cepa L.). Twenty-eight to 84 day-old onion plants were treated with NH + :NO~" ratios ranging from 0 to 100% of each ionic species in one mM solutions in a ...
Plant and Soil 171: 289-296, 1995. © 1995 KluwerAcademic Publishers. Printed in the Netherlands.
Effect of NH4+ : NO 3 ratios on growth and nitrogen uptake by onions * C. A b b ~ s 1, L.E. Parent 1, A. K a r a m 1 and D. Isfan 2 1ERSAM, Department of Soil Science, Paul-ComtoisBuilding Laval University, Sainte-Foy, Quebec, Canada, GI K 7P4 and 2Research Station, Ministry of Agriculture and Agrifood of Canada, 2560 Hochelaga Blvd., Sainte-Foy, Quebec, Canada, GIV 2J3 Received 3 November 1993. Accepted in revised form 15 October 1994
Key words: Allium cepa L., ammonium, kinetics, N influx, nitrate, onion
Abstract The modelling of ion uptake by plants requires the measurement of kinetic and growth parameters under specific conditions. The objective of this study was to evaluate the effect of nine NH + :NO 3 ratios on onions (Allium cepa L.). Twenty-eight to 84 day-old onion plants were treated with NH + :NO~" ratios ranging from 0 to 100% of each ionic species in one mM solutions in a growth chamber. Maximum N influx (Imax) was assessed using the N depletion method. Except at an early stage, ionic species did not influence significantly Irnax,the Michaelis constant (Km) and the minimum concentration for net uptake (Cmin). Imax for ammonium decreased from 101 to 59 pmole cm -2 s- 1 while ]maxfor nitrate increased from 26 to 54 pmole cm -2 s- 1 as the plant matured. On average, Km and Cmi, values were 14.29/aM, and 5.06/aM for ammonium, and 11.90/aM and 4.54/aM for nitrate, respectively. In general, the effect ofNH + :NO 3 ratios on root weight, shoot weight and total weight depended on plant age. At an early stage, maximum plant growth and N uptake were obtained with ammonium as the sole source of N. At later stages, maximum plant growth and N uptake were obtained as the proportion of nitrate increased in the nutrient solution. The was no apparent nutrient deficiency whatever NH + :NO 3 ratio was applied, although ammonium reduced the uptake of cations and increased the uptake of phosphorus.
Abbreviations: Imax-influx maximum, Kin-concentration in solution corresponding to half-maximum influx, Cmin-concentration in solution below which influx ceases. Introduction The soil solution contains both nitrate and ammonium ions in proportions varying with N fertilizer, soil type and season (Haynes and Goh, 1978). Plants may have seasonal preference for ionic species depending on growth stage. For most agricultural crops, nitrate is the main source ofN (Rao and Rains, 1976). The preference for nitrate over ammonium varies with plant species, plant developmental stage and several environmental factors (Gamiely et al., 1991). Nitrogen uptake models generally assume that transport processes are little affected by nitrification. Such simplification was believed to underestimate nitrogen uptake * The research was supported by the Natural Sciences and Engineering Research Council of Canada.
by plants (Barber, 1984). Barber and Cushman (1981) proposed but did not validate a mathematical model for simultaneous N uptake from both ammonium and nitrate, assuming that N was supplied to roots by mass flow and diffusion. Only ammonium uptake was further simulated using N-serve as nitrification inhibitor (Anghinoni and Barber, 1988). Nitrogen uptake may be affected by plant preference for N ionic species, antagonisms between ionic species, and plant age. Schrader et al. (1972) and Warncke and Barber (1973) showed that the N ionic species did not influence N absorption rate by corn seedlings or N influx into corn roots. DeaneDrummond and Glass (1983) found that NH4-N inhibition of NO3-N influx into barley seedlings was affected by previous NO3-N treatment. Lycklama (1963)
290 observed that NH4-N absorption by ryegrass seedlings was affected by NO3-N to a small extent, but that NO3-N absorption was inhibited to a large extent by NH4-N. Fried et al. (1965) found that NH4-N uptake by rice seedlings was larger than NO3-N uptake when both ions were present in equal amounts. Minotti et al. (1969) and Rao and Rains (1976) found that NO3-N uptake by wheat and barley seedlings was inhibited by NH4-N. Nitrate uptake by containerized Douglas fir seedlings was also inhibited by ammonium when both ionic species were present (Kamminga-van Wijk and Prins, 1993). The effects of various proportions of NO3-N and NH4-N on vegetable crops received little attention. Plant nutrition as a whole also depends to some extent on nitrate or ammonium nutrition. Enhanced translocation and accumulation of cations under nitrate nutrition is frequently observed (Deane-Drummond and Glass, 1983). Haynes and Gob (1978) found that potassium uptake by wheat roots was reduced by ammonium. Phosphorus uptake generally increased when NH4-N replaced NO3-N as nitrogen carrier (Serna et al., 1992). Thus, varying NH+:NO3 ratios in the root environment over time may affect N uptake kinetics, nutrient acquisition and plant growth. An integrated knowledge of those parameters could improve N uptake models applied to soil-plant systems. The objective of this study was to determine the effect of various NH + :NO~ ratios in the nutrient solution on onion growth, N uptake parameters and nutrient acquisition.
