The Uptake of Nitrate by Lolium perenne from Flowing Nutrient Solution

Transcription

1 Journal of Experimental Botany, Vol. 29, No. 109, pp , April 1978 The Uptake of Nitrate by Lolium perenne from Flowing Nutrient Solution I. EFFECT OF NO^ CONCENTRATION C. R. CLEMENT, M. J. HOPPER, AND L. H. P. JONES The Grassland Research Institute, Hurley, Berkshire, SL6 SLR Received 7 July 1977 ABSTRACT Experiments with simulated swards of perennial ryegrass {Lolium perenne L.) show the relationship between NOj concentration in flowing nutrient solution, nitrate uptake, plant growth, and the chemical composition of roots and shoots. Rates of uptake exceeding 1 g N m~ 2 d"' were maintained at NOj concentrations in solution down to 0-02 mg N I" 1. Short-term studies confirmed that at such low concentrations the plants were able to maintain rates of uptake of about 85% of maximum. Between 0-2 and 200 mg N 1~' the concentration of NOj in solution had little effect on rate of uptake or plant growth. With NOj at 1000 and 2000 mg N I" 1 there was a marked reduction in weight of the shoots and, more particularly, in the length and tensile strength of the roots. There were several significant trends in mineral composition of the plants (notably in S, Ca, Mg) which were apparently correlated with increasing concentration of NOj in solution. INTRODUCTION Plant roots experience a wide range of NO^ concentrations in the soil solution. An upper limit, set only by the solubility of nitrate salts in aqueous solution, will occur in the vicinity of dissolving granules of fertilizer. A lower limit is less easy to define because a number of physical and biological processes, including those associated with uptake by plant roots, may be involved in lowering the concentration of NO7 in the soil solution. Warncke and Barber (1974) report that bromegrass {Bromus inermis Leyss.) reduced the concentration of NOj in a nutrient solution to about 0-02 mg N 1~'. If this is accepted as a lower limit then the NO7 concentrations to which the roots of agricultural crops are exposed can extend from 0-02 to mg N I" 1, i.e to //M. The roots of grassland plants may experience concentrations approaching the upper end of the range when nitrogen fertilizers are applied to the surface of the soil, and concentrations approaching the low end when uptake is high and exceeds supply. Experimental evidence is, however, largely restricted to the middle of this range. One of the most extensive studies (Alberda, 1965) examined the effect of NOj" concentration on perennial ryegrass over the range 1-12 to 210 mg N I" 1, i.e to 15 mm. The present investigations were aimed at studying the uptake of NO^~ by perennial ryegrass from solutions with concentrations of NO7 in the range 0-02 to 2000 mg N I- 1 (1-43 to /M).

