Planta (Bed.) 127, 133--147 (1975) 9 by Springer-Verlag 1975
Patterns of Nitrogen Utilization in the Soybean Philip S. T h i b o d e a u a n d E r n e s t G. J a w o r s k i Monsanto Company, 800 N. Lindbergh Boulevard, St. Louis, Missouri 63166, USA Received 27 May; accepted 17 July 1975 Summary. The patterns of nitrate uptake, nitrate reductase activity in the leaves, and nitrogen fixation by the nodules were investigated in field-grown soybeans (Glycine max (L.) Merr.) over the growing season. The level of nitrate-reductase activity generally paralleled the concentration of nitrate in the leaf tissue over the entire growing season. A precipitous drop in both parameters was noted within 2-3 weeks after flowering. These parameters decreased by 80-95% at mid-pod fill, a stage where ovule (seed) development was in the logarithmic growth phase, placing a heavy demand on the plant for both energy and fixed nitrogen. The activity of nitrogen fixation of soybean root nodules bore a reciprocal relationship to that of nitrate reductase. The maximum levels of nitrogen fixation were reached at early pod fill when nitrate reductase activity had dropped to 25% of maximum activity. A rapid loss of nitrogen fixation activity occurred shortly after bean fill was initiated, again at a time when the ovules were developing at maximal rates. The total protein content of soybean leaves increased over the season to a maximum level at mid-pod fill. This was followed by a 50% drop over the next 3-week period when the plants approached senescence. This drop corresponded to that found for nitrogen fixation. A similar pattern was noted for watersoluble proteins in the leaf. These studies suggest that there is a close and competitive relationship between the processes of nitrate reduction and nitrogen fixation, with the latter process dominating as the major source of fixed nitrogen after the plants have flowered and initiated pods. At this transitional stage, both soil and environmental effects could cause pertrubation in these processes that could lead to a nitrogen stress causing flower and pod abscission. The rapid decay of nitrogen fixation at the time of midpod fill also suggests a competition between roots (nodules) and pods for available photosynthate. This competition appears to lead to the breakdown of foliar proteins and senescence.
Introduction T h e s o y b e a n offers a challenge as source of t h e f u t u r e p r o t e i n needs of m a n a n d a n i m a l s since t h e p r o t e i n c o n t e n t of s o y b e a n seeds is one of t h e highest k n o w n a n d y e t t h e yields t e n d to be r e l a t i v e l y low c o m p a r e d t o m a n y o t h e r i m p o r t a n t crops. D e s p i t e e x t e n s i v e studies on t h e n u t r i t i o n a l a n d c u l t u r a l p r a c t i c e s for soybeans, progress in i m p r o v i n g yields has n o t been d r a m a t i c . Genetic studies h a v e y i e l d e d some progress, b u t m a j o r b r e a k t h r o u g h s in s o y b e a n s c o m p a r a b l e to those achieved in corn h y b r i d s can n o t be a n t i c i p a t e d . I t is however k n o w n t h a t t h e y i e l d of s o y b e a n s can be m u c h higher ( > 7 0 0 0 kg/ha) t h a n c u r r e n t a v e r a g e s across t h e U n i t e d S t a t e s which r a n g e from 1750 to 2100 kg/ha. Yields of 4 9 0 0 6300 k g / h a h a v e b e e n r e p o r t e d ( S c o t t a n d Aldrich) b u t w i t h o u t defining t h e
134
P.S. Thibodeauand E. G. Jaworski
reason for these high yields. It can be assumed, however, that the genetic potential exists for yields 3-4 times the current average. While average corn yields have steadily increased over the past 20 years, soybean yields have remained virtually unchanged at 1750-2100kg/ha. The basis for this difference, while in part due to difficulties in breeding selfpollinating plants, can also be attributed to a lack of response to fertilizers by this leguminous crop. The response of soybeans to direct fertilizations is known to be inconsistent at best. The response of the soybean plant to nitrogen fertilization is confounded by the ability of the plant to utilize both nitrate and atmospheric nitrogen. Nitrogen fixation by the root nodules is normally initiated 3-5 weeks after planting (Hardy et al., 1971). Thus the initial nitrogen requirements of the seedlings and young plant must be met through utilization of nitrogen from the seed and from the soil. Nitrate is considered the primary source of nitrogen available from the soil. The importance of nitrogen in attaining maximum yields has been emphasized by Lathwell and Evans (1951). They found that the yield of soybeans was closely correlated with the amount of nitrogen accumulated throughout the life cycle. Grain yield was determined by the number of pods retained by the plant and this in turn was determined by the level of nitrogen available during the bloom period. The relationship between available nitrogen and yield was also borne out by the high correlation coefficients for total nitrogen and dry weight reported by Erdman and Means (1952). It is widely accept that the symbiotic mechanism is inadequate for maximum yields, and this fact indicates the possibility of fruitful research in nitrogen fertilization of soybeans. Hardy et al. (1971) have shown that as the supply of bound nitrogen is increased the contribution of the symbiotic bacteria decreases. Fertilization rates high enough to compensate for symbiotic inefficiencies have been attempted. Where such rates were used (Mederski, 1958; Lyons and Early, 1952; Lawn and Brun, 1974a) they have not resulted in spectacular grain yields; usually an increase of less than 100 kg/ha was obtained. No satisfactory physiological explanation for these results has been reported. If the input of nitrogen to the soybean plant is to be maximiT,ed the mechanisms regulating the utilization of nitrogen from soil and from the atmosphere must be understood. So far, nitrogen metabolism in the soybean plant was studied in segments. Seasonal profiles of nitratc-reductase activity in soybean leaves of soil-grown plants, reported by Harper and I-Iageman (1972) and Harper et. al. (1972), demonstrated a rapid decline in the reduction of nitrate shortly after full bloom. Nodule growth and activity in soil-grown plants, as determined by Hardy et al. (1968) and Weber et al. (1971), occur largely after flowering 1, with the major contribution of fixed atmospheric nitrogen observed during the period of pod fill. The two processes appear then to be successive events each contributing nitrogen at specific periods in the development of the soybean plant. The present study was initiated to establish the pattern of nitrate uptake, nitrate reduction, and nitrogen fixation in field- and greenhouse-grown soybeans, and to assess the possible interrelationships between the sources of nitrogen. 1 100% floweringrefers to the presence of at least one flower on all plants. Early, mid-, and late pod fill refer to stages when mean seed dry weightsare less than 50, 51-100, and greater than 100 mg respectively.