Materials and methods
Pre-treatments of onions seedlings Onion seedlings (Allium cepa L. CV. Norstar 210B) were grown in nutrient solutions in a growth chamber for 28 to 84 day under the following conditions: 220 C/18° C and 16 h/8 h day/night cycles, 15 klux light intensity using white fluorescent lamps, and 65% RH. The experimental design was a split-plot comprising five plant ages (28, 42, 56, 70 and 84 days) as main plots and nine NH + :NO~" ratios as subplots: 4:0, 3:1, 2:1, 3:2, 2:2, 2:3, 1:2, 1:3 and 0:4 to give a total N concentration of 1 mM. There were three replications. Onion seeds were sown in fiats containing moist vermiculite. Four uniform seedlings were transplanted two weeks later into 4.2-L pots and placed in the growth chamber. The pots were covered with perforat-
ed PVC lids. Seedlings were grown in nutrient solutions for two weeks before kinetic tests began. Nutrient solutions contained 46/zM H3BO3, 9.0 #M MnSO4 • H20, 0.8 #M ZnSO4 • 7H20, 0.3/JM CuSO4 • 5H20, 0.5 #M NaMoO4 • 2H20, 75 mM FeSO4 • 7H20, 1.0 mM MgSO4, 1.5 mM CaSO4 and 1.0 mM KH2PO4 (Warncke and Barber, 1973). NH4-N and NO3-N were applied as (NH4)2SO4 and Ca(NO3)2 • 4H20, respectively. Solutions were changed every two days. Nutrient solutions were stirred constantly by air bubbling. To minimize pH drop and to balance Ca, 150 #M to 1 mM CaCO3 was added to nutrient solutions in order to maintain Ca concentration at 2.5 mM (Gamiely et al., 1991; Goyal and Huffaker, 1986). Concentration of SO4-S in solutions was maintained at 3 mM by adding CaSOa.2H20. Solution pH was adjusted to 6 daily using 1 M KOH or 1 M HC1.
Kinetic studies At each growth stage, plants were transferred into 0. I mM CaSO4 solutions one day before measurement of uptake parameters (Warncke and Barber, 1973). Roots were rinsed thoroughly with distilled water and held in the open air for 1 min to drain excess solution. Roots were placed into a diluted solution containing the same NH + :NO3 ratios but giving 0.1 mM of total N, i.e. one tenth of the initial concentration. Net uptake of NHa-N and NO3-N was determined by measuring nutrient depletion in solution over six hours. Five mL aliquots were collected at 30 min intervals and deep-frozen for later nitrate and ammonium analyses. Solution loss due to sampling, transpiration and evaporation was replaced by deionized water to maintain constant volume of solution. Nitrogen concentrations in solution samples were measured colorimetrically by a modified Technicon method (Vezina et al., 1992) for ammonium and by the Feller et al. (1971) procedure for nitrate.
Plant parameters After completion of the kinetic studies, plants were separated into roots and shoots. Fresh and dry (70°C) weights were determined. Dry tissues were ground in a Wiley mill, wet-digested using a mixture of HNO3 and HC104, and analyzed for S by turbidimetry (Blanchar et al., 1965), for P by colorimetry (Olsen and Sommers, 1982) and for K, Ca and Mg by atomic absorption spectrophotometry (Isaak and Kerber, 1971). Total N was determined by the modified reduced iron Kjeldahl
291 method (Cataldo et al., 1974). Root length (L) was estimated by the line intercept method (Tennant, 1975) using half of root fresh weight. Mean root radius (r0) and root surface area (RSA) were calculated from root volume, root fresh weight and root length according to Barber (1984). Total N uptake by onions (Table 2) was calculated by multiplying N concentrations in shoot and roots by the dry matter weight. Data were analyzed using the GLM procedure (SAS Institute, 1990). The Scott-Knott cluster analysis method was used for separating means as described by Gates and Bilbro (1978).
