correlated with the [NH,+], (Tables I11 and IV). This result agrees with the suggestion that continuous NH4+efflux may be a common feature of net NH4+ uptake by roots of higher plants (Morgan and Jackson, 1988a). N efflux (either NH4+ or NO3-) has been reported to be quite significant, particularly at elevated concentrations of N (Morgan et al., 1973; Breteler and Nissen, 1982). Indeed, Deane-Drummond and Glass (1983a, 1983b) suggested that nitrate efflux might regulate net uptake by means of a type of pump and leak mechanism. By contrast, Lee and colleagueshave emphasized the importance of influx in the regulation of net uptake of nitrate, although nitrate efflux was equivalent to almost 40% of nitrate influx in barley roots (Lee and Clarkson, 1986; Lee and Drew, 1986). Morgan and Jackson (1988a, 1988b) also found that a sizable net efflux of endogenous 14NH4+occurred upon transfer to 15NH4+solutions in wheat, oat, and barley adequately supplied with nitrate. However, an exact parallel between root ammonium concentrations and net 14NH4+efflux was not observed. Although plasmalemma influx determines the maximum rate of net uptake (Lee and Clarkson, 1986), efflux certainly makes a significant contribution to detemining net uptake. Because of its short half-life of decay, 13Nis unsuitable for the determination of vacuolar parameters by efflux analysis. Nevertheless, the combination of 13NH4+efflux analysis and the CEC separatnon of I3Nproducts has enabled us to estimate $cv using two methods. Both results give values for dCy in the range from 1 to 1.5 pmol g-' fresh weight h-'. Method I is based on the esíimated 13NH4+accumulation during 30 min of loading, whereas method I1 involved use of S, values estimated minute by minute from a knowledge of the tlI2 of cytoplasmic exchange (see 'Materials and Methods"). There-

fore, method I1 is probably more refined than the value derived from method I. These values are somewhat lower than those obtained by efflux analysis in onion (Mac:klon et al., 1990); however, the Macklon study was undertaken at 2 m~ [NH4+],, compared with our analyses undertaken with GlOO plants at 100 p~ NH4+. The differences may also reflect the methodology and plant species employed. The NH4+Pools in Roots

In the present study, the values of Q, were in the range from 2.38 to 6.85 pmol g-' fresh weight for roots grown with different levels of NH4+ (Table IV). Fentem et al. (1983b) reported a value of 3.2 pmol g-' fresh weight in 9-d-old barley roots grown in 1 m NH4'. For barley, wheat, and oat grown in NO3- or N-free conditions, the value of INH4+], was in the range of 0.4 to 2 pmol g-' fresh weight (hdorgan and Jackson, 1988a, 1988b, 1989). However, when plants grown in NI&+ or in Nos- were pretreated with 0.5 to 1.5 mM [NH4+],for various periods of time, the values of Q, were high and varied from 6 to 35 pmol g-' fresh weight (Morgan and Jackson, 1988a; Lee and Ratcliffe, 1991). The relatively low intracellular NH4+ content, particularly under steadystate conditions, may reflect the efficiency of NH4+iissimilation (Goyal and Huffaker, 1984). Irrespective of the [NH4+J,provided during the growth period, the bulk of absorbed NH4+ was localized in the vacuole (Table IV). Nevertheless, because of the large size of the vacuole, the values of [NH4+], were significantly lower than those of the [NH4+],(Table IV). Increasing [NH4+],from 2 to 1000 p~ caused [NH4+],to increase more than 111-fold, whereas [NH4+],increased by only 2-fold. CytoplasmicNH4+ concentrations of rice roots estimated in the present study (Table IV) were in the range of reported values for wheat, maize, barley, and onion (Fentem et al., 1983b; Cooper and Clarkson, 1989; Macklon et al., 1990; Lee and Ratcliffe, 1991). On the basis of NMR studies of NH4+distribution in root tip of maize, cytoplasmic [NH4+]ranging from 3 to 438 p ~ 4were reported (Roberts and Pang, 1992). However, in that study lower values might have been expected, since root tip!j were excised from 2-d-old maize seedlings and maintained without an exogenous source of NH4+ during estimation of [NH4+], by NMR. Our indirect estimation of [NH4+],provided a range from 2.6 to 5.8 mM for G2, G100, and GlOOO plants (Table IV). Using I5N, Macklon et al. (1990) reported a similar range (3.9-10.9 mM) for [NH4+], in cortical cells of onion roots. Slightly higher values (15-36 mM) for [NH4+],were estimated in maize roots by 14N-NMRspectroscopy (Lee and Ratcliffe, 1991). Model of 13NH4+Uptake

by

Rice Plants

Despite the widespread use of compartmental analysis to investigate compartmentation of nonmetabolized iom, e.g. C1- (Cram, 1968), Na+ (Jeschke and Jambor, 1981), and K' (Memon et al., 1985), relatively few studies have been undertaken using metabolizable ions such as Po43- (Lef'ebvre and Clarkson, 1984), NO3- (Presland and McNaughton, 1984; Lee and Clarkson, 1986; Siddiqi et al., 1991), S04'(Cram, 1983), and NH4+ (Macklon et al., 1990). Presland and


