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Effects of Nitrate and Light Intensity on Photosynthesis and Nitrogen Fixation in Alfalfa Plants
Effects of Nitrate and Light Intensity on Photosynthesis and Nitrogen Fixation in Alfalfa Plants MIGUEL A. HERRERA 1), EULOGIO J. BEDMAR2), and JOSE OUVARES2) 1) On study leave from Facultad de Ciencias Agronomicas, Veterinarias y Forestales. Universidad de Chile. Santiago, Chile 2) Departamento de Microbiologfa. Estacion Experimental del Zaidfn. Csrc. Apartado 419. 18008-Granada, Spain Received September 12, 1986 . Accepted October 21, 1986
Summary Alfalfa plants (Medicago sativa, L. cv. Aragon) inoculated with Rhizobium meliloti were grown with a N-free-nutrient solution in controlled environmental chambers. After 5 weeks plants were placed under 2 irradiance regimes (120 and 60/LE· m -2. S-I) and different N concentrations were supplied to treatment plants as Ca(N03h. Rates of acetylene-dependent ethylene production and CO2 uptake were determined 2 days later. The addition of 1.75 and 3.5 mM N0 3 - to plants in each level of irradiance decreased both nitrogenase activity and CO 2 assimilation regardless of the light intensity. The addition of 7 and 14 mM N0 3 - resulted in an enhanced leaf photosynthesis. Simultaneously, the C 2H 2 reduction rate by nodulated plants at 60/LE was lower than that of N 2-dependent plants, while plants at 120/LE had similar acetylene reduction values. Higher levels of combined nitrogen, 28 and 56 mM N0 3 - , reduced nitrogenase activity of plants under both of the two light intensities and CO2 uptake by plants at 60/LE. However, current photosynthesis was not affected in plants at 120/LE. These results are interpreted as showing that alfalfa plants can utilize nitrate to improve CO 2 assimilation without inhibiting N2 fixation when plants are under conditions in which light is not saturating.
Key words: Medicago sativa, Rhizobium meliloti, acetylene reduction, CO2 uptake.
Introduction The fact that nitrogen fixation by nodulated leguminous plants depends upon photosynthesis has been provided by short and long term CO 2 enrichment studies (Hardy and Havelka, 1976; Phillips et aI., 1976). Although symbiotic nitrogen fixation by legumes reduces their requirements for soil N, the full potential for plant growth and yield cannot be accomplished in the absence of combined nitrogen (Hageman, 1979). Nitrate, the most common form of nitrogen absorbed by plant roots growing in cultivated soils, inhibits N2 fixation activity of nodules (Gibson, 1976; Rigaud, 1981). However, the mechanism by which nitrogenase activity is adversely affected by nitrate is still not clear. Growth light intensity has also been shown to cause changes in the efficiency with which CO 2 and N2 are reduced (Gibson, 1976). Most studies on this subject have dealt with CO2-enrichment studies (Bethlenfalvay and Phillips, 1977; Williams and Phillips, 1980).
J. Plant. Physiol. Vol.
128. pp. 467-472 {1987}
468
MIGUEL A.
HERRERA,
EULOGIO J.
BEDMAR,
and JosE OLIVARES
3
.
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56
Nitrate concentration (mM)
Fig. 1: Effect of N03 - concentration on the nitrogenase activity of alfalfa plants under 120 (0) and 60 (.) JLE' m - 2 • S - 1 oflight intensity. Data are given ± SE (n = 12).
Few data are available on the response of both net photosynthesis and nitrogenase activity of plants under relatively low light conditions to the application of varying nitrate concentrations. The present study was conducted to ascertain the effects of different levels of nitrate on photosynthesis and N2 fixation of alfalfa plants grown at photosynthetic photon flux densities which do not saturate photosynthesis.
