NASA Contractor Report 177534 

~ 

Efficiencv of N Use by Wheat as a 

Function-of Influx and Efflux of NOSR. C. Huffaker 

M. Aslam 

M. R. Ward 

Plant Growth Laboratory, University of California, Davis, California 

Prepared for 

Ames Research Center 

July 1989 

CONTRACT NCC2-99 

NASA 

National Aeronautics and 

Space Administration 

Ames Research Center 

Moffett Field, California 940% 
EFFICIENCY OF N USE BY WHEAT AS A FUNCTION OF 

INFLUX AND EFFLUX OF NO3- 

R.C. Huffaker, M. Aslam, M.R. Ward, University of California, Davis, CA 

ABSTRACT 

Since N assimilation is one of the most costly functions of a plant, its 

efflux before assimilation results in a serious energy cost and loss in 

efficiency which could decrease yields. Efficient crop production is critical 

to CELSS. Our objective is to determine the extent of efflux of the N species 

NO3-, NH4+, NO2- We 

found that 

Efflux/Influx (E/I) of NO3- was greater in darkness (35%) than in light (14%) 

and the ratio greatly increased with increased substrate NO3-, (up to 45% at 

10 mM). It seems advantageous to use the lowest possible nutrient 

concentration of NO3-. 

(competitive inhibitor of NO3- uptake) for effluxed NOg- was assessed and its 

toxicity determined. 

and urea after uptake, and possible means of regulation. 

NO3- ’ efflux became serious as its substrate level increased. 

The feasibility of using C103- as a trapping agent 

INTRODUCTION 

Crop production during extended space flights requires the development 

of procedures leading to the optimum use of the available energy. Optimizing 

the utilization of N by crop plants represents an area where significant 

progress can be made in the CELSS program. 

It is well recognized that ion uptake requires ATP and electron flow. 

The cost of NO3- assimilation is particularly high not only due to the energy 

requirement of uptake but also because of the need for 10 electrons to reduce 

it to the level of glutamate. 

balance, since each reduction of NO3- to the level of NH4+ produces an OH-. 

In addition are the costs of maintaining pH 

Thus it is not surprising that estimates of respiratory costs of ion 

absorption range up to 50% of total root respiration and 20% of total plant 

respiration (1). 

Recent reports in the literature indicate that NO3- efflux can begin 

very rapidly after its influx (2,3,4,5,6). In addition, prolonged leakage of 

NO3- occurs from root storage pools dependent upon the N-status of the roots 

I 
(7,8,9,10,11). Lee and Clarkson (6) estimated that efflux, at external 

concentrations above 1 mM, could account for up to 40% of the influxed NO3-. 

Thus NO3- efflux could represent a significant additional cost to an already 

energy costly assimilatory pathway. 

of NO3- efflux. 

NO3- utilizes ATP, which could greatly add to the energy cost of NO3- 

assimilation. 

Little is known concerning the regulation 

Recent work of Briskin (12) showed evidence that efflux of 

Measurement of NOg- efflux has been hampered by the lack of easy, rapid, 

and less costly analytical techniques for detecting the very low concentrations involved in a process with a half-life (tlI2) of minutes. The use of 

I5N as a tracer is laborious and has problems of sensitivity. I3N has only a 

10 min half-life, must be used at the site of generation (cyclotron), and is 

very costly to use. We developed an HPLC method which has the sensitivity, 

ease and low cost required. 

agent for NO3-. 

In addition, we evaluated C103- as a trapping 

This report presents results estimating NO3- influx, efflux, and net 

uptake across several mechanisms of NO3- uptake. 

MATERIALS AND METHODS 

Plant growth. Wheat seedlings were grown hydroponically in a 1/4 

strength Hoagland's solution for 8 days in an environmentally controlled 

growth chamber at 400 pE/mLsec, at 18"C, and 80% relative humidity (13,14). 

On the 7th day they were transferred into 1/4 strength Hoagland's (loading 

solution) containing NO3- at the concentration to be used in the efflux study 

(specified in each experiment). 

Measurement of NOg- efflux. After removal from the loading solution, 10 

seedlings were placed in 300 ml of the efflux solution for various periods of 

time (specified below) containing 0.06 mM Pi at pH 5.8, 0.2 mM CaS04, and with 

or without C103- at the specified concentrations. The seedlings were rinsed 
for 2 sec in 300 ml of efflux solution, then placed in 60 ml of efflux 

solutions for the 

and the amount of 

Measurement 

following times: 10 sec, 30 sec, 1, 

NO3- released at each time from the 

of NOg- and C103-. These compounds 

2, 5, 10, 15, and 20 min 

roots was determined. 

were measured by HPLC as 

described previously for NO3- (13,141. 

a UV monitor. 

