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 the Closed Ecology Life Support System (CELSS). The objective is to determine the extent of efflux of the N species NO3(-), NH4(+), NO2(-), and urea after uptake, and possible means of regulation. Researchers found that NO3 efflux became serious as its substrate level increased. Efflux/Influx (E/I) of NO3(-) was greater in darkness (35 percent) than in light (14 percent), and the ratio greatly increased with substrate NO3 (-), (up to 45 percent at 10 mM). It seems advantageous to use the lowest possible nutrient concentration of NO3(-). The feasibility of using ClO3(-) was assessed and its toxicity determined

Aslam, M.

Huffaker, R. C.

Ward, M. R.

English

NASA Technical Reports Server

N91-3i778
EFFICIENCY OF N USE BY WHEAT AS A FUNCTION OF
o
INFLUX AND EFFLUX OF NO 3
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 +, NO 2- and urea after uptake, and possible means of regulation. We
found that NO3"'efflux became serious as its substrate level increased.
Efflux/Influx (E/I) of NO 3- was greater in darkness (354) than in light (144)
and the ratio greatly increased with increased substrate NO3- , (up to 454 at
I0 mbl). It seems advantageous to use the lowest possible nutrient
concentration of NO 3- The feasibility of using CI03" as a trapping agent
(competitive inhibitor of NO 3" uptake) for effluxed NO 3- was assessed and its
toxicity determined.
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 NO 3- assimilation is particularly high not only due to the energy
requirement of uptake but also because of the need for I0 electrons to reduce
it to the level of glutamate. In addition are the costs of maintaining pH
balance, since each reduction of NO 3- to the level of NH4 + produces an OH"
Thus it is not surprising that estimates of respiratory costs of ion
absorption range up to 504 of total root respiration and 20_ of total plant
respiration (i).
Recent reports in the literature indicate that NO 3" efflux can begin
very rapidly after its influx (2,3,4,5,6). In addition, prolonged leakage of
NO 3- occurs from root storage pools dependent upon the N-status of the roots
55
https://ntrs.nasa.gov/search.jsp?R=19910022464 2020-03-19T17:03:49+00:00Z
(7,8,9,10,11). Lee and Clarkson (6) estimated that efflux, at external
concentrations above i mM, could account for up to 40% of the influxed NO3-.
Thus NO 3- efflux could represent a significant additional cost to an already
energy costly assimilatory pathway. Little is known concerning the regulation
of NO 3- efflux. Recent work of Briskin (12) showed evidence that efflux of
NO 3- utilizes ATP, which could greatly add to the energy cost of NO 3-
assimilation.
Measurement of NO 3" efflux has been hampered by the lack of easy, rapid,
and less costly analytical techniques for detecting the very low concentra-
tions involved in a process with a half-llfe (tl/2) of minutes. The use of
15N as a tracer is laborious and has problems of sensitivity. 13N has only a
i0 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. In addition, we evaluated CI03- as a trapping
agent for NO 3"
This report presents results estimating NO 3" influx, efflux, and net
uptake across several mechanisms of NO 3- 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 _E/m2sec, 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 NO 3" at the concentration to be used in the efflux study
(specified in each experiment).
Measurement of NO 3- efflux. After removal from the loading solution, i0
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 CaSO4, and with
or without CI03- at the specified concentrations. The seedlings were rinsed
58
for 2 sec in 300 ml of efflux solution, then placed in 60 ml of efflux
solutions for the following times: I0 sec, 30 sec, I, 2, 5, I0, 15, and 20 min
and the amount of NO 3- released at each time from the roots was determined.
Measurement of NO 3- and C103-. These compounds were measured by HPLC as
described previously for NO 3- (13,14). CI03" was also measured at 210 nm with
a UV monitor.
Uptake rates of NO 3" and C103-. Uptake rates were determined as
previously described by determining rates of depletion of NO 3- from substrate
