Root zone temperature sensitivity of nitrogen fixing and nitrate-supplied soybean [Glycine max (L.) Merr. cv maple arrow] and lupin (Lupinus albus L. cv ultra) plants

Environmentaland ExperimentalBotany, Vol. 34, No. 2, pp. 117 127, 1994

0098-8472(93)E0009--F

F'ergamon

Copyright © 1994 Elsevier Sci. . . . . Ltd Printed in Great Britain. All rights reserved 0098~472/94 $6.00 + 0.00

R O O T ZONE T E M P E R A T U R E S E N S I T I V I T Y OF N I T R O G E N F I X I N G A N D N I T R A T E - S U P P L I E D SOYBEAN [GLYCINE MAX (L.) M E R R . cv MAPLE A R R O W ] A N D L U P I N (LUPINUS ALBUS L. cv U L T R A ) PLANTS T. LEGROS and D. L. SMITH Plant Science Department, McGill University, Macdonald Campus, 21111 Lakeshore Road, Ste Anne de Bellevue, Qu+bec, Canada H 9 X 3V9

(Received 12 July 1993; accepted in revisedform 29 November 1993) LEGROS T. ANn SMITH D. L. Root zone temperature sensitivity of nitrogen fixing and nitrate-supplied soybean [Glycine max (L.) Merr. cv Maple Arrow] and lupin (Lupinus albus L. cv Ultra) plants. ENVIRONMENTAL AND EXPERIMENTALBOTANY 34, 117--127, 1994. Soybean [Glycine max (L.) Merr.] is a subtropical legume that is now often grown in cool temperate areas. U n d e r these conditions, low root zone temperatures (RZT) may adversely affect N2 fixation and therefore the growth and development of soybean plants. Lupin (Lupinus albus L.), is well adapted to cool temperate areas. Soybean exports N from root nodules as ureides, while lupin exports N as amides. The water solubility of ureides declines sharply with temperature while the solubility of amides is much less affected. Experiments were established to test the effect of suboptimal R Z T on N accumulation, N partitioning and dry matter partitioning by N2-fixing and mineral Nsupplied soybean and lupin plants. In the first experiment, soybean plants were grown at four constant R Z T : 10, 15, 20 and 25°C with constant 25°C air temperature. Four nitrogen (N) treatments were applied within each R Z T ; plants were fertilized with a complete N-free nutrient solution supplemented with either 0, 7.1 or 14.2 m M N, or received 14.2 m M N at the onset of the experiment with this level being gradually decreased to zero over the first 15 days of the experiment. In a second experiment, soybean and lupin were grown at 10, 15, 20 and 25°C R Z T and received either 0 or 14.2 m M N. Nitrogen accumulation and partitioning were more affected by low R Z T in N2-fixing than in mineral N-supplied soybean and lupin plants. Dry matter accumulation of N2-fixing soybean plants was also more sensitive to low R Z T than was that of mineral N-supplied soybean plants. The slower development of the N2-fixing soybean plants at R Z T less than 25°C was attributed to poor nodule development at temperatures in the 10°C range and to low nitrogenase activity at temperatures greater than 15°C. Data from the final harvest indicated that photosynthate shortage may have played a role in the reduced N2 fixation. Lupin had a higher tissue N concentration than soybean at low R Z T and a lower root to shoot per cent N ratio, but N 2 fixation in lupin seemed to be about as sensitive to low R Z T as that of soybean.

Key words: Glycine max (L.) Merr., Lupinus albus L., soybean, lupin, low root zone temperature, N 2 fixation,

nitrate uptake.

Abbreviations: DAP = days after planting, LSD = least significant difference, N P R = nodule to plant ratio, R G R = relative growth rate, R S N R = root to shoot N ratio, R S R = root to shoot ratio, R Z T = root zone temperature, SLW = specific leaf weight. 117