Plasmalemma

I

Ceil

Figure 5. Proposed model for ammonium uptake and compartmentation in rice roots. The bold values in parentheses are estimated fluxes of absorbed 13NH4+(pmol g-' fresh weight h-'). The

percentages represent the relative distributions of 13NH4+among the compartments as a proportion of the isotope entering the cell during the 30-min loading. bOc,From outside plasmalemma to cytoplasm; aASs, assimilation rate; &O, from cytoplasm to outside degradation rate; bCv,from cytoplasm to vacplasmalemma; bDEC, uole; bVc,from vacuole to cytoplasm; @cx, metabolite translocation from root to shoot; bxc,metabolite translocation from shoot to root. Fluxes accompanied by (?) indicate fluxes for which data are not available from the present study.

McNaughton (1984) postulated the existence of four compartments (three in the roots and one in the shoot) based upon the distribution of 13N among these tissues in maize plants. Using 15NH4+efflux analysis with excised onion roots, the compartmental parameters for superficial, Water Free Space, Donnan Free Space, cytoplasm, and vacuole were identified (Macklon et al., 1990). The present study has characterized two intracellular compartments and one extracellular compartment for I3NH4+in rice roots. The biochemical fractionation approach was also used to identify different compartments for NH4+assimilation. By using 15NH4+,three compartments were found corresponding to different cell types and a storage pool in barley roots (Fentem et al., 1983a) or different organelles (Rhodes et al., 1980). Spatial differences in the activities of enzymes involved in NH4+assimilation are also found along the root (Fentem et al., 1983a). In addition to this form of heterogeneity, there are distinct isozymes of glutamine synthetase located within the cytosol and within plastids (Miflin and Lea, 1980). Much less information is available conceming the partitioning of newly absorbed ammonium between these compartments, particularly conceming the partitioning between metabolized and unmetabolized fractions in the root and translocation to the shoot. In the present experiments, nearly 90% of absorbed 13N remained in the roots, of which 80% was in the cation form (13NH4+)after 30 min of loading (Table VI). Among the metabolized 13N pools in roots, significant quantities of absorbed 13N(10%) were translocated to shoots during the experiment (Table VI), and analysis of this 13Nby ion-exchange chromatography (Table I) revealed a virtual

1257

absence of 13NH4+.The remaining metabolized fractions consisted of 5.5% that failed to be held on the CEC, presumed to be amino acids andlor soluble protein, and 3.9% that was not soluble and remained associated with the root debris. Calculations derived from results of both efflux and chemical analyses showed that unmetabolized NH4+in the cytoplasm (QJ constituted only 8% of Qi for G2 roots and 30% for GlOO and GlOOO roots (Table IV). Using GlOO plants as an example, a model describing the spatial and biochemical compartmentation of newly absorbed NH4+ uptake by rice roots is given in Figure 5 . About 24% of unmetabolized NH4+ was allocated to the cytoplasm and 76% to the vacuole. Based on the influx of 13NH4+into roots, 41 and 20% of 13N remained in the cytoplasmic and vacuolar compartments, respectively, along with 20% that was effluxed and 19% that was assimilated. Of the 19% assimilated, roughly half (10% of influx) was translocated to shoots. This assimilation rate was based on total 13Ntransported across the plasmalemma and may underestimate the true assimilation rate because during the loading period the cytoplasmic 13NH4+pool would not have reached steady state. ACKNOWLEDCMENTS

We would like to express our sincere appreciation to Jamail Mehroke, Mala Fernando, Michael Adams, Tamara Hurtado, Salma Jivan, and Xiaoge Chen for assistance and discussions; Dr. J.E. Hill (University of Califomia, Davis) for the generous gift of rice seeds; and to TRIUMF (Vancouver, B.C., Canada) for 13N.

Received May 4, 1993; accepted August 24, 1993. Copyright Clearance Center: 0032-0889/93/103/1249/10. LITERATURE CITED

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