2 454 Clement, Hopper, and Jones Nitrate uptake by L. perenne MATERIALS AND METHODS Two experiments, each lasting for 8 weeks, were conducted, one in summer and the other in autumn. Perennial ryegrass (Lolium perenne L. cv. S23) was the experimental plant and in both experiments nitrogen was supplied in flowing nutrient solution only as NOT at 6 controlled concentrations, viz. 0-02, 0-2, 20, 200, 1000, and 2000 mg N I" 1. In experiment 1 the seeds were sown on 20 June, and the 6 concentrations of NOj were set and controlled from 18 July until the plants were harvested on 15 August. In experiment 2 the seeds were sown on 7 August, NOT treatments began on 4 September, and plants were harvested 4 weeks later on 2 October. Both experiments were conducted in a glasshouse with natural lighting. Air temperature was controlled at approximately 25 C day and 15 C night. The system of flowing solution culture in which the experimental plants were grown has been described by Clement, Hopper, Canaway, and Jones (1974). Briefly, this system comprises plant culture units within which solution is circulated through 24 culture vessels of 1-2 I capacity arranged in parallel. The culture vessels have screw-caps each of which holds 6 growth tubes with TABLE 1. Composition of flowing nutrient solutions Weeks from sowing NOT (mg N 1" ') so 2 - (mgs Ca 2+ I" 1 ) (mgl- 1 ) Other nutrients 0 (mg I" 1 ) Pretreatment period 10-2 Treatment period P 1-55 K 100 Mg 2-43 Fe 0-06 B 0-05 Mn 0-05 Cu 0002 Zn 0005 Mo 0005 Cl 006 " Supplied during pretreatment and treatment periods, i.e. 8 week experimental period. nylon mesh at their lower ends. This mesh is below the level of the circulating solution and provides support for the experimental plants which are grown from seed in the growth tubes. One plant culture unit was used for each NOT concentration (treatment). The arrangement of the culture vessels in a unit is such that the spatial distribution of the plants simulates a grass sward (Plate 1A) with an area of 0-8 m 2 ; this enables yield and ion uptake to be calculated on an area basis. Within each culture vessel the vertical flow of nutrient solution was held constant at 2-4 mm s" 1. Arnon solution, as described by Hewitt (1966) but diluted to give the concentrations shown in Table 1, was used as the basal nutrient solution. At 4 weeks after sowing the seed this solution was modified to give the 6 concentrations of NOj. Nitrate was supplied as Ca(NO 3 ) 2, and to minimize the effect of differences in Ca 2+ concentration this ion was also supplied as CaSO 4 at a high level for all treatments (Table 1). The nutrient solution was drained from each culture unit and replaced at 2 week intervals. The system of flowing solution culture includes an automatic monitoring and control assembly which was used to control the activity of both NOj and H + in the nutrient solution as follows. An ion-selective electrode coupled with a calomel reference electrode was used to control NOT at 0-02, 0-2, 20 and 200 mg N 1"' by activating nutrient pumps which supplied NO7, as HNO 3, to replace that taken up by the plants. At 1000 and 2000 mg N I~' NOT uptake by plants caused only a small proportionate decline in solution concentration, and periodic manual analysis and addition were adequate to maintain the set concentration. A combined glass/reference electrode was used to rnntrnl nh by activating a solenoid which effected delivery of a Ca(OH) 2 solution when the ph of the circulating solution fell below 6-0. All nutrients other than nitrogen were supplied by adjusting

3 Clement, Hopper, and Jones Nitrate uptake by L. perenne 455 the rate of delivery of metering pumps, according to the results of periodic analysis of the flowing nutrient solution. The concentration of NO7 in each plant culture unit was automatically recorded on paper tape at intervals of 20 min throughout the treatment period together with the amount of HN0 3 delivered by each nutrient pump. The total amounts of NO7, delivered to replace that removed by the plants from the flowing nutrient solution, were checked periodically by weighing the individual reservoirs which supplied each nutrient pump. Daily mean rates of uptake were computed from these records. At harvest the plants were removed from the culture vessels (see Plate 1B) and separated into shoots and roots. The shoots were weighed, dried at 100 C, and reweighed. The roots were rinsed in deionized water, blotted dry, and weighed; subsamples were retained for morphological and mechanical examination. For the latter purpose individual adventitious roots were removed fresh from the plants and a load applied to measure the weight required to break the root at a point about 30 mm below the base of the shoot. The remainder of the roots were dried at 100 C and weighed. Ground samples of the dried shoots and roots were analysed for total N (includes NO7) using a Coleman nitrogen analyser (model 29A), for NOj and, in experiment 1, for total S, SO*", and K, Ca, Mg, Fe, Cu, Mn, and Zn. RESULTS Nitrate uptake The cumulative amounts of NOj supplied to replace that taken up by the plants are plotted in Fig. 1. To avoid a confusion of points on the diagrams only the results from 2 concentrations of NO7 are given for each experiment, namely the lowest (0-02 mg N I" 1 ) and that above which there was no further increase in N uptake (20 mg N I" 1 ). In both experiments, the rate of uptake at the latter concentration exceeded 1 g N m~ 2 d" 1 for periods of several days and, in experiment 1, which was conducted in summer, this rate was maintained on average from 5 weeks after sowing until the end of the experiment. Over this period of 3 weeks there was no evidence that the rate increased with the increasing size of the plants. At the lowest concentration there was a reduction in NO7 uptake but, even so, the rate still exceeded 1 g N m~ 2 d" 1 on several occasions during the course of each experiment. The ability of plants to maintain a high rate of NO7 uptake at low and decreasing concentration in solution was also examined using the ion-monitoring and control assembly. For this purpose the concentration of NOj supplied by the nutrient pumps was adjusted so that a single operation of these pumps provided sufficient NO^" for approximately 2 h, during which depletion was monitored at intervals of 20 min. A typical depletion curve (Fig. 2) shows that as the NO7 concentration in solution fell from 0-13 to 0-02 mg N I" 1 the rate of uptake remained almost constant. This apparent constancy was examined more precisely by comparing the fall in NO^" concentration during the first 20 min interval (a b) with that during the last 20 min interval (c d) in a 2 h period of depletion. Mean rates of uptake calculated from 122 consecutive depletion curves were, respectively, and ± mg N m~ 2 min" 1 for the first and last 20 min intervals. The difference between these values represents a decline of only about 15% in the rate of NOj uptake coincident with a fall in solution concentration from 0-13to0-02mg.Nl- 1. Weight of shoots and roots The dry weight of shoots is shown in Fig. 3. Although the weight of plants grown in summer (experiment 1) was about twice that of plants grown in autumn