Nutrient influx was assumed to follow MichaelisMenten kinetics as described by Claassen and Barber (1974) and Barber (1984):
t [ Imax(C-Cmin) ] V [K-~(-(~--C'~)]
(1)
where C is the concentration of either NH +. or NO 3 in solution (#M), t is time (s), L is root length (cm) and V is the volume of solution in the pot (cm3); Imaxis the maximum ammonium or nitrate influx (pmole cm-2 s- 1); Km is the ammonium or nitrate concentration in solution (~tM) when influx is equal to Imax/2; Cmin is the minimum concentration in solution below which net influx is null (#M). In this paper nutrient influx was expressed per unit root surface area (RSA) instead of root length (Claassen and Barber, 1974). Hence, dC dt-
RSA [ Imax(C - Cmin) ] V [K-~'---~C---~n)I'
NH4+:NO3" o t~
4:0
3O
e
3:1 and 2:1
•
3:2 to 2:3
20 n3 !0
7 0 28
42 Plant
56
70
age
(day)
84
Fig. 1. Effectof NH+ :NO3 ratiosin solutionon total dry matter of onionsat fivegrowthstages.
Mathematical models
dE dt-
4o
(2)
Ammonium and nitrate data were fitted to Equation 2 using the Levenberg-Marquardt method in the Mathematica package (Novak, 1992). Nitrate and ammonium uptake were corrected for N removed by sampling.
Results and discussion Effect of NI-l~4 :NO 3 ratios on dry matter production The effect of NH + :NO 3 ratios on root weight, shoot weight and total weight generally depended on plant age (Fig. 1). After 28 days of growth, dry matter production was highest with the 4:0 NH+:NO~'0 ratio
and decreased thereafter. After 42 days of growth, NH4+ :NO 3 ratios < 1:2 produced smallest dry matter. After 56 days of growth and later on, total dry matter was largest with NH + :NO 3 ratios between 3:2 and 0:4. Thus, a shift in optimal NH+:NO3 ratios occured between 42 and 56 days of growth. In the 28 to 84-day interval, root and shoot dry matter production was smallest with the 4:0 ratio and largest with 1:2 to 0:4 ratios (Table 1). Similarly, Gamiely et al. (1991) found that, for 74 day-old onion plants, nitrate increased shoot and root weight, leaf area and bulb weight of onions as compared to ammonium; the largest bulb weight was obtained with NH + :NO~ ratios between 1:3 and 3:1. Since nearly all of the assimilated ammonia is translocated to the shoot as amino acids, amides and related compounds, ammonium assimilation has larger carbohydrate requirement than nitrate because of the need for carbon skeletons in the synthesis of nitrogenous compounds in the roots (Marschner, 1986). Root length and surface area were also influenced significantly and similarly by NH + :NO 3 ratios in relation to plant age (Fig. 2). After 28 days of growth, root length and surface area were largest with the 4:0 NH + :NO 3 ratio. After 56 days, root length and surface were greatest with the 2:3 to 0:4 ratios, and smallest with the 4:0 ratio. There was a significant effect of NH + :NO 3 ratios on rate of growth in length and surface area (Table 1). Rates were highest with NH+:NO3 ratios not exceeding 2:3. Rate of root growth was enhanced as nitrate proportions increased, thus providing more capacity to absorb nitrate. Ionic species and plant age did not influence significantly mean root radius (r0). Average r0 was 0.262 + 0.2 mm.