Patterns of Nitrogen Utilization
135
Materials and Methods Greenhouse Culture of Non.nodulated Soybeans. Soybeans, Glycine max (L.) Merr. cv. Dare, were obtained from Hummert Seed Co., St. Louis and grown hydroponically in gravel, 2 plants per 15 cm pot. The nutrient solution contained per 100 1 13.6 g KH2P04, 34 g K2S04, 107 g Ca(NOa)2.H20, 47.9 g MgSO4-7H20, 6.6 g (NH~)2SO~, 1.0 g Sequestercne (Fe-S-330, Geigy Chemical Corp.), 91 mg YmCI2 9H20, 57 mg H3BO 3, 44 mg ZnSO~. 7H20 , 4 mg CuSO~ • 5H20, and 2.5 mg Na2MoOa. 2H20. Bleeding Sap. Xylem exudate for measurement of nitrate uptake by roots of greenhonsegrown soybean plants was collected as follows. The plants were cut just below the cotyledons. The pot containing the roots and stem stumps was placed in a humid atmosphere in a sealed desiccator containing about 0.65 cm of water. Droplets of exudate formed on top of the stem stumps and were collected in a calibrated 50-~zl capillary pipet. The exudates were frozen until they could be analyzed for content of nitrate. Young plants tend to "bleed" more readily than older ones. Samplings were always made in the early morning. Nitrate Determination. The procedure of Lowe and Hamilton (1967) was followed. Nodules from the roots of soybean plants were collected and washed repeatedly in cold distilled water. The nodules were macerated in 0.1 M potassium succinate buffer, p H 6.8 (5 ml). The resulting slurry was squeezed through two layers of cheesecloth and the exudate centrifuged at 5000 • g for 5 min. The supernatant was discarded and the pellet, containing the bacteroids, was resuspended in the original volume of buffer and transferred to a glass-Teflon pestle homogenizer. The slurry was homogenized by hand for about 1 min. The suspension was flushed with nitrogen, sealed, and stored in ice. A standard curve was prepared to check the enzyme. 0.4 ml of KNO~ solution containing 0.4-8.0 tLg NOa- was placed in a tube with 0.4 ml of potassium succinate buffer, p H 6.8, and 0.1 ml water. 0.1 ml of enzyme was added and the mixture incubated at 45 ~ for 30 rain. The reaction was stopped by the addition of 1.0 ml of 1% sulfanilamide in 3 N HC1. After mixing 1.0 ml of 0.01% N(1-naphthyl)ethylenediamine was added and the mixture again mixed. After being allowed to stand for about 20 rain at room temperature, the samples were centrifuged at i0000 • g for 10 rain and the clear supernatant read at 540 nm. A blank was prepared for each level by adding the sulfanilamide prior to the enzyme. If this sequence was not followed, the blank values tended to be erratic. I t is not only important to keep the enzyme preparation cold, but to maintain an atmosphere of nitrogen over the enzyme at all times. Samples of bleeding sap were analyzed by dilution into the proper range (0.5-4 ~g NO aper 0.1 ml) with distilled water. Assays were performed on two different dilutions of each sample, and separate blanks were prepared for each. The test solution was added to 0.4 ml of potassium succinate buffer, and water was added to make a final volume of 1 ml after addition of the enzyme. The assay was then performed as described above. Cell-/tee Enzyme Assays. Tissue for the assay was rinsed in cold tap water followed by cold deionized water. I t was then stored on ice until used. If the leaves were over 3 cm 2 in area the veins were removed. Stem and root tissue was chopped with a razor blade. After powdering the tissue in a mortar containing liquid nitrogen, it was homogenized with 0,1 M Tris buffer, p H 7.5 containing 1 mM cysteine for 15 s in a conical glass homogenizer. A buffer-tissue ratio of 6:1 was used. The homogenate was squeezed through four layers of cheesecloth and one layer of Miracloth. The filtered homogenate was centrifuged at 1500 • g for 10 min. A 3-ml aliquot was then removed from the centrifuge tube for use in the nitrite-reduetase assay. The remaining solution was recentrifuged at 20000 • for 20 min. A second 3-ml aliquot was removed for use in the nitrate-reductase assay. This procedure permitted the simultaneous assay of both enzymes from one preparation of tissue. Nitrite reductase activity was determined by the method of Chroboczek-Kelker and Filner (1971) and nitrate reduetase activity by the procedure of Paneque et al. (1965). Fidd-grown Soybean Plants. Soybeans, cv. Wayne, pretreated with commercial soybean inoculant (Nitragin Co., Milwaukee, Wise., USA), were planted May 18 in 76-cm rows. The Ray silt loam was pretreated with Lasso | (Monsanto Co., St. Louis, Mo., USA) and Amiben (Amchem Products, Inc., Ambler, Penn., USA) herbicides each at 2.2 kg/ha. The soil was also fertilized with N, 11 kg-ha, and P and K each at 110 kg/ha.
136
P . S . Thibodeau and E. G. Jaworski
The study plot contained twenty 30.5-m rows. Alternate rows were not sampled to insure uniform shading of experimental rows. The rows involved in the study were subdivided into 3.7 m sections and the sections were numbered and randomized. Plants from three sections were sampled. Measurable rainfall in June was 8.0 cm, July, 13.6 cm, and August, none. Irrigation was not employed. Harvesting o/the Plants. Eight or more adjacent plants of approximately uniform size were dug in a clod from each of the three sections. The clod contained an area about 15 cm on either side of the row, about 5 em of row on either side of the plants, and was 20 to 25 cm deep. For the assay of leaf nitrate-reductase activity, level of nitrate, and determination of protein, two or more uniform plants were selected and cut at the soil level. The topmost fullyexpanded leaf from each of the two plants were placed in a paper towel and surrounded with ice until the enzymatic assay could be run. Time between harvest and assay was less than 30 min. During a given week, two such samplings were made. In alternate weeks a profile of nitrate-reductase and level of nitrate was done using all viable leaves from two plants of each of three sections. Corresponding leaves from the two plants, numbered from the bottom of the plant, were combined for assay. The root systems of the remaining plants were assayed for nitrogen-fixing ability. The plants were cut at soil level, the entire clod of soil was submerged in water, and the roots carefully washed free of soil. Depending on the moisture content of the soil, washing required up to 3 min. Lea/Nitrate-reductase Activity. The intact tissue assay of Jaworski (1971) was used to measure enzymatic activity. A disc, 1.5 cm in diameter, was cut from each leaflet of the two trifoliolate leaves. The 6 discs were weighed, placed into vials containing 5 ml of medium, the vials were sealed, and incubated in the dark at 25 ~ for 60 min. The incubation medium consisted of 0.i M phosphate buffer, pH 7.5, 0.02 M potassium nitrate, and 5% n-propanol. Blank incubations minus tissue were run identically. ~itrite released into the medium after 60 min was determined by the addition of 1.0 ml each of 1% sulfanilamide in 3 M hydrochloric acid and 0.02% N-l-naphthylethylenediamine hydrochloride. The reaction mixture was diluted if necessary and its optical density measured at 540 nm. Nitrate Determination in Leaves. The method of assay was that of Lowe and Hamilton (1967). Following the removal of discs for the measurement of nitrate-rednctase activity, the remainder of the two trifoliolate leaves was dried, weighed, and milled. A 500-rag sample was homogenized with 10 ml of hot deionized water, centrifuged at 20000• for 20 min, and an aliquot of the supernatant fluid was assayed for nitrate. Nitrate Determination in Soil Samples. Triplicate samples of soil were obtained. Each soil bore was 30 cm deep. Samples, 10 g each, of dried soil were extracted with 10 ml of hot deionized water, filtered, and washed on the filter with a second 10-ml volume of hot water. Aliquots of the filtrate were assayed for nitrate. Lea] Protein Content. The content of soluble protein in leaves was estimated in an aliquot of the supernatant fluid prepared for the leaf nitrate assay by the method of Lowry et al. (1951). In addition, Kjeldahl nitrogen analyses were performed on samples of milled leaf tissue. The values obtained were converted to percent protein per gram of fresh leaf materal, assuming that the values represented protein nitrogen only and that the water content of the leaves was uniformly 83 %. Nitrogen Fixation. The basic procedure for the measurement of nitrogen fixation by whole root systems was that of ~Iardy et al. (1968), but substantial modifications were made to better adapt the assay to field conditions. Washed nodulated roots were blotted and divided for duplicate assay of three or more roots each. The roots were placed in glass (mason) jars, ca. 0.51 (1 pint) size, with screw-type lids previously punched and fitted with a 7-ram serum-bottle stopper. Through the septum 50 ml of air in the incubation jar was withdrawn and replaced with 55 ml of scrubbed acetylene (Hardy et al., 1968). The final concentration of acetylene in the incubation was 11-12% depending on the volume occupied by the roots. Incubations were allowed to proceed for 60 min at 25 ~ The incubation atmosphere was sampled using a plain 10-ml Vacutainer tube (Becton-Dickinson and Co., Rutherford, ~ . J . , USA). There was no significant loss of ethylene