C103- was also measured at 210 nm with 

Uptake rates of NO3- and C103-. Uptake rates were determined as 

previously described by determining rates of depletion of NO3- from substrate 

solutions, then fitting the rate curves to best fit curves by polynomial 

analysis using a computer (13). 

RESULTS 

Mechanisms of NO3- uptake. The results in Figures 1 and 2 show several 

mechanisms for NO3- uptake. 

determined by either step up or step down, on continuous depletion experiments; Figures 1 and 2 are the results of continuous depletion experiments. 

In Figure 1, one mechanism is readily seen between about 0.2 and 0.7 mM. This 

is commonly referred to as Mechanism I in the literature (14). The rates 

above 0.7 mM are largely undefined but are referred to in the literature as 

Mechanism 11. Another mechanism is indicated in Figure 1 at concentrations 

below 0.1 mM and it is readily seen when the data are plotted between 0 and 

0.1 mM (Fig. 2). 

Uptake as a function of N03- concentration can be 

Comparison of uptake of NO3- and C103-. The comparative uptake of NOgand C103- is shown in Figures 3 and 4 at initial concentrations of 0.5 and 1 

mM. 

(Fig. l), whereas depletion of C103- is not straight forward. 

take up C103- is continuously lost with time (Fig. 4). 

Wheat plants deplete the NO3- concentration very efficiently to near zero 

The ability to 

Toxic effects of C103- on NO3- uptake. The increasingly toxic effects 

of C103- on NO3- uptake with time is seen in Table 1. 

3 
Comparative effects of pretreatments of C103- and NO3- on their uptakes. 

i The comparative effects of pretreatments of C103- and NO3- on their uptakes 

are shown in Table 2. 

subsequent uptake of NO3-, whereas increasing time of pretreatments greatly 

decreased C103- uptake. 

Pretreatments varying in time had little effect on 

C103- as a competitive inhibitor of NO3- uptake. Double reciprocal 

plots of rates vs concentrations show evidence that C103- is a competitive 

inhibitor of NO3- uptake (Fig. 5). 

NO3- efflux. Figure 6 shows a typical example of a determination of 

NO3- efflux. 

literature showing two different early losses of NO3-, one with a t of less 

than 10 sec and another with a t of minutes. After one min, the second set 

of rates approximate an apparent first order reaction. 

gives an estimate of the rate of NO3- efflux. 

NO3-, efflux varied consistently between 2.0 and 2.5 pmol/gxh. 

Our results matched quite closely those reported in the 

1/2 

1/2 

Extrapolation to to 

At a concentration of 1 mM 

Effect of increasing concentrations of NO3- and C103- on NO3- efflux. 

Efflux in the presence of increasing concentrations of NO3- and C103- is shown 

in Figure 7. 

NO3- and C103-. 

As expected, efflux increased with increasing concentrations of 

Influx, efflux, and net uptake of NO3-. Comparative rates of the three 

kinetic components of NO3- absorption are shown in Table 3. 

greatly increased between 0.2 and 10 mM external NO3-, whereas net uptake 

remained about the same. 

increasing concentration of NO3-. 

Efflux and influx 

Efflux/influx increased from 15 to 45% with 

Effect of light and dark on influx, efflux, and net uptake of NO3-. 

NO3- efflux was similar in plants in darkness and in light; however, influx 

and net uptake were much greater in light (Table 4). 

the influxed NO3- was effluxed, while in light the proportion effluxed was 

reduced to 14%. 

Thus, in darkness 35% of 

I 4 
DISCUSSION 

It is important to continue to develop information concerning the 

mechanisms of uptake of nutrient ions both to understand the reactions of the 

plants to changing concentrations and also for planning optimum concentrations 

of nutrient solutions for maximum efficiency. 

Mechanisms of NO3- uptake. At least three mechanisms of NO3- uptake are 

present, one between 0 and about 0.05 to 0.08 mM with a K,,, of ca 0.012 to 

0.018 mM, and another between 0.1 and about 0.7 mM with a k, of ca 0.025 to 

0.04 mM (Figs. 1 and 2). The latter is the typical mechanism reported in the 

literature (14). At concentrations above 1 mM, the mechanisms are largely 

undefined and are difficult to determine because efflux becomes such an 

important component (discussed below). 