Materials and Methods Biological material Seeds of Medicago sativa, L. cv. Aragon were surface-sterilized with 2.5 % HgCh for 10 min, thoroughly rinsed with sterile water and germinated on wet filter paper in Petri dishes. After 3 days, seedlings were selected for uniformity and placed in 20x200mm test tubes (Olivares et aI., 1980). Tubes contained the N-free nutrient solution described by Rigaud and Puppo (1975). Plants (5 plants per tube) were inoculated with 1 ml of a Rhizobium meliloti (strains GR4) suspension (108 cells/ml) from bacteria cultured on yeast mannitol slopes and grown in controlled environmental chambers under 16/8h light/dark cycle, 24/18C, 50% relative humidity and a photosynthetic photon flux density (400-700nm) of 120ILE' -2· S -1. After 5 weeks, tubes were divided into two batches, one of them remaining under the conditions described above and the other placed at 60 JLE . m - 2 . S-I. Twelve tubes under each irradiance were proj. Plant. Physiol. Vol. 128. pp. 467-472 {1987}
Nitrate and irradiance effects on alfalfa
469
vided with Ca(N0 3h at 0, 1.75,3.5,7,14,28 and 56mM final concentrations. Control plants received CaCho After 48 h, plants from each nutrient and light regime were moved to the laboratory for determination of CO2 uptake and C2H 2 reduction. Determination ofCO2 uptake and nitrogenase activity
The CO2 assimilation rate was determined from the [14C}incorporated into organic material when 14COrenriched air was supplied to the leaves (Ba I4C03, specific activity 185 MBq . mol- I, Amersham) through a Perpex photosynthetic chamber at a flow rate of 4 mI· S-I (L6pez-Gorje et al., 1985) and at irradiance levels corresponding to the light treatments. The leaves were then digested with 30% (w/v) HN0 3 and, after addition of an emulsifier·scintillation mixture (Packard, Instagel), the 14C-incorporated was measured by scintillation counting (Packard, Tri-Carb). For the nitrogenase activity determinations, the tubes were sealed with rubber stoppers and the ethylene production was measured as previously reported (Bedmer and Olivares, 1980). All experiments were repeated twice under the experimental conditions described.
Results and Discussion The selection of the growth light conditions used in the present study was made in a previous screening using Nrdependent plants and was based on the fact that the rate of apparent N2 fixation in plants grown at 120 ",E was similar to the rate in plants at 60 ",E (Fig. 1). Under these light intensities, the rate of leaf net photosynthesis was 3fold greater for the plants at the higher irradiance (Fig. 2). Thus, under such conditions, neither the current photosynthesis nor the partitioning of photosynthate were limiting symbiotic nitrogen fixation. The addition of relatively low levels of combined nitrogen such as 1.75 and 3.5 mM N0 3 - decreased C 2H 2 reduction and CO 2 uptake by plants in each irradiance treatment (Figs. 1 and 2). The sensitivity of nitrogen fixation to nitrate has been related to a competition between nitrogenase and nitrate reductase for photosynthate (Oghoghorie and Pate, 1971), to a depressed nodule and bacteroid respiration (Mahon, 1977; Trinchant and Rigaud, 1981), to a direct effect of nitrite on nitrogenase activity (Kennedy et al., 1975; Trinchant and Rigaud, 1980) and to an effect on the loading of amino acids or other nitrogen compounds into the xylem (Streeter, 1985). The finding that low levels of nitrate diminished net photosynthesis seems to suggest that nitrogenase activity was inhibited through an impaired carboxylation process rather than a limitation in the availability of photosynthate. This effect may be attributed to the competition for electrons and ATP between the CO 2 and N0 2- reduction processes in the chloroplast. Such inhibitory effects have been shown in nitrogen-deficient cells of Chiarella (Thomas et aI., 1976) and in Anabaena and Radospirillum growing in the presence of NH4 + under low light conditions (Yoch and Gotto, 1982). Short exposures of 24 to 48 h duration to combined nitrogen inhibited nitrogen fixation activity of nodules maintained in hydroponic conditions at high (500 - 600 ",E) levels of irradiation (Rigaud, 1976; Gibson and Pagan, 1977). However, our results showed that the addition of 7 or 14 mM N0 3 - to alfalfa plants did not affect nitrogenase activity in plants at 120 ",E, while the rate of nitrogen fixation in plants at 60 ",E was lower than that of the control plants (Fig. 1). These nitrate con-
J. Plant. Physiol.
Vol. 128. pp. 467-472 (1987)
470
3
u.
--
MIGUEL
A.
HERRERA, EULOGIO
J. BEDMAR, and JosE OLIVARES
0.4
CJI
.c N
0
u
0.3
-0 E E
.
.!!!