solutions, then fitting the rate curves to best fit curves by polynomial
analysis using a computer (13).
RESULTS
Mechanisms of NO 3" uptake. The results in Figures i and 2 show several
mechanisms for NO 3- uptake. Uptake as a function of NO 3- concentration can be
determined by either step up or step down, on continuous depletion experi-
ments; Figures I and 2 are the results of continuous depletion experiments.
In Figure I, 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 II. Another mechanism is indicated in Figure i at concentrations
below 0.i mM and it is readily seen when the data are plotted between 0 and
0.i mM (Fig. 2).
Comparison of uptake of NO 3- and CI03-, The comparative uptake of NO 3"
and CI03- is shown in Figures 3 and 4 at initial concentrations of 0.5 and 1
mM. Wheat plants deplete the NO 3- concentration very efficiently to near zero
(Fig. I), whereas depletion of CI03- is not straight forward, The ability to
take up CI03" is continuously lost with time (Fig. 4).
Toxic effects of 0103 - on NO 3" uptake. The increasingly toxic effects
of CI03" on NO 3- uptake with time is seen in Table I.
5Y
Comparative effects of pretreatments of CI0 3" and NO 3- on their uptakes.
The comparative effects of pretreatments of CI0 3- and NO 3" on their uptakes
are shown in Table 2. Pretreatments varying in time had little effect on
subsequent uptake of NO3", whereas increasing time of pretreatments greatly
decreased CIO 3" uptake.
CI0 3- as a competitive inhibitor of NO 3" uptake. Double reciprocal
plots of rates vs concentrations show evidence that CI0 3- is a competitive
inhibitor of NO 3" uptake (Fig. 5).
NO 3" efflux. Figure 6 shows a typical example of a determination of
NO 3- efflux. Our results matched quite closely those reported in the
literature showing two different early losses of NO3 °, one with a tl/2 of less
than I0 sec and another with a tl/2 of minutes. After one min, the second set
of rates approximate an apparent first order reaction. Extrapolation to t o
gives an estimate of the rate of NO 3- efflux. At a concentration of I mM
NO3" , efflux varied consistently between 2.0 and 2.5 _mol/gxh.
Effect of increasing concentrations of NO 3- and CI03" on NO 3- efflux.
Efflux in the presence of increasing concentrations of NO 3- and CI03- is shown
in Figure 7. As expected, efflux increased with increasing concentrations of
NO 3- and CI03-.
Influx, efflux, and net uptake of NO3-. Comparative rates of the three
kinetic components of NO 3- absorption are shown in Table 3. Efflux and influx
greatly increased between 0.2 and i0 mM external NO3-, whereas net uptake
remained about the same. Efflux/influx increased from 15 to 45% with
increasing concentration of NO 3"
Effect of light and dark on influx, efflux, and net uptake of NO3-.
NO 3- efflux was similar in plants in darkness and in light; however, influx
and net uptake were much greater in light (Table 4). Thus, in darkness 35% of
the influxed NO 3- was effluxed, while in light the proportion effluxed was
reduced to 14%.
58
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 NO 3" uptake. At least three mechanisms of NO 3- uptake are
present, one between 0 and about 0.05 to 0.08 mM with a K m of ca 0.012 to
0.018 mM, and another between 0.i and about 0.7 mM with a k m 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 I mM, the mechanisms are largely
undefined and are difficult to determine because efflux becomes such an
important component (discussed below).
Comparison of uptake of NO 3- and C103-. In a determination of NO 3-
efflux, very low concentrations of NO 3- are present in the external solution.
At these low levels of NO 3- (see Fig. 3), the wheat plant can very efficiently
absorb the NO 3" as it is effluxed into the external solution. Hence, CI03-
has been used as a trapping agent for the effluxed NO 3-
Although much work has been reported on the effects of Cl03- on NO 3-
uptake, the analytical procedures reported to separate ClO 3- from NO 3" in the
solutions were in some cases non specific, i.e., ion electrodes (3). Radio-
active CI03- was also used (3) which presents problems of low specific
activity and sticking to glassware. We developed an HPLC method which
effectively separates NO 3- 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 Cl03" on NO 3- uptake (15).