Comparison of uptake of NO3- and C103-. In a determination of NO3- 

efflux, very low concentrations of NO3- are present in the external solution. 

At these low levels of NO3- (see Fig. 3), the wheat plant can very efficiently 

absorb the NO3- as it is effluxed into the external solution. 

has been used as a trapping agent for the effluxed NO3-. 

Hence, C103- 

Although much work has been reported on the effects of C103- on NO3- 

uptake, the analytical procedures reported to separate C103- from NO3- in the 

solutions were in some cases non specific, i.e., ion electrodes (3). Radioactive C103- was also used (3) which presents problems of low specific 

activity and sticking to glassware. We developed an HPLC method which 

effectively separates NO3- from C103- and both can be measured simultaneously. 

In addition, much of the reported literature did not discriminate between 

kinetic effects and toxic effects of C103- on NO3- uptake (15). 

Toxic effects of C103-. The results showed that toxicity symptoms, as 

shown by decreased rates of uptake of NO3- and C103-, were apparent after 1 h 

(Fig. 4, Tables 1 and 2). Toxic effects of C103- were greater toward its own 

5 
uptake than towards NO3- uptake. 

during the 20 min period of the efflux experiments. 

We found that little toxicity occurred 

C103- as a competitive inhibitor of NO3- uptake. The results verified 

that C103- was a competitive inhibitor of NO3- uptake as earlier reported (3) 

(Fig. 5). 

NO3- and C103- since proportionately much larger concentrations of C103- were 

required to inhibit NO3- uptake. 

trapping agent for effluxed NO3- for short time experiments. 

Apparently the NO3- transporter discriminated effectively between 

In summary, C103- could be used as a 

NO3- efflux. The method for determining efflux was based on some 

excellent work done by Lee and Clarkson (6) and Shone and Flood (16) who used 

cereal roots to determine the kinetic parameters required to measure efflux of 

NO3-. 

film attached to the roots from the external solution and from the root free 

space was ca 7 sec. In addition, their results showed that the t for 

cytoplasmic release of NO3- was ca 4 min. 

time resulted in a linear regression line after 1 min, since at that time the 

first two kinetic parameters had passed through ca 9 half-lives. 

results, the relative contributions of these parameters would be very small in 

They found that the combined t of release of NO3- in the surface 1/2 

1/2 

A semilog plot of efflux rate vs 

In our 

relation to the amount of NO3- efflux. The rates of efflux we measured 

similar to those reported for cereals by workers using 13N-N03- at NO3- 

concentrations of 1.5 to 5 mM (3,4,6). 

Effect of increasing concentrations NO3- and C103- on NO3- efflux 

expected, efflux increased with increasing concentrations of NO3- (Fig. 

are 

As 

7). 

In the region of Mechanism I1 of uptake (0.2 mM), efflux was a significant 

deterrent to N use efficiency. 

Net NO3- uptake remained quite constant as external NO3- increased beyond 0.2 

mM. This occurred because, although influx increased, efflux correspondingly 

increased. The ratio of efflux/influx, expressed as a percentage, increased 

up to 45% at 10 mM NO3-. 

Here 15% of the NO3- influxed was effluxed. 

These results help explain results from Raper's 

b 
I. 

laboratory which showed that net uptake changed little as NO3- concentrations 

increased to high levels (11). 

significant levels of efflux at increasing concentrations of NO3-. 

These workers also presented evidence for 

Effect of light and dark on influx, efflux, and net uptake of NO3-. 

Since efflux is a function of the concentration of NO3- in the cytoplasm, 

light and dark periods had a profound effect on the ratio of efflux/influx 

(Table 4). Although efflux was the same in light or dark, influx was greatly 

increased in light along with the rate of NO3- reduction (17,18). Therefore, 

the relative percentage of NO3- effluxed is much less in light. 

Effect*of increasing concentrations C103- on NO3- efflux. Higher rates 

of NO3- efflux were detected as C103- increased in the efflux solution (Fig. 

7). 

an unknown effect. 

sufficiently large to be somewhat uncertain. 

result. 

This could be because of C103- serving as a trapping agent or because of 

The increase in efflux rate with 5 and 10 mM C103- was 

We are currently evaluating this 

Effect of NH4+ on NO3- efflux. The literature is confusing on this 

issue with results of little if any effect of NH4+ on NO3- efflux (5,6), and 

of large effect (4). We have not yet examined the interactions of the 

different N species on NO3- efflux. 