Patterns of Nitrogen Utilization
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fl'om samples stored in these tubes for as long as 6 days. The sampled atmosphere was subsequently assayed for ethylene by gas-chromatographic analysis using a stainless steel column, 2.7 m x 3.2 mm O.D. packed with Porapak R (Waters Assoc., Framingham, Mass., USA). The column and injector-port temperature was 50~ and detection was accomplished with a hydrogen-flame ionization detector. The retention time of ethylene was 3.5 rain and that of acetylene was 4.25 rain. The data were expressed either as the amount of ethylene produced per root system per hour, or as the amount produced as a function of nodule weight. The first notation is not an absolute measure since loss of nodules is inevitable in handling. However since this loss can be expected to be reasonably constant, this notation can be regarded as a minimum. estimate of the nitrogen available to a soybean plant at a time of day of maximal activity. Several comparisons were made between results obtained when incubations were conducted in air-acetylene or in argon-oxygen-acetylene. When conducting incubations in the inert atmosphere, the lids of the incubation jars were twice punched and sealed with 7-ram serum-bottle stoppers. After sealing the roots in the jar, a mixture of 25% oxygen-75% argon (Matheson Gas Products, Joliet, Ill., USA) was introduced at the bottom of the jar from a 10-cm neeedle inserted through one of the septa. A second needle pierced the second septum for escaping gases. The gases were exchanged for 2 rain at 400 ml/min. The remaining procedure was as described for incubations conducted in air-acetylene. Grouping o] Data. For a better estimate of the means, the results of field assays normally obtained twice weekly were grouped and plotted as the mean and standard deviation of all observations on that week. Results
Diurnal Variability. D i u r n a l v a r i a t i o n was m e a s u r e d t h r o u g h o u t the daylight hours to d e t e r m i n e the period of m a x i m u m n i t r a t e reduetase a n d lfitrogen fixing activities a n d to define the m o s t appropriate assay period for m i n i m i z i n g errors i n t r o d u c e d b y this variation. Both p a r a m e t e r s were m e a s u r e d 3 days after flowering h a d occurred. N i t r a t e - r e d u c t a s e activity, as shown i n Fig. 1, increased from 2.3 ~mol of n i t r a t e reduced g-~ h -1 prior to 9 a.m. to a p l a t e a u of 3.8 ~mol between a p p r o x i m a t e l y 10:30 a.m. to a t least 2 p.m. (14:00). The specific a c t i v i t y of n i t r o g e n fixation b y the nodules, expressed as n m o l C2H4 h -1 mg -1 nodule, was d e t e r m i n e d over the same period of t i m e (Fig. 2). Comparable assays were performed using 0.1 atmosphere of acetylene i n air or i n a n a r g o n - o x y g e n mixture. No significant d i u r n a l t r e n d was observed i n the t i m e s p a n studied, n o r was a consistent effect of air i n the i n c u b a t i o n m i x t u r e noted. The effectiveness of acetylene
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in competing with nitrogen for the active site of the enzyme m a y simply reflect the 60-fold greater solubility of acetylene in water relative to nitrogen, as previously noted b y H a r d y et al. (1973). Based on these results all harvests of tissue were made between 10:30 a.m. and 2 p.m. (14:00). Nitrate Uptake and Nitrate Reductase. The content of nitrate in the youngest fully-expanded trifoliolate leaves was determined at weekly intervals throughout the growing season. Initial values, as seen in Fig. 3, were high, ranging from an average of 120 to 160 ~mol NO 3 g -1 dry tissue. Thus the plants were capable of absorbing nitrate from the soil at a rapid rate during the period of vegetative growth. As the plants approached the reproductive stage (flowering), a rapid decline in the content of nitrate was noted. When all plants were flowering (100% flowering), nitrate levels had dropped to 40-60 ~mol g-1 dry weight and b y early pod fill these values were less than 10 ~mol g-1. Nitrate-reduetase activity (Fig. 4) was fairly constant at what appeared to be a maximal level from the early seedling stages until 2-3 weeks after flowering. At this point, a rapid decrease in enzymatic activity was noted. The nitrate concentrations decreased prior to the drop in enzymatic activity. The data imply t h a t nitrate-reductase (an inducible enzyme) was fully induced b y levels of tissue tissue nitrate ranging from 40 to 160 tzmol g-1 but that a rapid decline occurred as the concentration of inducing substrate fell below 40 ~mol g-1. These data are somewhat difficult to interpret because the higher the enzymatic activity per unit mass of tissue, the lower m a y be the nitrate concentration due to its conversion to nitrite. Nevertheless, there is a definite parallel between nitrate concentration and nitrate-reductase activity; this is clearly seen in Fig. 5 which shows the relationship between nitrate levels and nitrate reductase for re-
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presentative leaves. Each newly expanded leaf up to the onset of pod fill exhibited maximal nitrate-rednctase activity followed b y parallel declines in both levels of nitrate and nitrate-rednetase activity. I t is not clear why such declines occur. The depletion of soil nitrate is not the cause since analyses of soil after harvest of the mature crop indicated the presence of more than 65 kg of nitrate per ha. Since the uptake of nitrate b y roots is an active, energy-reqniring process it is conceivable, as will be seen later, t h a t energy m a y be limiting uptake of nitrate during the latter stages of development. With reduced nitrate uptake, a reduction in nitrate-reductase activity would be anticipated. I t is not likely t h a t nitrate is reduced at the root level since roots of plants grown in the greenhouse, in hydroponics, have been found to contain relatively low levels of nitrate-reduetase even under conditions when enzyme induction (high nitrate in the nutrient medi~) would be expected to be high. One possible explanation could be t h a t the root nodules, containfllg Rhizobium bacteroids known to reduce nitrate, are reducing nitrate to ammonia, as suggested recently b y Russell et al. (i974). The relative contribution of nitrate reduction in the nodules to total nitrogen in the plant remains to be evaluated.
Patterns of Nitrogen Utilization
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Table 1. Nitrate and nitrite reductase activities of soybean leaves a and nitrate levels in the bleeding sap from non-nodulated plants at different stages of development Weeks after planting
4 5 6 7 8 9 11 20
Stage of developmentb
4 trifoliolates 6 trifoliolates 8 trifoliolates Flowering Pod set Pod development Pod fill Pod fill
Nitrate reduetase, ~mol nitrate reduced/gm/h
Nitrite reduetase, ~mol nitrite redueed/gm/h
NOs- in bleeding sap, ~xg/50~l
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A deelhm in leaf nitrate-reduetase activity 2-3 weeks after flowering was also observed in greenhouse, hydroponically grown, nomnodulated plants raised on a constant level of inorganic nitrogen. Nitrate-reduetase activity as shown in Table 1 decreased as the plants developed with significant drops in activity at, or shortly after pod set. I t m a y be seen t h a t the ratio of nitrite- to nitrate-reduetase activities are quite high, indicating a very high capacity for the reduction of the nitrite ion. Analyses of nitrate in sap showed a decreased trend in movement up the stem despite its continued availability in the nutrient. Nodule Nitrogen Fixation. Nitrogen fixation in soybean root nodules was also monitored through much of the growing season. Nodule development is illustrated in Fig. 6. A significant mass of nodules is not formed until the plants are 5-6 weeks old. At this stage, corresponding to 100% flowering, nodule growth becomes exponential. I t reaches a m a x i m u m when the soybean pods have fully elongated and are beginning to fill. The nodules deteriorate rapidly at the time the plants begin to senesee. Fig. 7 shows t h a t the specific activity of nitrogenase in the nodules is high even when the nodule mass per plant is very low. This specific activity reaches a maximM plateau of 16-20 nmol acetylene reduced h -1 mg -1 of nodules at an early stage, persists until mid-pod fill, and then declines rapidly much earlier t h a n the decline shown for the nodule weights in Fig. 6. When the nitrogen-fixing activity is plotted on a per-plant basis (Fig. 8), it is seen t h a t the period of maximal nitrogen fixation is relatively short. However, since the rate of increase in nitrogen fixation is exponential between the flowering and early pod-fill stages (activity doubled in approximately 8-9 days), considerable quantities of fixed nitrogen are supplied to the plant. Based on seasonal acetylene reduction assays, it is estimated t h a t about 25-35% of the total nitrogen in the plant m a y be derived from nitrogen fixation in the nodules (Harper et al. 1971).