1/1
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1.75
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28
i
56
Nitrate concentration (mM)
Fig. 2: Effect of N03 - concentration on the net photosynthesis of alfalfa plants under 120 (0) and 60 (.) fLE· m - 2. S -1 of light intensity. Data are given ± SE (n = 12).
centrations increased the photosynthetic CO 2 uptake in plants at both growth light conditions (Fig. 2). Most N03 - in alfalfa is reduced in the leaves (Vance and Heichel, 1981). Then, it is possible that after 48 h of growth the reduction of nitrate may have resulted in an increased intracellular metabolic pool which enhanced plant net photosynthesis. Improved rates of CO2 assimilation counteracted the inhibition of N2 fixation by N03 - in plants at 120/lE, but it was not sufficient to abolish the depressive effect on acetylene reduction activity in plants at 60/lE (Fig. 1). Partial relief of the inhibition of N2 fixation by N03 - with increasing growth light intensities has been previously reponed in pea plants (Bethlenfalvay and Phillips, 1977). Since increases in net photosynthesis were not followed by increases in nitrogenase activity, the results indicate that either part of the photosynthate was being retained in the shoots rather than being partitioned to the roots and nodules or that the photosynthate was not capable of supporting simultaneously both nitrogen fixation and nitrate reduction. These results sustain the case presented by Oghoghorie and Pate (1971) that nitrate reduction competes with nitrogenase for products of photosynthesis.
J. Plant. Physiol.
Vol. 128. pp. 467-472 {1987}
Nitrate and irradiance effects on alfalfa
471
Finally, rates of apparent nitrogen fixation drastically decreased when plants under both light intensities were provided with 28 or 56 mM N0 3 - (Fig. 1). Nitrate also depressed net CO2 uptake in plants at 60 ",E, but did not affect photosynthesis of plants under 160 ",E (Fig. 2). The inhibition of nitrogenase activity was probably due to any of the mechanisms that repress N2 fixation mentioned before. Ammonia accumulation within the roots has been shown to uncouple A TP synthesis (Hewitt, 1975) and therefore can be responsible for the inhibition of the nitrogenase enzyme. Ammonia also has a toxic effect on leaves when present in excess of its assimilation. CO 2 uptake by N0 3 - -treated plants was depressed in plants at 60/LE. However, this effect was not observed in plants under 120/LE, indicating that products of photosynthesis were available to prevent ammonia accumulation in leaves and the decay in CO2 assimilation. The fact that intermediate levels of N0 3 - (7 and 14 mM) did not decrease acetylene reduction activity in alfalfa root nodules is of interest because many agricultural plants grow in mixed crops where the nitrogen fixation process can be impaired by conditions of partial shade or the presence of nitrate in the soil solution. Acknowledgements The authors thank Prof. J. Lopez-Gorje for useful advice during 14C determinations. This work was supported by the Comision Asesora de Investigacion Ciendfica y Tecnica (Spain) grant No. 1764/82.
References BEDMAR, E. J. and J. OLIVARES: Effect of chemical inhibitors of photorespiration on nitrogenase activity in nodulated alfalfa plants. Planta 150, 299-302 (1980). BETHLENFALVAY, G. J. and D. A. PHILLIPS: Ontogenetic interactions between photosynthesis and symbiotic nitrogen fixation in legumes. Plant Physio!. 60, 419-421 (1977). - Interactions between symbiotic nitrogen fixation, combined-N-application and photosynthesis in Pisum sativum. Physio!. Plant. 42, 119-123 (1978). GIBSON, A. H.: Recovery and compensation by nodulated legumes to environmental stress. In: NUTMAN, P. S. {ed.}: Symbiotic Nitrogen Fixation in Plants. Internat. Bio!. Programme Vo!' 7. pp. 385-403. Cambridge University Press. Cambridge {1976}. GIBSON, A. H. andJ. D. PAGAN: Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium. Planta 134, 17 -22 {1977}. HAGEMAN, R. H.: Integration of nitrogen assimilation in relation to yield. In: HEWITT, E. 1. and C. V. CUTTING {eds.}: Nitrogen Assimilation of Plants. Proc. VI Long Ashton Symposium. pp. 591-611. Academic Press, New York {1979}. HARDY, R. W. F. and U. D. HAVELKA: Photosynthate as a major factor limiting nitrogen fixation by field-grown legumes with emphasis on soybeans. In: NUTMAN, P. S. {ed.}: Symbiotic Nitrogen Fixation in Plants. Internat. Bio!. Programme Vo!' 7. pp. 421-439. Cambridge University Press. Cambridge (1976). HEWITT, E. J.: Assimilatory nitrate-nitrite reduction. Annu. Rev. Plant Physio!. 26, 73 -100 (1975). KENNEDY, J. R., J. RIGAUD, and J. R. TRINCHANT: Nitrate reductase from bacteroids of Rhizo· bium japonicum: enzyme characteristics and possible interaction with nitrogen fixation. Biochim. Biophys. Acta 397, 24-35 {1975}. LOPEz-GORJE, J., O. LASTRA, A. CHUECA, and M. LACHICA: Use of photosynthetic parameters for the diagnosis of copper deficiency in Pinus radiata seedlings. Physiol. Plant. 65, 508-512 (1985).