Ramifications. As the concentration of NO3- increases in the external 

solution, efflux becomes an increasingly important energy cost to the plant. 

Not only is energy needed for NO3- influx, it now appears that NO3- efflux may 

utilize ATP (12). This results in an almost doubling of the cost of 

absorption at higher concentrations of NOg-. 

that a nutrient solution for crop growth in CELSS should be optimized at the 

lowest concentrations possible. In addition, it is increasingly critical to 

determine the regulation of efflux of N compounds since it is not known how 

NO3-, NH4+, NO2-, and urea influence each others efflux. Will total efflux 

On this basis, it would seem 

7 
decreaee if the tbtal cahcihtration cif N in the wtrieht solukion lrl made up 

of the four spaciem instead af only NOg- and NH4+P 
REFERENCES 

1. Veen BW 1980 Energy cost of ion transport. In: Genetic engineering of 

osmoregulation, Eds, DW Rains, RC Valentine, A Hollaender. Plenum Press, 

New York, NY 

2. Deane-Drummond CE, ADM Glass 1982 Nitr e uptake into barley (Hordeum 

vulgare) plants. A new approach using “C103- as an analogue for NO3-. 

Plant Physiol 70:50-54 

3. Deane-Drummond CE, ADM Glass 1983a Short-term studies f nitrate uptake 

into barley plants using ion-specific electrodes and 38C103-. I. Control 

of net uptake by NO3- efflux. Plant Physiol 73:lOO-104 

into barley plants using ion-specific electrodes and 38C103‘. Regulation 

of NO3- efflux by NH4+. Plant Physiol 73:105-110 

4. Deane-Drummond CE, ADM Glass 1983b Short-term studies f nitrate uptake 

5. Glass ADM, RG Thompson L Bordeleau 1985 Regulation of nitrate influx in 

13 - barley. Studies using 

6. Lee RB, DT Clarkson 1986 Nitrogen-13 studies of nitrat fluxes in barley 

roots. I. Compartmental analysis from measurements of f3N efflux. J 

NO3 . Plant Physiol 77:379-381 

Exptl Bot 37:1753-1767 

7. Jackson WA, KD Kwik, RJ Volk 1976a Nitrate uptake during recovery from 

nitrogen deficiency. Physiol Plant 36:174-181 

8. Jackson WA, KD Kwik, RJ Volk, RG Butz 1976b Nitrate influx and efflux by 

intact wheat seedlings: effects of prior nitrate nutrition. Planta 

132~149-156 

9. Lee RB, MC Drew 1986 Nitrogen-13 studies of nitrate fluxes in barley 

roots. 11. Effect of plant N-status on the kinetic parameters of nitrate 

influx. J Exptl Bot 37:1768-1779 

10. MacKown CT, WA Jackson, RJ Volk 1982 Restricted nitrate influx and 

reduction in corn seedlings exposed to ammonium. Plant Physiol 69:353- 

359 

11. Tolley-Henry L, CD Raper, TC Granato 1988 Cyclic variations in nitrogen 

uptake rate of soybean plants: effects of external nitrate concentration. J Exptl Bot 39:613-622 

12. Briskin DP 1988 Nitrate transport in plasma membrane vesicles from red 

beet (Beta vulgaris L.) storage tissue. Plant Physiol (Supple) 86:475 

13. Goyal SS, RC Huffaker 1986a A novel approach and a fully automated 

transport simultaneously by intact wheat seedlings. Plant, Cell and 

Environ 9:209-215 

microcomputer-based system to study kinetics of NO3-, NO2-, and NH4 + 

14. Goyal SS, RC Huffaker 1986b The uptake of NO3-, NO2-, and NH4+ by intact 

wheat (Triticum aestivum) seedlings. I. Induction and kinetics of 

transport systems. Plant Physiol 82:1051-1056 

9 
15. Guy M, G Zabala, P Filner 1988 The kinetics of chlorate uptake by XD 

tobacco cells. Plant Physiol 86:817-821 

16. Shone MGT, AV Flood 1985 Measurement of free space and sorption of large 

molecules by cereal roots. Plant, Cell and Environ 8:309-315 

17. Aslam M, RC Huffaker 1982 In vivo nitrate reduction in roots and shoots 

of barley (Hordeum vulgare L.) seedlings in light and darkness. Plant 

Physiol 70:1009-1013 

18. Aslam M, RC Huffaker 1984 Dependency of nitrate reduction on soluble 

carbohydrates in primary leaves of barley under aerobic conditions. 