118

T. LEGROS and D. L. SMITH

ammonia uptake was much less sensitive than nitrate. (3i MATTHEWS and HAYES(18) found that TI~E fixation of N2 by the Bradyrhizobium japon- low R Z T restricted growth of uninoculated icum-soybean (Glycine max L.) symbiosis is econ- soybean mineral-N-supplied plants and inocuomically and environmentally important for the lated soybean plants grown in N-free medium. agricultural production of soybean. (26'28/As a sub- The plants relying solely on symbiotic N2 fixation tropical legume, soybean is best adapted to areas were much more affected than mineral-N-supwith long and warm growing seasons. Similarly, plied plants. However, as their findings were based its symbiont Bradyrhizobium japonicum requires on two separate experiments, one for mineral-Nwarm temperatures for optimum development supplied plants and one for N2-fixing plants, with and activity. !16;~The poor adaptability of soybean quite different rooting media in the two experito cool soils may be the primary yield-limiting ments, it is difficult to conclude with certainty factor in short-season areas. (2ui It has been dem- that the growth of N2-fixing soybean plants is onstrated that suboptimal soil root zone tem- more affected by low R Z T than that of mineralperatures (RZT) negatively affect nodulation in N-supplied plants. a number of legumes. !4'8'12) For soybean, variaA complex relationship exists between roots bility in sensitivity to R Z T may reflect the range and shoots of plants. A model proposed by RAPER of optima for particular soybean-Bradyrhizobium el al. !~°i shows a balanced interdependence genotype combinations] 16) Optima for soybean between shoot C supply and root N supply. At N2 fixation are most often reported to be around lower R Z T , root and shoot respiration decline 25°C. Below this temperature, the time to pro- while the net photosynthesis is less affected. The duce functional nodules increases and nodule photosynthates produced but not respired weight and number decreases. DuKE et al. ~6! accumulate in the leaves, largely as starch. (27)Low reported nodulation failure at 13°C, and MAT- R Z T also affects leaf area development through THEWS and HAYES/ia) reported similar results at changes in plant internal osmotic pressure, tl! and 10°C. Though some nitrogenase activity (acetylcausing N to be retained in the root tissues rather ene reduction) has been detected at temperatures than being partitioned to young growing tissues. as low as 3°C, '5/ nitrogenase activity is reported The latter results in N remobilization from older to decrease slowly with temperature between 25 tissue to maintain plant growth. (27iThe reduction and 16°C, and then rapidly between 16 and in leaf area then restricts root growth through 13°C. !6'~5)The more rapid decline was attributed diminished photosynthate production. to a sharp increase in the energy of activation of As the current literature contains no conclusive nitrogenase at lower temperatures. WALSI~ and comparison of the effects of R Z T on N2-fixing and LAYZELL (27) estimated that net nitrogenase mineral N-supplied soybean, an experiment was activity was reduced by 253/o between 25 and 15°C. However, at lower temperatures, this was conducted to study the effect of low R Z T on N partially compensated for by an increase in the accumulation and partitioning in soybean plants developing with a range of N levels, including 0 relative efficiency ofnitrogenase. (15i Soybean plants export most of the N fixed in N, and their effects on growth and dry matter nodules as ureides. !I9'23) Lupin, like a wide range partitioning. In addition, there are no published of other legumes, exports N from nodules as comparisons of cool R Z T effects on soybean (a amides. (25i The solubility of ureides in water is ureide exporter) and lupin (an amide exporter). low and decreases sharply as temperature Thus a second experiment compared the effects declines, while the solubility of amides in water is of suboptimal R Z T on N accumulation and N much higher and is much less affected by decreas- partitioning by N2-fixing soybean and lupin. Two ing temperature. Thus low root temperature may hypotheses were tested: (1) N2-fixing soybean is also limit the rate of export of fixed N from more sensitive to low R Z T than mineral N-supsoybean nodules. (25/ plied soybean, and (2) because of the low soluNitrate and ammonia uptake have also been bility of ureides at low temperatures, lupin is less reported as temperature sensitive, although sensitive to low R Z T than soybean. INTRODUCTION