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Patterns of Nitrogen Utilization
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As the pods begin to fill, a rapid decline in nitrogen-fixing activity per plant occurs. I t is at this stage that the ovules approach the exponential growth stage (shown in Fig. 9) and may well act as competing sinks for photosynthate. Since the above declines occur at a point when the ovules are synthesizing protein at a rapid rate (Fig. 9), it may be anticipated that the demand for nitrogen must be net by still some other mechanism. The data in Fig. 10 show that total leaf nitrogen (and water soluble proteins) declines. The filling pods may trigger a breakdown in leaf protein to obtain the fixed nitrogen needed for additional protein synthesis. The concomitant result of this process is a gradual deterioration of leaf function leading to senescence and seed maturation. Discussion The capacity of soybean plants to take up nitrate ions from the soil appears to be maximal during the early vegetative stages of development. In fact, it would appear that this capacity exceeds the ability of the plant to form nitrate-reductase. This may however be more apparent than real since the "true" enzyme level may be limited in its function by the available reductant (NADI-t). Studies by Liu and Hadley (1970) have demonstrated that both nodnlated and non-nodulated isolines of eight cultivars of soybeans possessed higher nitrate-reductasc levels than those found in our studies. While a general decline in enzymatic activity was noted by Lin and Hadley, it was not as dramatic as found for the two cultivars,
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W a y n e a n d Dare, used in our studies. A basic difference resides in t h e e n z y m a t i c assays used. L i u a n d H a d l e y i s o l a t e d t h e e n z y m e from leaves a n d a s s a y e d i t in t h e presence of excess levels of r e d u c t a n t (i.e. N A D H ) . Our a s s a y measures n i t r a t e -
Patterns of Nitrogen Utilization
145
reductase activity in situ and reflects the level of both enzyme and endogenous reductant, and should represent more closely the actual situation within the plant at a given time. These results tend to emphasize the importance of photosynthesis which supplies ATP, NADH and photosynthate. The diurnal curve of nitrate-reductase activity shown in Fig. 1 does not cover as extensive a period of time as others have studied since the objective for obtaining the data was to define an optimum time for measuring nitrate-reduetase under field conditions. Nevertheless it is consistent with data published by Bowerman and Goodmann (1971) for ryegrass and by Harper and Itageman (1972) for soybeans which both covered 24-h periods. Nitrate-reductase activity in Fig. 1 increased by 60% from 9 a.m. to noon. While nitrogen fixation in the nodule is dependent on photosynthate, diurnal fluctuations may not be as dramatic as those of nitrate-reductase. Results in Fig. 2 show little change in nodule (nitrogenase) specific activity between 9 a.m. (9:00) and 2 p.m. (14:00). The data are consistent with those of Hardy et al. (1968) when the mean observations for all nodulated roots of plants 16-27 days post-flowering are compared. The temporal behavior of nitrate-reductase in soybeans may offer a potential for manipulating the pattern of nitrate-reductase activity to obtain higher yields. This potential would exist early during vegetative growth. Enhancement of nitratereductase activity at this stage could result in a larger, more vigorous plant with a greater ability to support pods and a larger source of translocatable nitrogen for pod fill as nodule activity declines. Late applications of nitrogen fertilizer have been largely ineffectual as a means for increasing the availability of soybean nitrogen and yields. Our results with both hydroponically and field-grown soybeans demonstrate an ever decreasing ability of the plant to take up nitrate effectively even when the availability of that ion is constant. The pattern of nitrogen fixation demonstrated in our studies parallels both the qualitative and quantitative patterns published by Hardy et al. (1968) for Wayne soybeans grown in Delaware on light sandy soil. Our report augments that of Hardy et al. (1968) and the recent one of Lawn and Brun (1974b) by the measurement of both leaf nitrate-reductasc activity and levels of nitrate in the same plants. The reciprocal relationship between nitrate-reductase and nitrogen fixation is most interesting, particularly when consideration is given to the physiological state of the plant at the time this transition occurs. Environmental stresses that could affect either or both of these two vital processes for fixing nitrogen into amino acids (e.g. shade, heat, drought) could precipitate pod abscission and loss in yield potentiM. The basis for the reciprocal relationship can only be surmised, but it appears reasonable to suggest that the energy requirements (photosynthate, ATP, NADH) for nitrate reduction and nitrogen fixation are sufficiently great to preclude their simultaneous operation at maximal activity. As the root nodules develop in total mass, they may enhance the transport of photosynthate to the nodules and thus reduce the energy available for the uptake of nitrate by the root. From the data of Harper and Hageman (1972) a comparable reciprocal relationship can be observed for hydroponically grown plants by superimposing their seasonal profiles for nitrate-reductase and nitrogen fixation. 4 Planta (Bed.)
146
P.S. Thibodeau and E. G. Jaworski
The p r e m a t u r e decline in t h e nitrogen-fixing a c t i v i t y of t h e nodules which occurs prior to m i d - p o d fill f u r t h e r s u p p o r t s t h e concept of t h e i n a d e q u a c y of s y m b i o t i c nitrogen-fixation. The effects of s u p p l e m e n t a l lighting a n d CO 2e n r i c h m e n t on nodule a c t i v i t y a n d yield r e p o r t e d b y L a w n a n d B r u n (1974b) a n d H a r d y a n d I t a v e l k a (1973) i n d i c a t e t h a t the decline in nodule a c t i v i t y is also r e l a t e d to a decreasing a v a i l a b i l i t y of energy to t h e nodules. The correlation in t i m e b e t w e e n t h e decline in nodule a c t i v i t y a n d t h e decreasing levels of leaf n i t r o g e n implies t h a t as t h e a v a i l a b i l i t y of nitrogen for p o d fill from t h e nodules declines, t h e growing seed scavenges nitrogen from leaf proteins, a process which u l t i m a t e l y leads to p l a n t senescence. Grateful acknowledgement is made to Martha Ballard, Norbert Block, Donna Farley, and Joyce Fry for their assistance in these studies.
References Bowerman, A., Goodmann, P. J. : Variation in nitrate reductase activity in Lolium. Ann. Bot. 35, 353-366 (1971) Chroboczek-Kelker, H., Filner, P. : l%egulation of nitrite reduetase and its relationship to the regulation of nitrate reductase in cultured tobacco cells. Biochim. biophys. Acta (Amst.) 252, 69-82 (1971) Erdman, L. W., Means, V. M. : Use of total yield for predicting nitrogen content of inoculated legumes grown in sund cultures. Soil Sci. 78, 231-235 (1952) Hardy, R. W. F., Burns, 1%.C., ttebert, 1%.R., Holsten, 1%.D., Jackson, E . K . : Biological nitrogen fixation: a key to world protein. In: Biological Fixation in natural and agricultural habitats, Plant and Soil, Spec. vol., p. 561-590 Lie, T. A., Mulder, E. G., eds. The Hague: ~ijhoff 1971. Hardy, 1%.W. F., Burns, R. C., ttolsten, R. D. : Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol. Biochem. 5, 47-81 (1973) Hardy, 1%.W. F., Havelka, U.D.: Symbiotic N2-fixation: multifold enhancement by CO2enrichment of field-grown soybeans. (Abstr.) Plant Physiol. 51, Suppl. p. 35 (1973) Hardy, R. W. F., Holsten, R. D., Jackson, E. K., Burns, 1%.C. : The acetylene-ethylene assay for N2-fixat,on: laboratory and field evaluation. Plant Physiol. 43, 1185-1207 (1968) Harper, J. E., I-Iageman, R. tt. : Canopy and seasonal profiles of nitrate reductase in soybeans Plant Physiol. 49, 146-154 (1972) ttarper, J . E . , Nicholas, J.C., Hageman, R. H. : Seasonal and canopy variation in nitrate reductase activity of soybean varieties. Crop Sci. 12, 382-386 (1972) Jaworski, E. G. : Nitrate Reductase assay in intact plant tissues. Biochem. biophys. Res. Commun. 43, 1274-1279 (1971) Lathwell, D . J . , Evans, C. E. : Nitrogen uptake from solution by soybeans at successive stages of growth. Agron. J. 43, 264-270 (1951) Lawn, R. J., Brun, W. A. : Symbiotic nitrogen fixation in soybeans. III. Effect of supplemental nitrogen and intervarietal grafting. Crop Sci. 14, 22-25 (1974)a Lawn, R. J., Brun, W. A. : Symbiotic nitrogen fixation in soybeans. I. Effect of photosynthetic source-sink manipulations. Crop Sci. 14, 11-16 (1974)b Liu, M. C., Hadley, H. tt. : Relationships of nitrate reductase activity to protein content in related nodulating and non-nodulating soybeans. Crop Sci. 11, 467-471 (1971) Lowe, R. N., Hamilton, J. L. : t~apid method for determination of nitrate in plant and soil extracts. J. Agr. Food Chem. 15, 349-361 (1967) Lowry, O. tt., Rosebrough, N. J., Farr, A. L., l~andall, R. J. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 198, 265-275 (1951) Lyons, J. C., Earley, E. B. : The effect of ammonium nitrate applications to field soils on nodulation, seed yield, and nitrogen and oil content of the seed of soybeans. Soil Sci. Soc. Amer., Proc. 16, 259-263 (1952)
Patterns of Nitrogen Utilization
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Paneque, A., DelCampo, F . F . , Ramirez, J.M., Losada, M.: Flavin nucleotide nitrate reductase from spinach. Biochim. biophys. Acta (Amst.) 109, 79-85 (1965) Russell, W. J., Johnson, D. 1~., Randall, D. D. : Nitrate reduction in soybean nodules and leaves in relation to Ne(C2tt2) fixation. Agron. Abstr., 1974 Annual ~ecting, p. 76 Scott, W. O., Aldrich, S. R. : Fertilizer for soybeans. In: Modern soybean production, p. 98, Barksdale, B., ed. Cincinnati, Ohio: The Farm Quarterly 1970 Weber, D. F., Caldwell, B. E., Sloger, C., Vest, H. G. : Some USDA studies on the soybeanrhizobium symbiosis. In: Biological nitrogen fixation in natural and agricultural habitats, Plant and Soil, Spec. vol., p. 293-304, Lie, T. A., Mulder, E. G., eds. The Hague: Nijhoff 1971
Patterns of Nitrogen Utilization in the Soybean Philip S. T h i b o d e a u a n d E r n e s t G. J a w o r s k i Monsanto Company, 800 N. Lindbergh Boulevard, St. Louis, Missouri 63166, USA Received 27 May; accepted 17 July 1975 Summary. The patterns of nitrate uptake, nitrate reductase activity in the leaves, and nitrogen fixation by the nodules were investigated in field-grown soybeans (Glycine max (L.) Merr.) over the growing season. The level of nitrate-reductase activity generally paralleled the concentration of nitrate in the leaf tissue over the entire growing season. A precipitous drop in both parameters was noted within 2-3 weeks after flowering. These parameters decreased by 80-95% at mid-pod fill, a stage where ovule (seed) development was in the logarithmic growth phase, placing a heavy demand on the plant for both energy and fixed nitrogen. The activity of nitrogen fixation of soybean root nodules bore a reciprocal relationship to that of nitrate reductase. The maximum levels of nitrogen fixation were reached at early pod fill when nitrate reductase activity had dropped to 25% of maximum activity. A rapid loss of nitrogen fixation activity occurred shortly after bean fill was initiated, again at a time when the ovules were developing at maximal rates. The total protein content of soybean leaves increased over the season to a maximum level at mid-pod fill. This was followed by a 50% drop over the next 3-week period when the plants approached senescence. This drop corresponded to that found for nitrogen fixation. A similar pattern was noted for watersoluble proteins in the leaf. These studies suggest that there is a close and competitive relationship between the processes of nitrate reduction and nitrogen fixation, with the latter process dominating as the major source of fixed nitrogen after the plants have flowered and initiated pods. At this transitional stage, both soil and environmental effects could cause pertrubation in these processes that could lead to a nitrogen stress causing flower and pod abscission. The rapid decay of nitrogen fixation at the time of midpod fill also suggests a competition between roots (nodules) and pods for available photosynthate. This competition appears to lead to the breakdown of foliar proteins and senescence.