4 456 Clement, Hopper, and Jones Nitrate uptake by L. perenne 30 r 20 Experiment 1 e z a. O Experiment L Weeks after sowing FIG. 1. Cumulative amounts of NOj supplied by the nutrient pumps to replace that taken up by simulated swards of perennial ryegrass from flowing nutrient solutions containing different concentrations of NOj., 002 mg N I" 1 ; O O, 20 mg N I" Z on 010 A a \ b solution u nutri c i«o \ \ 008 \ \ \ \ 002 \ d i * * U UU ) Time (min) FIG. 2. A typical curve of NOj depletion in flowing nutrient solution as a result of uptake by perennial ryegrass. Rates of uptake during the first and last 20 min intervals in a period of depletion were calculated by multiplying (a b) and (c u), respectively, by the volume o f solution in a plant culture unit.

5 Clement, Hopper, and Jones Nitrate uptake by L. perenne 457 (experiment 2), the form of the curve showing the response to increasing concentration of NO7 in solution was similar for both experiments. The maximum weight of shoots was reached at 20 mg N I"" 1. Below this concentration there was a gradual decline, with 90% of the maximum yield at 0-2 and 70% at 0-02 mg N 1~'. At 200 mg N I" 1 yield was similar to that at 20 but above 200 mg N I" 1 there was a marked reduction in shoot weight. 700 r o 0 L NOJ in nutrient solution (mg N I" 1 ) 2000 FIG. 3. Dry weight of shoots and roots of perennial ryegrass grown in flowing nutrient solutions containing NOj at controlled concentrations in the range 0-02 to 2000 mg N I" 1 (1-43 to ftm). A, experiment 1; A A, experiment 2. The dry weight of roots decreased with increasing concentration of NOj in solution (Fig. 3). At lower concentrations the roots were darker brown than at higher concentrations, and more finely divided. Although the weight of roots was not greatly reduced at the higher concentrations of NO7 there were marked reductions in length (Plate 1B) and in tensile strength (Fig. 4). There was no visual evidence of death of roots during the course of the experiments. Nitrogen in shoots and roots The effect of the concentration of NOj in the nutrient solution upon the amount of total N in shoots and roots is shown in Fig. 5. Both response curves are similar in form to those for the dry weight of the shoots (Fig. 3) but, in experiment 1, low

6 458 Clement, Hopper, and Jones Nitrate uptake by L. perenne concentrations of NOj reduced the quantity of N in the plant to a greater extent than yield. The concentrations of total N and of NOj, expressed on a dry weight basis, are shown in Fig. 6 for both shoots and roots. Increases in total N were mainly due to increases in the NO7 content of the plant. With increases in NO7 concentration in solution over the range 0-02 to 2000 mg N I" 1 the NO^" in the shoots increased from 0-38 to 1-48% N in experiment 1 and from 1-38 to 1-80% N in experiment 2. The concentration in the roots rose from 0-36 to 1-28% N in experiment 1 and from 1-68 to 2-33% N in experiment 2. II 1! 2 o. 5 If Ji NOjin nutrient solution (mg N 1"') FIG. 4. Tensile strength of the roots of perennial ryegrass grown in flowing nutrient solutions containing different concentrations of NOj. A, experiment 1; A A, experiment 2. Throughout the whole range of NOj concentration in solution the reduced (or metabolized) N, calculated as the difference between total N and N0 3 -N, remained substantially constant at 2-97 ± 0-08% N in the shoots and 2-19 ± 0-09% N in the roots (Fig. 6). Mineral composition of shoots and roots The dry matter and mineral contents of the shoots and roots from experiment 1 are given in Table 2. With increasing concentration of NOj in the nutrient solution the dry matter content of the shoots fell to a minimum of 12-4% at 20 and then rose to a maximum of 18-5% at 2000 mg N I" 1. The mineral composition of the shoots was within the range commonly found in grass grown in the field (Whitehead, 1966) and was not greatly influenced by NOj concentration in solution with two notable exceptions. First, the concentration of Mg decreased and Ca increased with each increase in NOj concentration. This is probably because the concentration of Ca 2+ in the nutrient solution was increased with increasing NOj concentration (Table 1). Second, the concentration of total S, and 2000