LOW ROOT-ZONE TEMPERATURE, N 2 FIXATION AND SOYBEAN GROWTH MATERIALS AND M E T H O D S

Experiment 1 Seeds of the Canadian soybean cv Maple Arrow (maturity group 00) were sterilized with 70% alcohol and, immediately afterward, inoculated with B. japonicum inoculant (Nitragin inoculant, strain 532C, Nitragin Co., Milwaukee, Wis.). They were germinated and the seedlings grown for 10 days in vermiculite-filled trays (54 x 29 cm) at air and soil temperatures of 25°C with an 18-hr photoperiod and 300/IM m -2 s -l of photosynthetically active radiation provided by Coolwhite T M fluorescent tubes (General Electric, Montreal). Plantlets were selected for uniformity and transplanted singly into 13 cm pots, in a 1 : 1 sand/calcinated clay (turface) medium. Pots had been placed on a controlled environment growth bench (GB48, Conviron, Winnipeg, Canada), as a part of the apparatus described in the following paragraph, held at a constant air temperature of 25°C with the same photoperiod and light intensity as for seed germination, described above. The R Z T equipment consisted of four sets of plastic tanks (72 x 45 cm). In three of them, water was circulated around the 13 cm pots by pumps from three thermostatically controlled water baths; a _ 0.5°C precision was obtained. The pots in each tank were sealed, with water tight glue, to the bottom of the tank; a hole drilled in the bottom of the tank, below the pot, allowed the pots to drain. The R Z T treatments (10, 15, 20 or 25°C) were applied to 16 pots at a time. The experiment was repeated five times and each repetition was considered to be a replicate. The N treatment consisted of three constant levels of ammonium nitrate: 0 (low N), 7.1 (medium N), 14.2 mM N (high N), plus a fourth treatment in which the plants received 7.1 mM N at the onset of the experiment, this amount being gradually decreased to zero over 15 days (transient N). The N treatments were applied randomly within each R Z T treatment, and the R Z T treatments were randomly assigned to the tanks on the growth bench, making the experimental design a split plot with temperature as the whole plot factor and N level the subplot factor. All plants were watered daily with a N-free nutrient solution, 24 supplemented with ammonium nitrate at the

119

level indicated by the N treatment. At every watering, enough solution was added to cause the pot to drain, thereby flushing the pots. At the time of transplanting, a time zero harvest was made. Subsequent harvests were conducted on days 9, 18, 25 and 31 after transplanting. The various R Z T regimes were applied at transplanting and maintained until the plants were harvested. At each harvest, plants were separated into leaves, stems, roots and nodules; these were dried for 48 hr at 70°C and then ground to pass through a 1 mm screen using a Wiley mill (Thomas-Wiley Laboratory Mill, No. 4, Thomas Scientific, U.S.A.). At each harvest, the number of nodules was counted for each plant. Subsamples were taken for Kjeldahl analysis (Kjeltec system, Tecator AB, Hoganas, Sweden). Roots and shoots were treated separately. The root-toshoot N concentration ratio was calculated by dividing root N concentration by shoot N concentration (RSNR) from the results obtained. Leaf area was measured with an image analyzer (Delta T, Decagon Devices Ltd, Cambridge, U.K.). Using these results, we computed the following parameters: nodule to plant ratio (NPR), root to shoot ratio (RSR), specific leaf weight (SLW), root-to-shoot N concentration ratio (RSNR) and relative growth rate (RGR).

Experiment 2 The same experimental conditions and procedures were used in the second experiment. Lupin seeds were inoculated with Rhizobium lupini (Nitragin inoculant, Nitragin Co., Milwaukee, Wis.). Soybean seeds were inoculated as described above. Two plants were grown per pot, and all plants were harvested at one time, 25 days after transplanting. Lupin or soybean plants received either 0 (low N) or 14.2 (high N) mM N. Each species by N level combination was randomly distributed within each root temperature treatment.

Statistical analysis A split plot analysis of variance was performed using the SAS system, with R Z T as main plot units, and N and genotype treatments as subplot units. Each treatment was replicated five times. The least significant difference (LSD) test was used to separate means when there was no sig-

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T. LEGROS and D. L. SMITH

nificant R Z T by N interaction, and in cases of significant interactions, contrasts were used to compare subplot units within main plot units. RESULTS

With the exception of the R G R measurements, only the data of the last harvest, i.e. 31 days after transplanting, will be presented for the first experiment, as they are typical of the data collected at all other harvests. Nitrogen accumulation T e m p e r a t u r e and N level considerably affected nitrogen accumulation per plant. At 10°C R Z T , N2 fixation and NO3 utilization were severely, and similarly inhibited (Table 1). At 25 and 20°C R Z T , the N2-fixing (low N) plants contained 33 and 35%, respectively, as much N as high N plants, while at 15°C, low N plants contained