J Plant. Physiol. Vol.
128. pp. 467-472 {1987}
472
MIGUEL A. HERRERA, EULOGIO J. BEDMAR, and JosE OLIVARES
MAHON, J. D.: Root and nodule respiration in relation to acetylene reduction in intact nodulated peas. Plant Physio!' 60, 812-816 (1977). OGHOGHORIE, C. G. O. and J. S. PATE: The nitrate stress syndrome of the nodulated field pea (Pisum arvense L.). In: LIE, P. S. and E. G. MULDER (eds.) Biological Nitrogen Fixation in Natural and Agricultural Habitats, Plant and Soil Special Volume. pp. 185-202. Martinus Nijhoff, The Hague (1971). OLIVARES, J., J. CASADESUS, and E. J. BEDMAR: Method for testing degree of infectivity of Rhizobium meliloti strains. App!. Environ. Microbio!' 39, 967 - 970 (1980). PHILLIPS, D. A., K. D. NEWEll, S. A. HASSELL, and C. E. FELLING: The effect of CO2 enrichment on root nodule development and symbiotic N2 reduction in Pisum sativum L. Am. J. Bot. 63,356-362 (1976). RIGAUD, J.: Effet des nitrates sur la fixation d' azote par les nodules de Haricot (Phaseolus vulgaris L.). Physio!. veg. 14, 297 -308 (1976). - Comparison of the efficiency of nitrate and nitrogen fixation in crop yield. In: BEWLEY, J. D. (ed.) Nitrogen and Carbon Metabolism. pp. 17 -48. Martinus Nijhoff, The Hague (1981). RIGAUD, J. and A. PuPPO: Indole-3-acetic acid catabolism by soybean bacteroids. J. Gen. Microbio!' 88, 223-228 (1975). STREETER, J. G.: Nitrate inhibition of legume nodule growth and activity. II. Short term studies with high nitrate supply. Plant Physio!. 77, 325-328 (1985). THOMAS, R. J., C. R. HIPKIN, and P. J. SYRETT: The interaction of nitrogen assimilation with photosynthesis in nitrogen-deficient cells of Chlorella. Planta 133, 9-13 (1976). TRINCHANT, J. C. and J. RIGAUD: Nitrite inhibition of nitrogenase from soybean bacteroids. Arch. Microbio!. 124,49-54 (1980). - - Acetylene reduction and respiration of bacteroids isolated from frenchbeans receiving nitrate. Physio!. Plant. 53, 511-517 (1981). VANCE, C. P. and G. H. HEICHEL: Nitrate assimilation during vegetative regrowth of alfalfa. Plant Physio!. 68, 1052- i056 (1981). YOCH, D. C. and J. W. GOTTO: Effect of light intensity and inhibitors of nitrogen assimilation on NH4 + inhibition of nitrogenase activity in Rhodospirillum rubrum and Anabaena sp. J. Bacterio!' 151, 800-806 (1982). WILLIAMS, L. E. and D. A. PHILLIPS: Effect of irradiance on development of apparent nitrogen fixation and photosynthesis in soybean. Plant Physio!. 66, 968-972 (1980).
j. Plant. Physiol. Vol. 128. pp. 467-472 (1987)
Summary Alfalfa plants (Medicago sativa, L. cv. Aragon) inoculated with Rhizobium meliloti were grown with a N-free-nutrient solution in controlled environmental chambers. After 5 weeks plants were placed under 2 irradiance regimes (120 and 60/LE· m -2. S-I) and different N concentrations were supplied to treatment plants as Ca(N03h. Rates of acetylene-dependent ethylene production and CO2 uptake were determined 2 days later. The addition of 1.75 and 3.5 mM N0 3 - to plants in each level of irradiance decreased both nitrogenase activity and CO 2 assimilation regardless of the light intensity. The addition of 7 and 14 mM N0 3 - resulted in an enhanced leaf photosynthesis. Simultaneously, the C 2H 2 reduction rate by nodulated plants at 60/LE was lower than that of N 2-dependent plants, while plants at 120/LE had similar acetylene reduction values. Higher levels of combined nitrogen, 28 and 56 mM N0 3 - , reduced nitrogenase activity of plants under both of the two light intensities and CO2 uptake by plants at 60/LE. However, current photosynthesis was not affected in plants at 120/LE. These results are interpreted as showing that alfalfa plants can utilize nitrate to improve CO 2 assimilation without inhibiting N2 fixation when plants are under conditions in which light is not saturating.