7 Clement, Hopper, and Jones Nitrate uptake by L. perenne 459 NOj in nutrient solution (mg N 1 PLATE 1. A. Foliar canopy of perennial ryegrass grown for 6 weeks in flowing nutrient solution containing NOj at 20 mg N 1~'. B. Perennial ryegrass plants grown for 8 weeks in flowing nutrient solutions containing NOj at controlled concentrations in the range 0-02 to 2000 mg N I" 1 (1-43 to fm) in particular of the SOJ" fraction, fell with increasing NOj concentration in solution. DISCUSSION From 2 weeks after the start of treatments, the plants in each culture unit provided a full foliar canopy of about 300 mm height over an area of 0-8 m 2 (Plate 1A). Total insolation, measured outside the glasshouse during the treatment period of 4 weeks, was 472 and 296 MJ m~ 2 for experiments 1 and 2 respectively. The

8 460 Clement, Hopper, and Jones Nitrate uptake by L. perenne maximum weights of shoots at harvest were equivalent to 6 t dry matter ha ' in experiment 1 and 3 t dry matter ha" 1 in experiment 2. Such amounts are similar to those found in field crops of grass in mid-summer and in late autumn respectively. The weight of shoots in experiment 1 represents a mean growth rate of 20 g m~ 2 d" 1 over the 4 week treatment period, which is close to the maximum rate for ryegrass (Coleman and Lazenby, 1975) and similar to the maximum for several other crops (Sibma, 1968). Likewise the maximum rate of NOj uptake (equivalent to 10 kg N ha" 1 d" 1 ) is similar to that which may be calculated from the results of fertilizer experiments with grass in the field and in potted soils (Hunt, 1966; o 10 0 L NOj in nutrient solution (mg N I" 1 ) FIG. 5. Amounts of total N in shoots and roots of perennial ryegrass grown in flowing nutrient solutions containing different concentrations of NOj. A, experiment 1; A A, experiment 2. Henzell, Martin, Ross, and Haydock, 1964). Such rates of uptake were maintained in both experiments throughout the 2 weeks prior to harvest (Fig. 1). These observations suggest that conditions in experiment 1 were near optimal for growth and NOj uptake. Apart from the lower level of insolation, conditions in experiment 2 were similar to those in experiment 1. Although there was a reduction in the weight of shoots at the lowest and highest concentrations of NO^" in the nutrient solution, it is notable that over a 1000-fold range, i.e. from 0-2 to 200 mg N I" 1, the weight of shoots varied by less than 10% and the roots by little more. These results are consistent with those from other experiments in which plants grown in stirred or flowing solutions with NO7 concentration maintained at about 1 mg N I" 1 at the root/solution interface gave near maximum yield (Alberda, 1965; Cox and Reisenauer, 1973). Changes in the mineral contents of the plants were also small over this range (Table 2), apart from some increase in NOj which was present in the shoots in concentrations considerably above that required for maximum yield (Hyltnn. Williams. Ulrich, and Cornelius, 1964; Cowling and Lockyer, 1970) but within the range (0-26 to