16% as much N as high N plants, indicating a greater sensitivity o f N 2 fixation than NO3 uptake at the intermediate temperature, 15°C. T h e responses of N and dry matter accumulation to low R Z T were not different for lupin and soybean (data not shown). Tissue N concentration All plants of the 10°C R Z T plus the low and transient N plants of the 15°C treatment showed obvious N deficiency symptoms (leaf yellowing) during the experiment and had low tissue N concentrations (Table 1). At all temperatures, symbiotic plants had lower N concentrations than plants supplied mineral N, with the N concentration difference between the two N levels decreasing as R Z T increased from 15 to 25°C. For Experiment 2 (Table 2), the concentration of N in the root and in the shoot are presented separately. As in Experiment 1, tissue N con-

Table 1. The effect of R Z T and nitrogen level on plant N accumulation, plant N concentration, and root-to-shoot per cent N ratio ( RS NR) of soybean ( cv Maple Arrow) 31 days after transplanting RZT (°C)

Plant total nitrogen (mg)

Plant nitrogen concentration (mg g ')

RSNR

10

Low N Transient N Medium N High N

14.7 23.8 42.5 46.0

11 13 18 20

3.31 2.57 2.06 2.14

15

Low N Transient N Medium N High N

35.8 64.3 194.3 229.3

20 19 40 40

1.62 1.72 1.16 1.40

20

Low N Transient N Medium N High N

162.8 199.9 410.8 461.5

33 28 41 46

1.14 1.24 0.86 0.86

25

Low N Transient N Medium N High N

209.6 295.1 472.9 630.7

34 33 38 47

0.83 0.92 0.76 0.71

238.5 114.1

9 7

0.38 0.33

LSD0.05a LSD0.05b

LSDa is for comparison of means at different levels of the whole plot factor (temperature) and LSDb is for comparison of means within a single level of the whole plot factor.

LOW ROOT-ZONE TEMPERATURE, N2 FIXATION AND SOYBEAN GROWTH

12!

Table 2. The effect of R Z T and N source on the root and shoot per cent N, and the root-toshoot N concentration ratio in soybean and lupin

Root nitrogen concentration (mgg ~)

Shoot nitrogen concentration (mgg ')

RSNR

Low N High N Low N High N

19 27 15 23

9 17 18 30

2.24 1.56 0.88 0.79

Low N High N Low N High N

29 40 21 26

20 28 27 38

1.46 1.43 0.79 0.68

Low N High N Low N High N

30 42 25 27

28 41 37 41

1.07 1.02 0.66 0.67

Low N High N Low N High N

31 33 26 18

28 36 37 37

1.11 0.91 0.69 0.48

7 6

6 5

0.46 0.30

RZT (°C) 10

Soybean Lupin

15

Soybean Lupin

20

Soybean Lupin

25

Soybean Lupin

LSD0.05a LSD005b

LSDa is for comparison of means at different levels of the whole plot factor (temperature) and LSDb is for comparison of means within a single level of the whole plot factor.

centration increased with increasing temperature and when plants were supplied mineral N; these results occurred for soybean and lupin. Lupin tolerated the low R Z T better than soybean, as evidenced by lower root N concentrations and higher shoot N concentrations at the cooler RZT, and responded well to added mineral N at 10 and 15°C only. At 25°C R Z T the mineral N-supplied lupin plants had a lower concentration of N in their root systems than Nz-fixing lupin plants. At 10°C R Z T low N lupin plants showed symptoms of N deficiency and had a lower N concentration than at higher RZT. Nitrogen allocation

A measure of the N allocation between the root and the shoot system was obtained by calculating the RSNR. When roots and shoots are grown at different temperatures, the relative N status

independent of weight of the above and below ground tissues can be conveniently compared. Nitrogen concentration provides an indication of degree of N limitation. The high values obtained for the R S N R at the lowest R Z T of Experiment 1 indicate that less N was exported from the root to the growing portion of the shoot than occurred at higher RZT. The high N concentration values in the root are due to a very low N concentration in the shoot (Table 1). The low N and transient N plants had a greater tendency to retain N in their root systems than the medium or high N plants at all root temperatures except 25°C, where RSNR values are similar for the two sets of plants. Even at 20°C, the RSNR of the low and transient N plants was significantly higher than the RSNR of the medium and high N plants. Although somewhat less pronounced, the data of the second experiment also indicate that at low