292 Table 1. Regression equation coefficients for the relation between root dry matter, shoot dry matter, total dry matter, root growth rate on the one hand and plant age on the other Treatment
Root dry matter
Shoot dry matter
Total dry matter
Root growth rate
NH4+ :NO 3
Az
A
A
Length x 1 0 4 c m s -1
4:0
0.009 fY
0.211 a
0.270 d
-5.164 a
0.314 d
-5.019 a
7.09 c
3:1
0.014 e
0.054 b
0.371 c
-9.582 b
0.381 c
-9.549 b
7.49 c
12.87 c
2:1
0.017 d
-0.073 c
0.409 c
-11.239 b
0.420 c
-11.279 b
8.18 b
13.63 b
3:2
0.018 d
-0.126 c
0.488 b
-13.554 c
0.500 b
-13.375 c
8.35 b
13.86 b
2:2
0.018 d
-0.054 c
0.517 b
-14.885 c
0.528 b
-14.866 c
8.56 b
14.35 b
B
B
B
Surface x 105cm2s-I 11.57 c
2:3
0.020 c
-0.126 c
0.534 b
-15.777 c
0.548 b
-15.867 c
10.27 a
16.35 a
1:2
0.022 b
-0.253 d
0.557 a
-17.523 d
0.572 a
-17.684 a
10.44 a
16.51 a
1:3
0.025 a
-0.360 e
0.586 a
-18.604 d
0.603 a
-18.627 a
10.00 a
15.89 a
0:4
0.024 a
-0.370 e
0.592 a
-19.201 d
0.608 a
-19.237 a
9.88 a
15.73 a
z Equation are Y = At + B,where Y = root,shoot or total dry matter, t = plant age in day. Y Means in each column followed by a different letter are significantly different (p _< 0.05) after the Scott-Knott mean separation procedure.
80
1200
Go 800
40
600
3o
400
20
28
42
56
70
84
28
Plant age
Fig. 2.
~ 42
56
70
84
(day)
Effect of NH4+ :NO 3 ratios in solution on length and surface of onion roots at five growth stages.
Effect of Nl-~4 :NO ~ ratios on absorption kinetics of ammnonium and nitrate The Imax, Km and Cmin values were determined for each plant age and NH + :NO~- ratio by measuring depletion of NH + and NO~ in solution over time and fitting Equation 2 to the data. The shape of depletion curves was similar among NH++ and NO{ treatments. Thus, depletion curves for nine NH + :NO 3 ratios were presented only for onion plants at mid-period (42 days) (Fig. 3). Rapid depletion indicated that no induction period was necessary for initial ammonium or nitrate uptake by onion roots whatever NH+:NO~ - ratio was applied. The values of Imax, Km and Cmin were derived as equation parameters. There was significant treatment
effect on Imax, Km and Cmin only at an early stage (Fig. 4). At the 28-day period, root Imax for the ammonium ion was significantly higher with the 4:0 ratio (101 pmole cm -2 s - l ) than with other ratios (59 pmole c m - 2 s - 1 in average). Relationships between Imax for ammonium (in pmole cm -2 s -1) and plant age (t, in days) were significantly quadratic (Fig. 4). For the 4:0 NH+:NO~ratio, the regression was: Imax = 214.70 -- 5.101 t + 0.030t 2, R 2 = 0.86 For other ratios, the regression was: Imax = 94.83 - 1.577 t + 0.008 t 2, R 2 = 0.96
293
NH 4 +
NO3 -
._. ]00 L
50
°oo T
el
T
4O
+
0 I
,
,
,
~o
,
,
,
5O
0
X
I I-'-
4:0
o
80
4O O -M
4~
O
.I00
N H 4 + :N O 3 -
14-I
Z
-:--:::
3O
6O
0 I--I ¢ ¢t
4O
0 I::1
2o
"t~
14 .U o u
20 ----
O U
i
0
60
I
120
i
i80
i
240
i
300
i
360
Time
Fig. 3.
o:o
10
0
60
120
!8(?
240
300
36(3
(min)
The depletion of NH+ -N and NO~--N in solution by 42-day onion plants as related to uptake time for different NH+ :NO3 ratios.
Vertical bars represent standard errors of the means.
Root Imax for nitrate increased linearly with plant age (Fig. 4) as follows:
ii0 " i00 " ~'~ i
~
Imax = 15.31 + 0.433 t, R z = 0.88 Edwards and Barber (1976), who investigated the effect of plant age and NH + :NO 3 ratios on N uptake by 2 to six-week-old corn plants, also found that Imax for ammonium decreased quadratically by plant age for various NH + :NO~ ratios. From day 28 to day 42, Imax for ammonium was considerably higher than Imax for nitrate. During the 42 to 56 day period, the Imax'S for ammonium and nitrate were similar. Thereafter Imax for nitrate exceeded considerably Imax for ammonium. The modelling of soil-plant systems should thus consider soil available ammonium and nitrate as well as plant age for improving the prediction of N uptake by crops.