9 Clement, Hopper, and Jones Nitrate uptake by L. perenne % NO 3 -N) reported for ryegrass grown in the field (Neilson, 1974) and in potted soils (Nowakowski, Cunningham, and Nielsen, 1965). At the lowest concentration of NOj in solution (0-02 mg N I" 1 ) the weight of shoots was 20% below the maximum in experiment 1 and 34% below the 4-0r L NO 7 m nutrient solution (mg N I" 1 ) 2000 FIG. 6. Concentrations of total N and of NO j in shoots and roots of perennial ryegrass grown in flowing nutrient solutions containing different concentrations of NO7. Shaded areas represent the reduced or metabolized N. Experiment 1: A, total N;, NO 3 -N; experiment 2: A A, total N;D D, NO 3 -N. maximum in experiment 2. This was accompanied by a marked fall in the concentration of NOj in shoots and roots in experiment 1 but not in experiment 2 (Fig. 6). Thus, the greater proportionate reduction in yield occurred with the higher NO7 level in the shoots at harvest. An explanation may be sought in the form of the curves showing cumulative daily rates of NOj uptake (Fig. 1). At the lowest

10 462 Clement, Hopper, and Jones Nitrate uptake by L. perenne concentration of NO 7 in solution the plants in experiment 1 had a greater rate of uptake during the first 2 weeks of the treatment period than did those in experiment 2. During the last 2 weeks the rates of uptake were similar in both experiments, and it would seem therefore that the difference in yield between the 2 experiments was due to a differing ability of the plants to take up NO7 from low concentration during the first 2 weeks. It follows that the similar rates of uptake during the last 2 weeks would lead to a higher concentration of NO7 in the smaller plants. One implication is that seedlings growing in low light may require higher concentrations of NO7 in the external solution than tho'se growing at high light or larger plants growing at low light. TABLE 2. Dry matter and mineral contents of shoots and roots of perennial ryegrass grown inflowing nutrient solutions containing different concentrations ofnoj NOj (mg N I" 1 ) Dry rnattpt HldLlCi (% fr. wt.) K Ca % dry wt. Totals SO 4 -S Mg Fe Cu Mn mgkg-'dry wt. Zn Shoots Roots The plant's ability to take up NO7 from solutions of low and decreasing concentration at an almost undiminished rate (Fig. 2) calls for comment. The measured concentrations almost certainly overestimate those at the site of uptake for two reasons. First, at a concentration of 0-02 mg N I" 1 over half the NO7 in the nutrient solution flowing into each culture vessel was taken up by the plants thus reducing the concentration in the outflow to <0-01 mg N I" 1. Second, because uptake by mass flow in the transpiration stream was negligible (<0-1 mg N m~ 2 d" 1 ) a considerable diffusive flux must be assumed. This would require a downward gradient in concentration across the boundary layer between the flowing solution and the root surface, and then across the cell wall to the site of transport through the plasmalemma (Polle and Jenny, 1971). If 0-01 mg N I" 1 is taken as the concentration of NOj at the site of uptake then it may be calculated, for experiment 2, that the ratio of the concentration ot N.O7 in