122

T. LEGROS and D. L. SMITH Table 3. Effect of root zone temperature and N levels on the relative growth rate (RGR) of soybean

RGR RZT (°C)

Growth period (days after transplanting) 0-9 10 18 19-25 26-31

10

Low N Transient N Medium N High N

0.114 0.132 0.137 0.133

0.059 0.058 0.065 0.056

0.024 0.050 0.034 0.065

0.024 0.061 0.059 0.049

15

Low N Transient N Medium N High N

0.127 0.126 0.150 0.140

0.053 0.089 0.084 0.062

0.028 0.080 0.087 0.125

0.053 0.060 0. I 13 0.077

20

Low N Transient N Medium N HighN

0.125 0.135 0.150 0.145

0.077 0.122 0.104 0.119

0.071 0.097 0.115 0.122

0.127 0.096 0.111 0.121

25

Low N Transient N Medium N High N

0.143 0.151 0.159 0.170

0.097 0.133 0.124 0.135

0.102 0.111 0.122 0.097

0.098 0.095 0.134 0.132

0.028 0.028

0.040 0.032

0.025 0.043

0.059 0.076

LSD0.0~a LSD005b

LSDa is for comparison of means at different levels of the whole plot factor (temperature) and LSDb is for comparison of means within a single level of the whole plot factor. R Z T plants supplied with less mineral N have the other growing periods (9-25, 26-31 DAP), higher R S N R values (Table 2), the R G R of plants relying only on N2 fixation was comparatively more affected at low R Z T than Lupin had a lower R S N R than soybean (Table 2), particularly at 10 and 15°C. Relative to the R G R of plants supplied with mineral N. Low soybean, the N concentration values for the roots N plants had R G R values of 62, 22 and 46% of oflupin remained low over the temperature range the R G R of medium or high N plants at 15°C, tested. It seems that the o p t i m u m value for the but R G R s of 72, 83 and 75% of the medium or R S N R of lupin was 0.65 to 0.70, as above this high N plants at 25°C, for all three growing periods. For each temperature, plants of the tranvalue, growth was not o p t i m u m (10 and 15°C low N treatment) and below this value (25°C sient N treatments had a R G R similar to the high N treatment) the roots and shoots did not medium or high N plants during the second and third growing period but R G R s similar to the low appear healthy. An o p t i m u m could not be determined for soybean from our experiment as the N plants during the fourth growing period. Only the data of the last harvest are presented 25°C treatment yielded the highest levels for fixfor the other growth parameters as they show the ing and mineral N-supplied soybean plants. same trends as those of all the preceding harvests. Relative growth rates Plant dry weight and leaf area were increased During the first growing period [nine to 18 days with both increasing temperature treatments and after planting (DAP)] the relative growth rate increasing N levels (Table 4). At 10°C, growth ( R G R ) was increased by about 15% due to the was poor at all N levels. The positive effect of addition of mineral N at all R Z T (Table 3). Over mineral N addition was more pronounced at 15°C

LOW R O O T - Z O N E TEMPERATURE, N~ F I X A T I O N AND SOYBEAN G R O W T H

123

Table 4. Effect of root zone temperatureon soybeangrowth underfour d~erent N levels RZT (°C)

Plant dry weight (g)

Leaf area (cm 2)

Root: shoot ratio

Leaf specific Nodule Nodule to weight Nodule weight plant ratio (mg cm '2) number (g) (g g ')