Introduction T h e s o y b e a n offers a challenge as source of t h e f u t u r e p r o t e i n needs of m a n a n d a n i m a l s since t h e p r o t e i n c o n t e n t of s o y b e a n seeds is one of t h e highest k n o w n a n d y e t t h e yields t e n d to be r e l a t i v e l y low c o m p a r e d t o m a n y o t h e r i m p o r t a n t crops. D e s p i t e e x t e n s i v e studies on t h e n u t r i t i o n a l a n d c u l t u r a l p r a c t i c e s for soybeans, progress in i m p r o v i n g yields has n o t been d r a m a t i c . Genetic studies h a v e y i e l d e d some progress, b u t m a j o r b r e a k t h r o u g h s in s o y b e a n s c o m p a r a b l e to those achieved in corn h y b r i d s can n o t be a n t i c i p a t e d . I t is however k n o w n t h a t t h e y i e l d of s o y b e a n s can be m u c h higher ( > 7 0 0 0 kg/ha) t h a n c u r r e n t a v e r a g e s across t h e U n i t e d S t a t e s which r a n g e from 1750 to 2100 kg/ha. Yields of 4 9 0 0 6300 k g / h a h a v e b e e n r e p o r t e d ( S c o t t a n d Aldrich) b u t w i t h o u t defining t h e
134
P.S. Thibodeauand E. G. Jaworski
reason for these high yields. It can be assumed, however, that the genetic potential exists for yields 3-4 times the current average. While average corn yields have steadily increased over the past 20 years, soybean yields have remained virtually unchanged at 1750-2100kg/ha. The basis for this difference, while in part due to difficulties in breeding selfpollinating plants, can also be attributed to a lack of response to fertilizers by this leguminous crop. The response of soybeans to direct fertilizations is known to be inconsistent at best. The response of the soybean plant to nitrogen fertilization is confounded by the ability of the plant to utilize both nitrate and atmospheric nitrogen. Nitrogen fixation by the root nodules is normally initiated 3-5 weeks after planting (Hardy et al., 1971). Thus the initial nitrogen requirements of the seedlings and young plant must be met through utilization of nitrogen from the seed and from the soil. Nitrate is considered the primary source of nitrogen available from the soil. The importance of nitrogen in attaining maximum yields has been emphasized by Lathwell and Evans (1951). They found that the yield of soybeans was closely correlated with the amount of nitrogen accumulated throughout the life cycle. Grain yield was determined by the number of pods retained by the plant and this in turn was determined by the level of nitrogen available during the bloom period. The relationship between available nitrogen and yield was also borne out by the high correlation coefficients for total nitrogen and dry weight reported by Erdman and Means (1952). It is widely accept that the symbiotic mechanism is inadequate for maximum yields, and this fact indicates the possibility of fruitful research in nitrogen fertilization of soybeans. Hardy et al. (1971) have shown that as the supply of bound nitrogen is increased the contribution of the symbiotic bacteria decreases. Fertilization rates high enough to compensate for symbiotic inefficiencies have been attempted. Where such rates were used (Mederski, 1958; Lyons and Early, 1952; Lawn and Brun, 1974a) they have not resulted in spectacular grain yields; usually an increase of less than 100 kg/ha was obtained. No satisfactory physiological explanation for these results has been reported. If the input of nitrogen to the soybean plant is to be maximiT,ed the mechanisms regulating the utilization of nitrogen from soil and from the atmosphere must be understood. So far, nitrogen metabolism in the soybean plant was studied in segments. Seasonal profiles of nitratc-reductase activity in soybean leaves of soil-grown plants, reported by Harper and I-Iageman (1972) and Harper et. al. (1972), demonstrated a rapid decline in the reduction of nitrate shortly after full bloom. Nodule growth and activity in soil-grown plants, as determined by Hardy et al. (1968) and Weber et al. (1971), occur largely after flowering 1, with the major contribution of fixed atmospheric nitrogen observed during the period of pod fill. The two processes appear then to be successive events each contributing nitrogen at specific periods in the development of the soybean plant. The present study was initiated to establish the pattern of nitrate uptake, nitrate reduction, and nitrogen fixation in field- and greenhouse-grown soybeans, and to assess the possible interrelationships between the sources of nitrogen. 1 100% floweringrefers to the presence of at least one flower on all plants. Early, mid-, and late pod fill refer to stages when mean seed dry weightsare less than 50, 51-100, and greater than 100 mg respectively.