Plant Physiol 75:623-628 

10 
Table 1. 

The initial concentration of substrate NO3 was 0.6mM. 

Materials and Methods for procedures. 

Effect of pretreatment of C103 on NO3 uptake. 

See 

............................................................ 

1 

2 

3 

7.3 

6.2 

5.3 

4.9 

5.3 

3.0 

2.3 

11 
PRETREATMENT (1mM) 

HOURS 

PRETREATMENT 

0 

3 

5 

6.3 

6.8 

3.5 

1.1 

0.5 

12 
Table 3. Efflux, net uptake and influx of NO3. See 

Materials 61 Methods for procedures. .......................................................... 

INFLUX, EFFLUX, AND NET UPTAKE OF NO3 

0.2 

1.0 

10.0 

1.1 

2.5 

7.7 

6.5 7.6 15 

8.5 11.0 23 

9.5 17.2 45 

13 
Table 4. Effect of light and dark treatments (24h) on NO 

efflux, net uptake and influx. 

procedures. 

See Material and Methods $or 

............................................................ 

EFFLUX NET INFLUX EFFLUX 

UPTAKE INFLUX 

TREATMENT 

(/J.rmol/gxh) % 

LIGHT* 1.3 7.9 9.2 14 

DARK 1.0 1.8 2.8 35 

* plants induced in 0.2m.M NO3 for 24h 

light or dark 

14 
FIGURE LEGENDS 

Fig. 1. NO3 uptake across Mechanisms I and 11. See 

Materials and Methods for procedures. 

Fig. 2. NO3 uptake across Mechanism I. See Materials and 

Methods for procedures. 

Fig. 3. Depletion of NO3 from 0.5 and l.0mM substrate 

solutions. See Materials and Methods for procedures. 

Fig. 4. Depletion of C103 from 0.5 and l.0mM substrate 

solutions. See Materials and Methods for procedures. 

Fig. 5. C10 as a competitive inhibitor of NO3 uptake. See 

Materials ana Methods for procedures. 

Fig.6. Semilog plot of NO3 efflux vs time. See Materials 

and Methods for procedures. 

Fig.7. Effect of C103 on NO3 efflux. See Materials and 

Methods for procedures. 

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22 
Report Documentation Page 

1. Report No. 

NASA CR 177534 

2. Government Accession No. 

4. Title and Subtitle 

19. Security Classif. (of this report) 

Unclassified Unclassified 23 

20. Security Classif. (of this page) 21. No. of pages 

Efficiency of N Use by Wheat as a Function of Influx and 

Efflux of NO3'. 

22. Price 

A0 2 

7. Author(s1 

R.C. Huffaker, M. Aslam, and M.R. Ward 

9. Performing Organization Name and Address 

Plant Growth Laboratory 

University of California, Davis 

Davis, CA 95616 

12. Sponsoring Agency Name and Address 

National Aeronautics and Space Administration 

Washington, D.C. 24056 

3. Recipient's Catalog No. 

5. Report Date 

July 1989 

6. Performing Organization Code 

8. Performing Organization Report No. 

10. Work Unit No. 

199-61-12 

11. Contract or Grant No. 

NCC2-99 

13. Type of Report and Period Covered 

Contractor Report 

14. Sponsoring Agency Code 

15. Supplementary Notes 

Point of Contact: MS 239-4, Moffett Field, CA 94035 

Robert D. MacElroy, NASA-Ames Research Center 

41 5-694-5573 or FTS 8-464-5573 

16. Abstract 

Since N assimilation is one of the most costly functions of a plant, its 

efflux before assimilation results in a serious energy cost and loss in effic 

which could decrease yields. Efficient crop production is critical to CELSS. 

ency 

Our objective is to determine the extent of efflux of the N species NO -",NHv+, 

NO2-, and urea after uptake, and possible means of regulation. 

of NO - was greater in darkness (35%) than in light (14%) and the ratio greatly 

increased with increased substrate NO -, (up to 45% at 10mM). It seems 

advantageous to use the lowest possibje nutrient concentration of NO -. 

feasibility of using C10 - as a trapping agent (competitive inhibit02 of NO - 

uptake) for effluxed NO was assessed and its toxicity determined. 

We fou2d that 

- efflux became serious as its substrate level increased. Efflux/lnflux (E/I) No 3 

The 

3 

3 

17. Key Words (Suggested by Author(s1) I 18. Distribution Statement 

Life support systems, CELSS, Plant 

product i v i ty Unclassified - Unlimited 

Subject category 54