10

Low" N Transient N Medium N High N

1.3 1.9 2.2 2.2

88 124 149 139

0.23 0.17 0.17 0.18

5.9 6.4 6.9 7.2

14 15 20 14

0.03 0.03 0.04 0.03

0.02 0.01 0.02 0.01

15

Low N Transient N Medium N High N

1.8 3.5 5.0 6.2

117 248 430 444

0.42 0.38 0.35 0.32

5.7 6.1 5.4 7.3

35 45 38 37

0.12 0.14 0.10 0.06

0.07 0.04 0.02 0.01

20

Low N Transient N Medium N High N

5.0 7.2 9.4 9.6

444 595 994 1292

0.35 0.37 0.33 0.30

5.6 5.5 4.5 3.9

97 119 80 51

0.31 0.37 0.15 0.09

0.06 0.05 0.01 0.01

25

Low N Transient N Medium N High N

6.0 10.3 12.3 13.1

617 1025 1362 1468

0.37 0.42 0.33 0.30

4.4 4.5 4.2 4.5

112 172 79 46

0.31 0.49 0.16 0.04

0.05 0.05 0.01 0.003

4.1 2.3

598 297

0.16 0.05

1.6 1.4

78 29

0.15 0.07

0.020 0.012

LSD005a LSD0.0.~b

LSDa is for comparison of means at different levels of the whole plot factor (temperature) and LSDb is for comparison of means within a single level of the whole plot factor.

than at 20 or 25°C. T h e low N plants a c c u m u lated 32, 53 a n d 4 7 % of the m e d i u m or high N p l a n t d r y weights at 15, 20 a n d 25°C, respectively, while the transient N plants a c c u m u l a t e d 63, 76 and 81% of the m e d i u m or high N p l a n t d r y weights at the same respective t e m p e r a t u r e s ( T a b l e 4).

Leaf area T h e positive effects of N a d d i t i o n on soybean leaf a r e a a n d p l a n t d r y weight were more pronounced at 15 than at 20 or 25°C, with the leaf a r e a of the low N plants representing 27, 41 and 4 4 % of the m e d i u m or high N plants' leaf area at 15, 20 and 25°C, respectively. T h e corresponding values for the transient plants were 57, 54 a n d 720/0 . T h e differences in p l a n t d r y weight a n d leaf area due to R Z T m a y be related to the specific leaf weight ( S L W ) , which was significantly higher at low temperatures. S L W increased when R Z T was decreased from 25 to 20°C for the low N and

transient N plants, while for m e d i u m a n d high N plants S L W did not increase until R Z T reached 15°C (Table 4).

Nodule development As seeds were inoculated at seeding, 10 days before the e x p e r i m e n t a l treatments were applied, some nodules were a l r e a d y present a n d in the last stages of d e v e l o p m e n t when plants were transferred to the 10°C treatment. However, comparison of the n u m b e r of nodules present at the zero time harvest and present at the final harvest showed that no nodules were initiated at 10°C and few at 15°C. M o r e nodules developed on the transient N plants than on the low N plants at 20 and 25°C b u t relative to p l a n t size low N plants had nodule mass equal to that of transient N plants ( T a b l e 4). Between 15 a n d 25°C, the nodule weight to p l a n t d r y m a t t e r ratio (NPR) decreased as the R Z T increased at the low N level. F o r low N plants, nodule n u m b e r was decreased substantially (Table 4) when c o m p a r e d between

[24

T. LEGROS and D. L. SMITH

15 and 20°C (64%) or between 20 and 25°C (20%); however, for the high N plants the corresponding decrease was much smaller (28 and 0%, respectively). DISCUSSION

Plant N accumulation and distribution

Soybean plants preferentially use mineral N over atmospheric N even under optimum temperature conditions for N~ fixation.!2'23) At suboptimal root temperatures, soybean nitrate uptake and N 2fixation decrease, (6'15ibut the data of MATTHEWS and HAYES(18! although not directly comparable across N levels, provided a preliminary indication that N2 fixation was more affected than mineral N uptake. Our data show clearly and within the confines of a single experiment that N accumulation was more restricted by low R Z T for N2-fixing soybean plants than for mineral-Nsupplied soybean plants, At 15°C, soybean plants growing in a medium supplemented with mineral N did not show symptoms of N deficiency (leaf colour pale green to yellow) while N2-fixing soybean plants growing at the same R Z T were severely deficient. WALSH and LAYZELL(27) and RUFTY el al. (21) noted that upon exposure to low RZT, soybean plants initially retain N in the root system leading to a decrease in N partitioning to the young shoots, and ultimately mobilization of N from the older leaves to allow new shoot growth. Our data indicated that this pattern was true for both N sources. Nitrogen partitioning was altered over a larger temperature range for plants relying on N 2 fixation than for mineral-N-supplied plants (see RSNR data of Tables 1 and 2). This may be due to two factors: (1) N accumulation and N concentration in N2-fixing plants was generally lower than in mineral N supplied plants, and (2) N concentration decreased with decreasing root temperature. It may be that the N concentration in the root has to be above a critical level before export to the shoot can take place. Given that the ureides exported from soybean nodules are much less soluble at lower R Z T than the amides exported from the nodules oflupin, (25/ N2-fixing soybean may be less able to export N from roots than N2-fixing lupin at low RZT. How-