Patterns of Nitrogen Utilization
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Materials and Methods Greenhouse Culture of Non.nodulated Soybeans. Soybeans, Glycine max (L.) Merr. cv. Dare, were obtained from Hummert Seed Co., St. Louis and grown hydroponically in gravel, 2 plants per 15 cm pot. The nutrient solution contained per 100 1 13.6 g KH2P04, 34 g K2S04, 107 g Ca(NOa)2.H20, 47.9 g MgSO4-7H20, 6.6 g (NH~)2SO~, 1.0 g Sequestercne (Fe-S-330, Geigy Chemical Corp.), 91 mg YmCI2 9H20, 57 mg H3BO 3, 44 mg ZnSO~. 7H20 , 4 mg CuSO~ • 5H20, and 2.5 mg Na2MoOa. 2H20. Bleeding Sap. Xylem exudate for measurement of nitrate uptake by roots of greenhonsegrown soybean plants was collected as follows. The plants were cut just below the cotyledons. The pot containing the roots and stem stumps was placed in a humid atmosphere in a sealed desiccator containing about 0.65 cm of water. Droplets of exudate formed on top of the stem stumps and were collected in a calibrated 50-~zl capillary pipet. The exudates were frozen until they could be analyzed for content of nitrate. Young plants tend to "bleed" more readily than older ones. Samplings were always made in the early morning. Nitrate Determination. The procedure of Lowe and Hamilton (1967) was followed. Nodules from the roots of soybean plants were collected and washed repeatedly in cold distilled water. The nodules were macerated in 0.1 M potassium succinate buffer, p H 6.8 (5 ml). The resulting slurry was squeezed through two layers of cheesecloth and the exudate centrifuged at 5000 • g for 5 min. The supernatant was discarded and the pellet, containing the bacteroids, was resuspended in the original volume of buffer and transferred to a glass-Teflon pestle homogenizer. The slurry was homogenized by hand for about 1 min. The suspension was flushed with nitrogen, sealed, and stored in ice. A standard curve was prepared to check the enzyme. 0.4 ml of KNO~ solution containing 0.4-8.0 tLg NOa- was placed in a tube with 0.4 ml of potassium succinate buffer, p H 6.8, and 0.1 ml water. 0.1 ml of enzyme was added and the mixture incubated at 45 ~ for 30 rain. The reaction was stopped by the addition of 1.0 ml of 1% sulfanilamide in 3 N HC1. After mixing 1.0 ml of 0.01% N(1-naphthyl)ethylenediamine was added and the mixture again mixed. After being allowed to stand for about 20 rain at room temperature, the samples were centrifuged at i0000 • g for 10 rain and the clear supernatant read at 540 nm. A blank was prepared for each level by adding the sulfanilamide prior to the enzyme. If this sequence was not followed, the blank values tended to be erratic. I t is not only important to keep the enzyme preparation cold, but to maintain an atmosphere of nitrogen over the enzyme at all times. Samples of bleeding sap were analyzed by dilution into the proper range (0.5-4 ~g NO aper 0.1 ml) with distilled water. Assays were performed on two different dilutions of each sample, and separate blanks were prepared for each. The test solution was added to 0.4 ml of potassium succinate buffer, and water was added to make a final volume of 1 ml after addition of the enzyme. The assay was then performed as described above. Cell-/tee Enzyme Assays. Tissue for the assay was rinsed in cold tap water followed by cold deionized water. I t was then stored on ice until used. If the leaves were over 3 cm 2 in area the veins were removed. Stem and root tissue was chopped with a razor blade. After powdering the tissue in a mortar containing liquid nitrogen, it was homogenized with 0,1 M Tris buffer, p H 7.5 containing 1 mM cysteine for 15 s in a conical glass homogenizer. A buffer-tissue ratio of 6:1 was used. The homogenate was squeezed through four layers of cheesecloth and one layer of Miracloth. The filtered homogenate was centrifuged at 1500 • g for 10 min. A 3-ml aliquot was then removed from the centrifuge tube for use in the nitrite-reduetase assay. The remaining solution was recentrifuged at 20000 • for 20 min. A second 3-ml aliquot was removed for use in the nitrate-reductase assay. This procedure permitted the simultaneous assay of both enzymes from one preparation of tissue. Nitrite reductase activity was determined by the method of Chroboczek-Kelker and Filner (1971) and nitrate reduetase activity by the procedure of Paneque et al. (1965). Fidd-grown Soybean Plants. Soybeans, cv. Wayne, pretreated with commercial soybean inoculant (Nitragin Co., Milwaukee, Wise., USA), were planted May 18 in 76-cm rows. The Ray silt loam was pretreated with Lasso | (Monsanto Co., St. Louis, Mo., USA) and Amiben (Amchem Products, Inc., Ambler, Penn., USA) herbicides each at 2.2 kg/ha. The soil was also fertilized with N, 11 kg-ha, and P and K each at 110 kg/ha.
136
P . S . Thibodeau and E. G. Jaworski
The study plot contained twenty 30.5-m rows. Alternate rows were not sampled to insure uniform shading of experimental rows. The rows involved in the study were subdivided into 3.7 m sections and the sections were numbered and randomized. Plants from three sections were sampled. Measurable rainfall in June was 8.0 cm, July, 13.6 cm, and August, none. Irrigation was not employed. Harvesting o/the Plants. Eight or more adjacent plants of approximately uniform size were dug in a clod from each of the three sections. The clod contained an area about 15 cm on either side of the row, about 5 em of row on either side of the plants, and was 20 to 25 cm deep. For the assay of leaf nitrate-reductase activity, level of nitrate, and determination of protein, two or more uniform plants were selected and cut at the soil level. The topmost fullyexpanded leaf from each of the two plants were placed in a paper towel and surrounded with ice until the enzymatic assay could be run. Time between harvest and assay was less than 30 min. During a given week, two such samplings were made. In alternate weeks a profile of nitrate-reductase and level of nitrate was done using all viable leaves from two plants of each of three sections. Corresponding leaves from the two plants, numbered from the bottom of the plant, were combined for assay. The root systems of the remaining plants were assayed for nitrogen-fixing ability. The plants were cut at soil level, the entire clod of soil was submerged in water, and the roots carefully washed free of soil. Depending on the moisture content of the soil, washing required up to 3 min. Lea/Nitrate-reductase Activity. The intact tissue assay of Jaworski (1971) was used to measure enzymatic activity. A disc, 1.5 cm in diameter, was cut from each leaflet of the two trifoliolate leaves. The 6 discs were weighed, placed into vials containing 5 ml of medium, the vials were sealed, and incubated in the dark at 25 ~ for 60 min. The incubation medium consisted of 0.i M phosphate buffer, pH 7.5, 0.02 M potassium nitrate, and 5% n-propanol. Blank incubations minus tissue were run identically. ~itrite released into the medium after 60 min was determined by the addition of 1.0 ml each of 1% sulfanilamide in 3 M hydrochloric acid and 0.02% N-l-naphthylethylenediamine hydrochloride. The reaction mixture was diluted if necessary and its optical density measured at 540 nm. Nitrate Determination in Leaves. The method of assay was that of Lowe and Hamilton (1967). Following the removal of discs for the measurement of nitrate-rednctase activity, the remainder of the two trifoliolate leaves was dried, weighed, and milled. A 500-rag sample was homogenized with 10 ml of hot deionized water, centrifuged at 20000• for 20 min, and an aliquot of the supernatant fluid was assayed for nitrate. Nitrate Determination in Soil Samples. Triplicate samples of soil were obtained. Each soil bore was 30 cm deep. Samples, 10 g each, of dried soil were extracted with 10 ml of hot deionized water, filtered, and washed on the filter with a second 10-ml volume of hot water. Aliquots of the filtrate were assayed for nitrate. Lea] Protein Content. The content of soluble protein in leaves was estimated in an aliquot of the supernatant fluid prepared for the leaf nitrate assay by the method of Lowry et al. (1951). In addition, Kjeldahl nitrogen analyses were performed on samples of milled leaf tissue. The values obtained were converted to percent protein per gram of fresh leaf materal, assuming that the values represented protein nitrogen only and that the water content of the leaves was uniformly 83 %. Nitrogen Fixation. The basic procedure for the measurement of nitrogen fixation by whole root systems was that of ~Iardy et al. (1968), but substantial modifications were made to better adapt the assay to field conditions. Washed nodulated roots were blotted and divided for duplicate assay of three or more roots each. The roots were placed in glass (mason) jars, ca. 0.51 (1 pint) size, with screw-type lids previously punched and fitted with a 7-ram serum-bottle stopper. Through the septum 50 ml of air in the incubation jar was withdrawn and replaced with 55 ml of scrubbed acetylene (Hardy et al., 1968). The final concentration of acetylene in the incubation was 11-12% depending on the volume occupied by the roots. Incubations were allowed to proceed for 60 min at 25 ~ The incubation atmosphere was sampled using a plain 10-ml Vacutainer tube (Becton-Dickinson and Co., Rutherford, ~ . J . , USA). There was no significant loss of ethylene