Toxic effects of Ci03-, The results showed that toxicity symptoms, as
shown by decreased rates of uptake of NO 3- and Ci03" , were apparent after 1 h
(Fig. 4, Tables 1 and 2). Toxic effects of Cl03- were greater toward its own
59
uptake than towards NO 3" uptake. We found that little toxicity occurred
during the 20 min period of the efflux experiments.
CI03" as a competitive inhibitor of NO 3- uptake, The results verified
that CIO 3- was a competitive inhibitor of NO 3" uptake as earlier reported (3)
(Fig. 5). Apparently the NO 3- transporter discriminated effectively between
NO 3" and CI03" since proportionately much larger concentrations of CI03" were
required to inhibit NO 3" uptake. In summary, CI03" could be used as a
trapping agent for effluxed NO 3- for short time experiments.
NO 3- 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-. They found that the combined tl/2 of release of NO 3- in the surface
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 tl/2 for
cytoplasmic release of NO 3- was ca 4 min. A semilog plot of efflux rate vs
time resulted in a linear regression line after i min, since at that time the
first two kinetic parameters had passed through ca 9 half-lives. In our
results, the relative contributions of these parameters would be very small in
relation to the amount of NO B- efflux. The rates of efflux we measured are
similar to those reported for cereals by workers using 13N-N03" at NO 3-
concentrations of 1.5 to 5 mM (3,4,6).
Effect of increasing concentrations NO 3- and CI03- on NO 3- efflux. As
expected, efflux increased with increasing concentrations of NO 3- (Fig. 7).
In the region of Mechanism II of uptake (0.2 raM), efflux was a significant
deterrent to N use efficiency. Here 15% of the NO 3- influxed was effluxed.
Net NO 3" uptake remained quite constant as external NO 3- increased beyond 0.2
nuM. This occurred because, although influx increased, efflux correspondingly
increased. The ratio of efflux/influx, expressed as a percentage, increased
up to 45% at i0 mM NO 3- These results help explain results from Raper's
60
laboratory which showed that net uptake changed little as NO 3- concentrations
increased to high levels (II). These workers also presented evidence for
significant levels of efflux at increasing concentrations of NO3".
Effect of light and dark on influx, efflux, and net uptake of NO3-.
Since efflux is a function of the concentration of NO 3- 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 NO 3" reduction (17,18). Therefore,
the relative percentage of NO 3- effluxed is much less in light.
Effect of increasing concentrations CI03- on NO 3- efflux, Higher rates
of NO 3- efflux were detected as CI03- increased in the efflux solution (Fig.
7). This could be because of CI03" serving as a trapping agent or because of
an unknown effect. The increase in efflux rate with 5 and I0 mM CI03" was
sufficiently large to be somewhat uncertain. We are currently evaluating this
result.
Effect of NH4 + on NO 3 efflux. The literature is confusing on this
issue with results of little if any effect of NH4 + on NO 3- efflux (5,6), and
of large effect (4). We have not yet examined the interactions of the
different N species on NO 3- efflux.
Ramifications. As the concentration of NO 3- increases in the external
solution, efflux becomes an increasingly important energy cost to the plant.
Not only is energy needed for NO 3- influx, it now appears that NO 3- efflux may
utilize ATP (12). This results in an almost doubling of the cost of
absorption at higher concentrations of NO3-. On this basis, it would seem
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 +, NO 2 , and urea influence each others efflux. Will total efflux
61
decrease if the total concentration of N in the nutrient solution is made up
of the four species instead of only NO 3- and NH4+?
62
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
i0.
ii.
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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
Deane-Drummond CE, ADM Glass 1982 Nitr_e uptake into barley (Hordeum
vulgare) plants. A new approach using _VCl03- as an analogue for NO3-.
Plant Physiol 70:50-54