ever, it must be noted that differences between the two species at low R Z T may be due to other differences between the two species, The only definitive way to isolate the effect of the type of N export product on N partitioning at low temperature would be the development and utilization ofisolines ofamide- and ureide-exporting legumes. We found that mineral-N-supplied soybean plants had lower a RSNR at low R Z T than N2-fixing lupin plants but that the overall pattern of N and dry matter accumulation was the same for soybean and lupin. The finding of no difference means that concerns about comparing species do not apply. The accumulation and partitioning of N by N2fixing soybean and lupin plants was more affected by low R Z T than were those of mineral-Nsupplied soybean and lupin plants. Based on the RSNR values, the optimum temperature for N partitioning to the shoot was lower for lupin than for soybean. This may have been due to the differences in water solubility of uriedes and amides at low RZT. It may also have been due to the fact that lupin is generally more cold tolerant than soybean and maybe less heat tolerant.(I7) Plant growth and dry matter allocation

R G R represents the efficiency with which a plant uses existing material to produce new material./1°/ The conditions under which plants are grown affect this efficiency. Root zone temperatures and N-levels had marked effects on RGR. The lowest values were for N2-fixing plants at low R Z T and the highest for mineral-N-supplied plants at high RZT. The transient N treatment was designed to avoid N deficiency occurring in N2-fixing plants when they had exhausted their seed N reserves but not yet developed their N2-fixing capabilities. Plants of this treatment exhibited RGRs similar to that of the mineral-Nsupplied plants during the first growing periods, and RGRs similar to the "N2-fixing" plants during the latter growth period, indicating that the early N deficiency in "N2-fixing" plants had no long term effects on their efficiency. Based on R G R studies, RYLE et al. (221 and KE~l~ et al. I~ concluded that there was a higher energy requirement for N 2 fixation than for combined N assimilation in soybean. Our results also showed lower

LOW ROOT-ZONE TEMPERATURE, N2 FIXATION AND SOYBEAN GROWTH growth rates for N2-fixing plants than mineralN-supplied plants, and showed that low R Z T combines with the higher energy cost for biologically fixed N than mineral N to further reduce growth. DUKE et al. 161 concluded that there was a large increase in the activation energy of nitrogenase below 15°C, and LAYZELL et al. !15) reported an increase in activation energy between 16 and 13°C with some, but not all strains o f B.japonicum studied. In addition, Kuo and BOERSMA(t4) reported that at about 17°C the cell membranes through which the water must pass to enter the plant are altered, leading to a water stress that may have a direct effect on the rate ofN 2 fixation. One of these factors, or the combination of both, may explain the very poor growth of the low N plants at 15°C. Leaf elongation is much more sensitive than net photosynthesis to restriction in water uptake due to a decrease in RZT. cl/Low R Z T also sharply reduces root respiration./6'27i However, per unit leaf area, photosynthetic rates are not strongly affected by low RZT. As a result, excess photosynthates are produced and accumulate in leaves, mainly as starch. This sink limitation was clearly shown by the higher SLW of the plants grown at 10 and 15°C RZT. At 20°C RZT, the SLW of the mineral-N-supplied plants was not different from the SLW of plants grown at 25°C RZT, while the SLW of the N2 fixing plant was 20% higher. It appears that symbiosis was sinklimited at the 20°C R Z T and that it may be fully functional only at a R Z T that is optimum for nitrogenase activity. Nodule development was affected by R Z T and combined N level. HOGLUND (9) described a bimodal growth response to fertilizer N added at the commencement of an experiment. He observed an initial rapid growth rate followed by a transient decrease in growth rate that occured when soil N became limiting to plant growth. This limitation occurred between the onset of nodule formation and the onset of N2 fixation. Following the onset of N2 fixation, growth rates increased. This bimodal effect was observed in our data with decreased growth rates occurring when seed N reserves were depleted. Plants grown at 15°C R Z T had a tendency to produce more nodule mass relative to their size than plants grown at