Patterns of Nitrogen Utilization
137
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Fig. 1. Partial diurnal curve of nitrate-reductase activity in the topmost fully expanded leaf of soybeans grown in the field. Enzymatic activity was measured in Ieaf discs of plants 51 days after planting at the times indicated. The data are expressed as the means • standard deviations of triplicate observations
fl'om samples stored in these tubes for as long as 6 days. The sampled atmosphere was subsequently assayed for ethylene by gas-chromatographic analysis using a stainless steel column, 2.7 m x 3.2 mm O.D. packed with Porapak R (Waters Assoc., Framingham, Mass., USA). The column and injector-port temperature was 50~ and detection was accomplished with a hydrogen-flame ionization detector. The retention time of ethylene was 3.5 rain and that of acetylene was 4.25 rain. The data were expressed either as the amount of ethylene produced per root system per hour, or as the amount produced as a function of nodule weight. The first notation is not an absolute measure since loss of nodules is inevitable in handling. However since this loss can be expected to be reasonably constant, this notation can be regarded as a minimum. estimate of the nitrogen available to a soybean plant at a time of day of maximal activity. Several comparisons were made between results obtained when incubations were conducted in air-acetylene or in argon-oxygen-acetylene. When conducting incubations in the inert atmosphere, the lids of the incubation jars were twice punched and sealed with 7-ram serum-bottle stoppers. After sealing the roots in the jar, a mixture of 25% oxygen-75% argon (Matheson Gas Products, Joliet, Ill., USA) was introduced at the bottom of the jar from a 10-cm neeedle inserted through one of the septa. A second needle pierced the second septum for escaping gases. The gases were exchanged for 2 rain at 400 ml/min. The remaining procedure was as described for incubations conducted in air-acetylene. Grouping o] Data. For a better estimate of the means, the results of field assays normally obtained twice weekly were grouped and plotted as the mean and standard deviation of all observations on that week. Results
Diurnal Variability. D i u r n a l v a r i a t i o n was m e a s u r e d t h r o u g h o u t the daylight hours to d e t e r m i n e the period of m a x i m u m n i t r a t e reduetase a n d lfitrogen fixing activities a n d to define the m o s t appropriate assay period for m i n i m i z i n g errors i n t r o d u c e d b y this variation. Both p a r a m e t e r s were m e a s u r e d 3 days after flowering h a d occurred. N i t r a t e - r e d u c t a s e activity, as shown i n Fig. 1, increased from 2.3 ~mol of n i t r a t e reduced g-~ h -1 prior to 9 a.m. to a p l a t e a u of 3.8 ~mol between a p p r o x i m a t e l y 10:30 a.m. to a t least 2 p.m. (14:00). The specific a c t i v i t y of n i t r o g e n fixation b y the nodules, expressed as n m o l C2H4 h -1 mg -1 nodule, was d e t e r m i n e d over the same period of t i m e (Fig. 2). Comparable assays were performed using 0.1 atmosphere of acetylene i n air or i n a n a r g o n - o x y g e n mixture. No significant d i u r n a l t r e n d was observed i n the t i m e s p a n studied, n o r was a consistent effect of air i n the i n c u b a t i o n m i x t u r e noted. The effectiveness of acetylene
138
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in competing with nitrogen for the active site of the enzyme m a y simply reflect the 60-fold greater solubility of acetylene in water relative to nitrogen, as previously noted b y H a r d y et al. (1973). Based on these results all harvests of tissue were made between 10:30 a.m. and 2 p.m. (14:00). Nitrate Uptake and Nitrate Reductase. The content of nitrate in the youngest fully-expanded trifoliolate leaves was determined at weekly intervals throughout the growing season. Initial values, as seen in Fig. 3, were high, ranging from an average of 120 to 160 ~mol NO 3 g -1 dry tissue. Thus the plants were capable of absorbing nitrate from the soil at a rapid rate during the period of vegetative growth. As the plants approached the reproductive stage (flowering), a rapid decline in the content of nitrate was noted. When all plants were flowering (100% flowering), nitrate levels had dropped to 40-60 ~mol g-1 dry weight and b y early pod fill these values were less than 10 ~mol g-1. Nitrate-reduetase activity (Fig. 4) was fairly constant at what appeared to be a maximal level from the early seedling stages until 2-3 weeks after flowering. At this point, a rapid decrease in enzymatic activity was noted. The nitrate concentrations decreased prior to the drop in enzymatic activity. The data imply t h a t nitrate-reductase (an inducible enzyme) was fully induced b y levels of tissue tissue nitrate ranging from 40 to 160 tzmol g-1 but that a rapid decline occurred as the concentration of inducing substrate fell below 40 ~mol g-1. These data are somewhat difficult to interpret because the higher the enzymatic activity per unit mass of tissue, the lower m a y be the nitrate concentration due to its conversion to nitrite. Nevertheless, there is a definite parallel between nitrate concentration and nitrate-reductase activity; this is clearly seen in Fig. 5 which shows the relationship between nitrate levels and nitrate reductase for re-
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presentative leaves. Each newly expanded leaf up to the onset of pod fill exhibited maximal nitrate-rednctase activity followed b y parallel declines in both levels of nitrate and nitrate-rednetase activity. I t is not clear why such declines occur. The depletion of soil nitrate is not the cause since analyses of soil after harvest of the mature crop indicated the presence of more than 65 kg of nitrate per ha. Since the uptake of nitrate b y roots is an active, energy-reqniring process it is conceivable, as will be seen later, t h a t energy m a y be limiting uptake of nitrate during the latter stages of development. With reduced nitrate uptake, a reduction in nitrate-reductase activity would be anticipated. I t is not likely t h a t nitrate is reduced at the root level since roots of plants grown in the greenhouse, in hydroponics, have been found to contain relatively low levels of nitrate-reduetase even under conditions when enzyme induction (high nitrate in the nutrient medi~) would be expected to be high. One possible explanation could be t h a t the root nodules, containfllg Rhizobium bacteroids known to reduce nitrate, are reducing nitrate to ammonia, as suggested recently b y Russell et al. (i974). The relative contribution of nitrate reduction in the nodules to total nitrogen in the plant remains to be evaluated.
Patterns of Nitrogen Utilization
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Table 1. Nitrate and nitrite reductase activities of soybean leaves a and nitrate levels in the bleeding sap from non-nodulated plants at different stages of development Weeks after planting
4 5 6 7 8 9 11 20
Stage of developmentb
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A deelhm in leaf nitrate-reduetase activity 2-3 weeks after flowering was also observed in greenhouse, hydroponically grown, nomnodulated plants raised on a constant level of inorganic nitrogen. Nitrate-reduetase activity as shown in Table 1 decreased as the plants developed with significant drops in activity at, or shortly after pod set. I t m a y be seen t h a t the ratio of nitrite- to nitrate-reduetase activities are quite high, indicating a very high capacity for the reduction of the nitrite ion. Analyses of nitrate in sap showed a decreased trend in movement up the stem despite its continued availability in the nutrient. Nodule Nitrogen Fixation. Nitrogen fixation in soybean root nodules was also monitored through much of the growing season. Nodule development is illustrated in Fig. 6. A significant mass of nodules is not formed until the plants are 5-6 weeks old. At this stage, corresponding to 100% flowering, nodule growth becomes exponential. I t reaches a m a x i m u m when the soybean pods have fully elongated and are beginning to fill. The nodules deteriorate rapidly at the time the plants begin to senesee. Fig. 7 shows t h a t the specific activity of nitrogenase in the nodules is high even when the nodule mass per plant is very low. This specific activity reaches a maximM plateau of 16-20 nmol acetylene reduced h -1 mg -1 of nodules at an early stage, persists until mid-pod fill, and then declines rapidly much earlier t h a n the decline shown for the nodule weights in Fig. 6. When the nitrogen-fixing activity is plotted on a per-plant basis (Fig. 8), it is seen t h a t the period of maximal nitrogen fixation is relatively short. However, since the rate of increase in nitrogen fixation is exponential between the flowering and early pod-fill stages (activity doubled in approximately 8-9 days), considerable quantities of fixed nitrogen are supplied to the plant. Based on seasonal acetylene reduction assays, it is estimated t h a t about 25-35% of the total nitrogen in the plant m a y be derived from nitrogen fixation in the nodules (Harper et al. 1971).