Deane-Drummond CE, ADM Glass 1983a Short-term studies _f nitrate uptake
into barley plants using ion-speclflc electrodes and 3 CI03- " I. Control
of net uptake by NO 3 efflux. Plant Physiol 73:100-104
Deane-Drummond CE, ADM Glass 19835 Short-term studies of nitrate uptake
36
into barley plants using ion-speciflc electrodes and ClO_-. Regulation
of NO 3 efflux by NH 4 . Plant Physiol 73:105-110
Glass ADM, RG Thompson13L Bordeleau 1985 Regulation of nitrate influx in
barley. Studies using NO3". Plant Physiol 77:379-381
Lee RB DT Clarkson 1986 Nitrogen-13 studies of nitrate fluxes in barley
' 13
roots. I. Compartmental analysis from measurements of N efflux. J
Exptl Bot 37:1753-1767
Jackson WA, KD Kwik, RJ Volk 1976a Nitrate uptake during recovery from
nitrogen deficiency. Physiol Plant 36:174-181
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
Lee RB, MC Drew 1986 Nitrogen-13 studies of nitrate fluxes in barley
roots. II. Effect of plant N-status on the kinetic parameters of nitrate
influx. J Exptl Bot 37:1768-1779
MacKown CT, WA Jackson, RJ Volk 1982 Restricted nitrate influx and
reduction in corn seedlings exposed to ammonium. Plant Physiol 69:353-
359
Tolley-Henry L, CD Raper, TC Granato 1988 Cyclic variations in nitrogen
uptake rate of soybean plants: effects of external nitrate concentra-
tion. J Exptl Bot 39:613-622
Briskin DP 1988 Nitrate transport in plasma membrane vesicles from red
beet (Beta vulgaris L.) storage tissue. Plant Physiol (Supple) 86:475
Goyal SS, RC Huffaker 1986a A novel approach and a fully automated
microcomputer-based system to study kinetics of NO3- , NO2" , and NH4 +
transport simultaneously by intact wheat seedlings. Plant, Cell and
Environ 9:209-215
RC Huffaker 19865 The uptake of NO3", NO2-, and NH4 + by intactGoyal SS,
wheat (Triticum aestivum) seedlings. I. Induction and kinetics of
transport systems. Plant Physiol 82:1051-1056
63
15.
16.
17.
18.
Guy M, G Zabala, P Filner 1988 The kinetics of chlorate uptake by XD
tobacco cells. Plant Physiol 86:817-821
Shone MGT, AV Flood 1985 Measurement of free space and sorption of large
molecules by cereal roots. Plant, Cell and Environ 8:309-315
Aslam M, RC Huffaker 1982 In vivo nitrate reduction in roots and shoots
of barley (Hordeum vulEare L.) seedlings in light and darkness. Plant
Physiol 70:1009-1013
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carbohydrates in primary leaves of barley under aerobic conditions.
Plant Physiol 75:623-628
B4
Table I. Effect of pretreatment of CIO 3- on NO3 ° uptake. The initial
concentration of substrate NO 3 was 0.6 raM.
[ClO 3- ]
ImM 5mM
Hours Pre- NO 3- Uptake
treatment (_mol/gxh)
0 7.3 7.3
1 6.2 5.3
2 5.3 3.0
3 4.9 2.3
Table 2. Effect of pretreatment of CIO 3- or NO 3- on their uptakes.
Hours Pre-
treatment
Pretreatment (i mM)
NO 3" CIO 3"
Uptake (at I mM)
(#mol/gxh)
6.3 3.5
I.i
6.8 0.5
66
°Table 3. Efflux, net uptake and influx of NO 3 • See Materials and
Methods for procedures.
EFFLUX, INFLUX, AND NET UPTAKE OF NO 3
Net Efflux
Efflux Uptake Influx Influx
[NO3-] _mol/gxh(mM)
0.2 I.i 6.5 7.6 15
1.0 2.5 8.5 II.0 23
i0.0 7.7 9.5 17.2 45
67
Table 4. Effect of light and dark treatments (24 h) on NO 3 efflux, net
uptake and influx. See Materials and Methods for procedures.
Treatment
Efflux Net Influx Efflux
Uptake
(#mol/gxh)
Light* 1.3 7.9 9.2 14
Dark 1.0 1,8 2.8 35
plants induced in 0.2 mM NO 3 for 24 h light or dark.
FIGURELEGENDS
-
Fig. I. NO 3 uptake from 0 to i000 #m. See Materials and Methods for
procedures.
Fig. 2. NO 3- uptake from 0 to I00 _m. See Materials and Methods for
procedures.
Fig. 3. Depletion of NO 3 from 0.5 and 1.0 mM substrate solutions. See
Materials and Methods for procedures.
Fig. 4. Depletion of CIO 3 from 0.5 and 1.0 mM substrate solutions. See
Materials and Methods for procedures.
Fig. 5. CiO 3" as a competitive inhibitor of NO 3- uptake. See Materials and
Methods for procedures.
Fig. 6. Semilog plot of NO 3- efflux vs time. See Materials and Methods for
procedures.
Fig. 7. Effect of CIO 3- on NO 3" efflux. See Materials and Methods for
procedures.
69
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Efficiency of N use by wheat as a function of influx and efflux of NO3

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