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higher RZT. This may be an attempt by the plant to compensate for lower nodule activity, although this kind of compensation is usually not complete. (25! As the plant is sink limited, this higher NPR may not occur at the expense of other growth processes and might, under natural conditions, allow the plant to take advantage rapidly of a rise in RZT. Whereas the work of MATTHEWSand HAYES (18) provided indirect evidence with data for N levels compared across different rooting media and different experiments, our experiments show clearly that low R Z T results in a more restricted growth for soybean plants relying only on N2 fixation than for soybean plants relying on mineral N. For instance, the plant dry weight of low N (N2-fixing) plants was 46, 52 and 29% that of the high N plants at 25, 20 and 15°C, respectively. However, caution should be taken when using those results to implement cultural practices as the conditions of the experiments were extreme, with 0 N fertility and constant RZT. The results of HARDING and SttEEHY (7) suggest that plants may respond differently to suboptimal temperatures when grown under varying root and shoot temperatures, typical of field conditions, than when grown under constant temperatures. For the nitrogen fixing (low N) plants, a comparison across temperature treatments reveals a 14-fold increase in total N accumulation between 10 and 25°C, and a six-fold increase between 15 and 25°C, while for plant dry weight the same comparisons show only 4.6- and 3.3-fold increases, respectively. As N accumulation for this treatment represents N2 fixation, the greater relative increase in N accumulation indicates that symbiotic N 2 fixation was more inhibited by low R Z T than was overall plant growth. In the same vein, a comparison between 10 and 25°C for nodule weight shows a 10.3-fold increase, much higher than that of dry matter (4.6), indicating that nodule development was a significant cause of the increase in nitrogen accumulation at the higher temperature. However, a comparison between 15 and 25°C for nodule weight reveals a 2.6-fold increase, quite similar to the increase for plant dry weight (3.3), suggesting that the increase in accumulated N by the final harvest between 15 and 25°C was largely due to improved nodule function.

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T. LEGROS and D. L. SMITH

U n d e r the conditions of our experiment, the growth differences between the N2-fixing a n d the m i n e r a l - N - s u p p l i e d soybean plants was due to poor nodule d e v e l o p m e n t below a b o u t 15°C, but due to lower nodule activity when c o m p a r e d between 15 and 25°C. T h e high specific leaf weights suggest that the low nodule activity was not the result of a p h o t o s y n t h a t e shortage a n d that the plants were sink limited. IMSANDE(II) concluded that increased N2 fixation was the key to increasing soybean yield. I f this proves to be true, the d e v e l o p m e n t of the soybean crop in areas with s u b o p t i m a l soil temperatures d u r i n g a portion or the totality of the growing season, will p r o b a b l y necessitate the selection of the B. japonicum-soybean symbiosis and not just the soybean host. I n s u m m a r y , our d a t a allow us to accept the hypothesis that N2-fixing soybean is more sensitive to low R Z T t h a n m i n e r a l - N - s u p p l i e d soybean. F u r t h e r , the d a t a suggest that at the lowest R Z T tested there was a restriction in nodule development, while at higher R Z T the restriction was with nodule activity. O u r findings cause us to reject the hypothesis that the low solubility of ureides at low t e m p e r a t u r e s causes N2-fixing soybean to be more sensitive to low R Z T than N2-fixing lupin, i m p l y i n g that the export of N from nodules as ureides is not the m a i n cause of low R Z T sensitivity for N2-fixing soybean plants.

Acknowledgements We thank the Natural Science and Engineering Research Council of Canada for financial support in the form of a postgraduate scholarship held by T. Legros, and an operating grant held by D. Smith. We also thank the conseil de la recherche en pficherie et en agroalimentaire du Qu6bec for financial support for a portion of this work, in the form of an operating grant held by D. Smith.

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