a
90-
80-
16 O
70
"
®
60"
'~
50"
8
40"
M ~
°
30"
20" 10" i 28
| 42
Plant
i 56
age
i 70
I 84
(day)
Fig. 4. Maximum influx (Imax) of NH4-N and NO3-N in onions as + related to NH 4 :NO3 ratios and plant age (r-I) Imaxof NH4-N for 3:1 to 1:3 NUt :NO3 ratios, (BB) Imaxof NH4-N for the 4:0 NH+ :NO~ratio and ( O ) Imax of NO3- N for all NHt :NO 3 ratios.
No significant effect of plant age on Km for ammonium and nitrate was detected. Average Km was 14.29
294 4- 1.26 # M for ammonium and 11.90 4- 1.12 /~M for nitrate. Km values averaged 12 #M for both ions throughout growth stages. Km values obtained in this study were within the range of Km values reported for other crops. Comparative Km values for nitrate or ammonium were as follows: 12 to 20 ~M for 2 to 6 week-old corn plants (Edwards and Barber, 1976), 15 to 25 # M for tall fescue and reed canary grass seedlings (Barber, 1984), 34 /~M for rape seedlings (Bhat et al., 1979), 33 #M; for ryegrass seedlings (Lycklama, 1963), 21 to 30 #M for corn seedlings (Van den Honert and Hooymanns, 1955), 62 to 75/~M for rice seedlings (Teo et al., 1992) and 4- to 9-week-old rice plants (Youngdahl et al., 1982) and 8-17/~M for Douglas fir seedlings (Kamminga-van Wijk and Prins, 1993). Onion roots generally showed higher affinity for N ions than other crops. Km values are low as compared to nitrate concentration in the soil solution, which could range from 3 to 15 mM (Barber, 1984). As a result, ionic N influx into onion plants should proceed close to Imaxuntil most of the nitrate have been depleted from soil solution. There was no significant effect of plant age on Groin for ammonium and nitrate. Average Cmin was 5.06 4- 0.80 #M for ammonium and 4.54 4- 0.61 #M for nitrate. Warncke and Barber (1974), who investigated four plant species, obtained Cmin values of 1.7, 2.7, 2.4 and 1.4 #M for seedlings of forage sorghum, grain sorghum, soybeans and bromegrass, respectively. Cmin values were 3 to 9/zM for 2- to 6-week-old corn (Edwards and Barber, 1976), 0.3 #M for rye seedlings (Olsen, 1950), 3.1 #M for rice (Teo et al., 1992). Thus, onions could deplete both ionic forms of nitrogen to a very low concentration in the soil solution and be effective sinks for nitrate. To achieve high uptake effÉciency, plants should have as high a Imax value and as low Km and Cmin values as possible. At an early stage of growth, onions preferred ammonium. At later stages, onions preferred nitrate. Accordingly, N uptake was greatest with higher NH4+:NO~- ratios at an early stage, and with lower NH4+ :NO 3 ratios at later- stages (Table 2). Effect o f N H + :NO 3 ratios on elemental concentrations
The NH + :NO 3 ratios affected nutrient concentrations in roots and shoots at the 84-day stage (Table 3). According to Bergmann (1988), critical ranges for nutrient sufficiency in the foliage of onions at midseason are: 20.0-30.0 mg N k g - 1, 2.5-4.0 mg P k g - l,
Table 2. Effect of NH4 + :NO3 ratios in solution on N
uptake by onions Treatment Plant age (day) NH+ :NO3 28 42 56 70 (mM/pot) 4:0 3:1 2:1 3:2 2:2 2:3 1:2 1:3 0:4 CVY
11.52 z 7.53b 7.11b 5.46b 5.55b 4.74b 4.15b 3.98b 3.05b
84
18.60a 21.75b 31.13d 36.50f 19.19a 23.51b 39.99c 52.06e 18.76a 24.29b 43.70c 58.19d 20.07a 34.36a 53.81 b 68.44c 19.20a 35.56a 57.56b 73.20b 18.06a 35.47a 58.71b 74.90b 11.27b 34.94a 57.53b 76.10b 10.08b 35.10a 65.13a 83.88a 8.69b 34.71 a 64.05a 85.76a 8.78%
z Means in each column followedby a different letter are significantlydifferent(p _