11 Clement, Hopper, and Jones Nitrate uptake by L. perenne 463 the roots to that in solution exceeded :1. This is undoubtedly an underestimate of the concentration gradient across the plasmalemma since the concentration in the roots was calculated on the assumption that all the water in the fresh root was within the symplast. Morgan, Volk, and Jackson (1973) proposed a model for NOj uptake by root cells which included components for active unidirectional influx and passive bidirectional influx and efflux. There is considerable evidence that efflux of NO^ can occur but, in the light of our results, its significance, under conditions involving large, fast growing plants with the ability to maintain high rates of uptake across large concentration gradients, is open to question. The high rates of uptake at low concentrations of NO j in solution are at some variance with the published value for the Michaelis constant (K m ) of 0-46 mg N I" 1 obtained from experiments with ryegrass (Lycklama, 1963). The use of flowing solution culture has served to emphasize that before. Michaelis-Menten kinetics, or similar theoretical analyses (Hodges, 1973), can be properly applied to observed relationships between the concentration of a nutrient in root media and uptake by the intact plant it is necessary to establish that supply to the site at which the appropriate uptake mechanisms operate is not limiting. This requirement is difficult to meet with fast-growing plants and the associated high rates of nutrient demand. It may perhaps be approached by growing plants in solutions with different rates of flow (Edwards and Asher, 1974) and measuring the rate of uptake when an increasing rate of flow has no further effect. High concentrations of ions in solution are known to affect the growth and metabolism of plants by (a) changing water relations, (b) specific ion toxicity, (c) reduced uptake and transport of other essential ions (Greenway, 1973). The osmotic potential of the solution containing NO7 at a concentration of 2000 mg N I" 1 was about 500 kpa. This is similar to the osmotic potential at which Magistad, Ayers, Wadleigh, and Gauch (1943) obtained a 30% reduction in yield of beet and an 80% reduction in yield of carrot. These authors noted that the reduction in yield was greatest when the plants were grown under conditions of low relative humidity and high evaporative stress. Although the temperature of the glasshouse in which our plants were grown was maintained at or below 25 C the relative humidity during the day averaged about 40%. There is evidence that, even at high relative humidity, the rate of extension of the leaves of perennial ryegrass falls considerably at soil water potentials below 700 kpa (Garwood, unpublished) and that under similar stress photosynthesis may be reduced by about 50% (Sheehy, Green, and Robson, 1975). Specific toxicity of the NO7 ion seems unlikely since its concentration in the plant was almost constant at solution concentrations of 200 mg N I" 1 and above (Fig. 6). Even at the highest concentration in solution the level of NO7 in the shoots (1-65% N) was, it may be noted, considerably less than that found in the tops of Italian ryegrass (2-04% N) grown in potted soils at the point of maximum yield (Nielsen and Cunningham, 1964; Nowakowski et al., 1965). Likewise there is no evidence that the uptake and transport of other nutrient ions within the plant were seriously affected by the high concentration of NOj in the culture solutions.

12 464 Clement, Hopper, and Jones Nitrate uptake by L. perenne ACKNOWLEDGEMENTS The authors wish to thank Messrs. R. J. Canaway and D. J. Hatch for technical assistance. LITERATURE CITED ALBERDA, TH., Neth. J. agric. Sci. 13, CLEMENT, C. R., HOPPER, M. J., CANAWAY, R. J., and JONES, L. H. P., 1974 J. exp. Bot. 25, COLEMAN, R. L., and LAZENBY, A., PI. Soil, 42, COWLING, D. W., and LOCKYER, D. R., J. agric. Sci., Camb. 75, Cox, W. J., and REISENAUER, H. M., PL Soil, 38, EDWARDS, D. G., and ASHER, C. J., Ibid. 41, GREENWAY, H., /. Aust. Inst. agric. Sci. 39, HENZELL, E. F., MARTIN, A. E., Ross, P. J., and HAYDOCK, K. P., Aust. J. agric. Res. 15, HEWITT, E. J., Sand and water culture methods used in the study of plant nutrition. Commonw. Bur. Hort. Plantn Crops, Tech. Commun. No. 22. Commonw. Agric. Bur., Farnham Royal. HODGES, T. K., Adv. Agron. 25, HUNT, I. V., Proc. 9th int. Grassld Congr. Sao Paulo. Pp HYLTON, L. O., JR., WILLIAMS, D. E., ULRICH, A., and CORNELIUS, D. R., Crop Sci. 4,16-9. LYCKLAMA, J. C, Ada. bot. neerl. 12, MAGISTAD, O. C, AYERS, A. D., WADLEIGH, C. H., and GAUCH, H. G., PI. Physiol., Lancaster, 18, MORGAN, M. A., VOLK, R. J., and JACKSON, W. A., Ibid. 51, NEILSON, F. J. A., N.Z. vet. J. 22,12-3. NIELSEN, K. F., and CUNNINGHAM, R. K., Proc. Soil Sci. Soc. Am. 28, NOWAKOWSKI, T. Z., CUNNINGHAM, R. K., and NIELSEN, K. F., /. Sci. Fd Agric. 16, POLLE, E. O., and JENNY, H., Physiologia PI. 25, SHEEHY, J. E., GREEN, R. M., and ROBSON, M. J., Ann. Bot. 39, SIBMA, L., Neth. J. agric. Sci. 16, WARNCKE, D. D., and BARBER, S. A., /. environ. Qual. 3, WHITEHEAD, D. C, Nutrient minerals in grassland herbage. Commonw. Bur. Past. Fid Crops, Mimeographed Publication No. 1/1966, Commonw. Agric. Bur., Farnham Royal.