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As the pods begin to fill, a rapid decline in nitrogen-fixing activity per plant occurs. I t is at this stage that the ovules approach the exponential growth stage (shown in Fig. 9) and may well act as competing sinks for photosynthate. Since the above declines occur at a point when the ovules are synthesizing protein at a rapid rate (Fig. 9), it may be anticipated that the demand for nitrogen must be net by still some other mechanism. The data in Fig. 10 show that total leaf nitrogen (and water soluble proteins) declines. The filling pods may trigger a breakdown in leaf protein to obtain the fixed nitrogen needed for additional protein synthesis. The concomitant result of this process is a gradual deterioration of leaf function leading to senescence and seed maturation. Discussion The capacity of soybean plants to take up nitrate ions from the soil appears to be maximal during the early vegetative stages of development. In fact, it would appear that this capacity exceeds the ability of the plant to form nitrate-reductase. This may however be more apparent than real since the "true" enzyme level may be limited in its function by the available reductant (NADI-t). Studies by Liu and Hadley (1970) have demonstrated that both nodnlated and non-nodulated isolines of eight cultivars of soybeans possessed higher nitrate-reductasc levels than those found in our studies. While a general decline in enzymatic activity was noted by Lin and Hadley, it was not as dramatic as found for the two cultivars,
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W a y n e a n d Dare, used in our studies. A basic difference resides in t h e e n z y m a t i c assays used. L i u a n d H a d l e y i s o l a t e d t h e e n z y m e from leaves a n d a s s a y e d i t in t h e presence of excess levels of r e d u c t a n t (i.e. N A D H ) . Our a s s a y measures n i t r a t e -
Patterns of Nitrogen Utilization
145
reductase activity in situ and reflects the level of both enzyme and endogenous reductant, and should represent more closely the actual situation within the plant at a given time. These results tend to emphasize the importance of photosynthesis which supplies ATP, NADH and photosynthate. The diurnal curve of nitrate-reductase activity shown in Fig. 1 does not cover as extensive a period of time as others have studied since the objective for obtaining the data was to define an optimum time for measuring nitrate-reduetase under field conditions. Nevertheless it is consistent with data published by Bowerman and Goodmann (1971) for ryegrass and by Harper and Itageman (1972) for soybeans which both covered 24-h periods. Nitrate-reductase activity in Fig. 1 increased by 60% from 9 a.m. to noon. While nitrogen fixation in the nodule is dependent on photosynthate, diurnal fluctuations may not be as dramatic as those of nitrate-reductase. Results in Fig. 2 show little change in nodule (nitrogenase) specific activity between 9 a.m. (9:00) and 2 p.m. (14:00). The data are consistent with those of Hardy et al. (1968) when the mean observations for all nodulated roots of plants 16-27 days post-flowering are compared. The temporal behavior of nitrate-reductase in soybeans may offer a potential for manipulating the pattern of nitrate-reductase activity to obtain higher yields. This potential would exist early during vegetative growth. Enhancement of nitratereductase activity at this stage could result in a larger, more vigorous plant with a greater ability to support pods and a larger source of translocatable nitrogen for pod fill as nodule activity declines. Late applications of nitrogen fertilizer have been largely ineffectual as a means for increasing the availability of soybean nitrogen and yields. Our results with both hydroponically and field-grown soybeans demonstrate an ever decreasing ability of the plant to take up nitrate effectively even when the availability of that ion is constant. The pattern of nitrogen fixation demonstrated in our studies parallels both the qualitative and quantitative patterns published by Hardy et al. (1968) for Wayne soybeans grown in Delaware on light sandy soil. Our report augments that of Hardy et al. (1968) and the recent one of Lawn and Brun (1974b) by the measurement of both leaf nitrate-reductasc activity and levels of nitrate in the same plants. The reciprocal relationship between nitrate-reductase and nitrogen fixation is most interesting, particularly when consideration is given to the physiological state of the plant at the time this transition occurs. Environmental stresses that could affect either or both of these two vital processes for fixing nitrogen into amino acids (e.g. shade, heat, drought) could precipitate pod abscission and loss in yield potentiM. The basis for the reciprocal relationship can only be surmised, but it appears reasonable to suggest that the energy requirements (photosynthate, ATP, NADH) for nitrate reduction and nitrogen fixation are sufficiently great to preclude their simultaneous operation at maximal activity. As the root nodules develop in total mass, they may enhance the transport of photosynthate to the nodules and thus reduce the energy available for the uptake of nitrate by the root. From the data of Harper and Hageman (1972) a comparable reciprocal relationship can be observed for hydroponically grown plants by superimposing their seasonal profiles for nitrate-reductase and nitrogen fixation. 4 Planta (Bed.)
146
P.S. Thibodeau and E. G. Jaworski
The p r e m a t u r e decline in t h e nitrogen-fixing a c t i v i t y of t h e nodules which occurs prior to m i d - p o d fill f u r t h e r s u p p o r t s t h e concept of t h e i n a d e q u a c y of s y m b i o t i c nitrogen-fixation. The effects of s u p p l e m e n t a l lighting a n d CO 2e n r i c h m e n t on nodule a c t i v i t y a n d yield r e p o r t e d b y L a w n a n d B r u n (1974b) a n d H a r d y a n d I t a v e l k a (1973) i n d i c a t e t h a t the decline in nodule a c t i v i t y is also r e l a t e d to a decreasing a v a i l a b i l i t y of energy to t h e nodules. The correlation in t i m e b e t w e e n t h e decline in nodule a c t i v i t y a n d t h e decreasing levels of leaf n i t r o g e n implies t h a t as t h e a v a i l a b i l i t y of nitrogen for p o d fill from t h e nodules declines, t h e growing seed scavenges nitrogen from leaf proteins, a process which u l t i m a t e l y leads to p l a n t senescence. Grateful acknowledgement is made to Martha Ballard, Norbert Block, Donna Farley, and Joyce Fry for their assistance in these studies.
References Bowerman, A., Goodmann, P. J. : Variation in nitrate reductase activity in Lolium. Ann. Bot. 35, 353-366 (1971) Chroboczek-Kelker, H., Filner, P. : l%egulation of nitrite reduetase and its relationship to the regulation of nitrate reductase in cultured tobacco cells. Biochim. biophys. Acta (Amst.) 252, 69-82 (1971) Erdman, L. W., Means, V. M. : Use of total yield for predicting nitrogen content of inoculated legumes grown in sund cultures. Soil Sci. 78, 231-235 (1952) Hardy, R. W. F., Burns, 1%.C., ttebert, 1%.R., Holsten, 1%.D., Jackson, E . K . : Biological nitrogen fixation: a key to world protein. In: Biological Fixation in natural and agricultural habitats, Plant and Soil, Spec. vol., p. 561-590 Lie, T. A., Mulder, E. G., eds. The Hague: ~ijhoff 1971. Hardy, 1%.W. F., Burns, R. C., ttolsten, R. D. : Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol. Biochem. 5, 47-81 (1973) Hardy, 1%.W. F., Havelka, U.D.: Symbiotic N2-fixation: multifold enhancement by CO2enrichment of field-grown soybeans. (Abstr.) Plant Physiol. 51, Suppl. p. 35 (1973) Hardy, R. W. F., Holsten, R. D., Jackson, E. K., Burns, 1%.C. : The acetylene-ethylene assay for N2-fixat,on: laboratory and field evaluation. Plant Physiol. 43, 1185-1207 (1968) Harper, J. E., I-Iageman, R. tt. : Canopy and seasonal profiles of nitrate reductase in soybeans Plant Physiol. 49, 146-154 (1972) ttarper, J . E . , Nicholas, J.C., Hageman, R. H. : Seasonal and canopy variation in nitrate reductase activity of soybean varieties. Crop Sci. 12, 382-386 (1972) Jaworski, E. G. : Nitrate Reductase assay in intact plant tissues. Biochem. biophys. Res. Commun. 43, 1274-1279 (1971) Lathwell, D . J . , Evans, C. E. : Nitrogen uptake from solution by soybeans at successive stages of growth. Agron. J. 43, 264-270 (1951) Lawn, R. J., Brun, W. A. : Symbiotic nitrogen fixation in soybeans. III. Effect of supplemental nitrogen and intervarietal grafting. Crop Sci. 14, 22-25 (1974)a Lawn, R. J., Brun, W. A. : Symbiotic nitrogen fixation in soybeans. I. Effect of photosynthetic source-sink manipulations. Crop Sci. 14, 11-16 (1974)b Liu, M. C., Hadley, H. tt. : Relationships of nitrate reductase activity to protein content in related nodulating and non-nodulating soybeans. Crop Sci. 11, 467-471 (1971) Lowe, R. N., Hamilton, J. L. : t~apid method for determination of nitrate in plant and soil extracts. J. Agr. Food Chem. 15, 349-361 (1967) Lowry, O. tt., Rosebrough, N. J., Farr, A. L., l~andall, R. J. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 198, 265-275 (1951) Lyons, J. C., Earley, E. B. : The effect of ammonium nitrate applications to field soils on nodulation, seed yield, and nitrogen and oil content of the seed of soybeans. Soil Sci. Soc. Amer., Proc. 16, 259-263 (1952)
Patterns of Nitrogen Utilization
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Paneque, A., DelCampo, F . F . , Ramirez, J.M., Losada, M.: Flavin nucleotide nitrate reductase from spinach. Biochim. biophys. Acta (Amst.) 109, 79-85 (1965) Russell, W. J., Johnson, D. 1~., Randall, D. D. : Nitrate reduction in soybean nodules and leaves in relation to Ne(C2tt2) fixation. Agron. Abstr., 1974 Annual ~ecting, p. 76 Scott, W. O., Aldrich, S. R. : Fertilizer for soybeans. In: Modern soybean production, p. 98, Barksdale, B., ed. Cincinnati, Ohio: The Farm Quarterly 1970 Weber, D. F., Caldwell, B. E., Sloger, C., Vest, H. G. : Some USDA studies on the soybeanrhizobium symbiosis. In: Biological nitrogen fixation in natural and agricultural habitats, Plant and Soil, Spec. vol., p. 293-304, Lie, T. A., Mulder, E. G., eds. The Hague: Nijhoff 1971