Key words: Medicago sativa, Rhizobium meliloti, acetylene reduction, CO2 uptake.
Introduction The fact that nitrogen fixation by nodulated leguminous plants depends upon photosynthesis has been provided by short and long term CO 2 enrichment studies (Hardy and Havelka, 1976; Phillips et aI., 1976). Although symbiotic nitrogen fixation by legumes reduces their requirements for soil N, the full potential for plant growth and yield cannot be accomplished in the absence of combined nitrogen (Hageman, 1979). Nitrate, the most common form of nitrogen absorbed by plant roots growing in cultivated soils, inhibits N2 fixation activity of nodules (Gibson, 1976; Rigaud, 1981). However, the mechanism by which nitrogenase activity is adversely affected by nitrate is still not clear. Growth light intensity has also been shown to cause changes in the efficiency with which CO 2 and N2 are reduced (Gibson, 1976). Most studies on this subject have dealt with CO2-enrichment studies (Bethlenfalvay and Phillips, 1977; Williams and Phillips, 1980).
J. Plant. Physiol. Vol.
128. pp. 467-472 {1987}
468
MIGUEL A.
HERRERA,
EULOGIO J.
BEDMAR,
and JosE OLIVARES
3
.
.tl
--.a ~
2
~
:I: N
U
-0
E c
>.
-.. > u
.... 1/1 C ell
01 0
L.
Z
0 i
0
1.75
3.5
7
14
28
56
Nitrate concentration (mM)
Fig. 1: Effect of N03 - concentration on the nitrogenase activity of alfalfa plants under 120 (0) and 60 (.) JLE' m - 2 • S - 1 oflight intensity. Data are given ± SE (n = 12).
Few data are available on the response of both net photosynthesis and nitrogenase activity of plants under relatively low light conditions to the application of varying nitrate concentrations. The present study was conducted to ascertain the effects of different levels of nitrate on photosynthesis and N2 fixation of alfalfa plants grown at photosynthetic photon flux densities which do not saturate photosynthesis.
Materials and Methods Biological material Seeds of Medicago sativa, L. cv. Aragon were surface-sterilized with 2.5 % HgCh for 10 min, thoroughly rinsed with sterile water and germinated on wet filter paper in Petri dishes. After 3 days, seedlings were selected for uniformity and placed in 20x200mm test tubes (Olivares et aI., 1980). Tubes contained the N-free nutrient solution described by Rigaud and Puppo (1975). Plants (5 plants per tube) were inoculated with 1 ml of a Rhizobium meliloti (strains GR4) suspension (108 cells/ml) from bacteria cultured on yeast mannitol slopes and grown in controlled environmental chambers under 16/8h light/dark cycle, 24/18C, 50% relative humidity and a photosynthetic photon flux density (400-700nm) of 120ILE' -2· S -1. After 5 weeks, tubes were divided into two batches, one of them remaining under the conditions described above and the other placed at 60 JLE . m - 2 . S-I. Twelve tubes under each irradiance were proj. Plant. Physiol. Vol. 128. pp. 467-472 {1987}
Nitrate and irradiance effects on alfalfa
469
vided with Ca(N0 3h at 0, 1.75,3.5,7,14,28 and 56mM final concentrations. Control plants received CaCho After 48 h, plants from each nutrient and light regime were moved to the laboratory for determination of CO2 uptake and C2H 2 reduction. Determination ofCO2 uptake and nitrogenase activity
The CO2 assimilation rate was determined from the [14C}incorporated into organic material when 14COrenriched air was supplied to the leaves (Ba I4C03, specific activity 185 MBq . mol- I, Amersham) through a Perpex photosynthetic chamber at a flow rate of 4 mI· S-I (L6pez-Gorje et al., 1985) and at irradiance levels corresponding to the light treatments. The leaves were then digested with 30% (w/v) HN0 3 and, after addition of an emulsifier·scintillation mixture (Packard, Instagel), the 14C-incorporated was measured by scintillation counting (Packard, Tri-Carb). For the nitrogenase activity determinations, the tubes were sealed with rubber stoppers and the ethylene production was measured as previously reported (Bedmer and Olivares, 1980). All experiments were repeated twice under the experimental conditions described.
Results and Discussion The selection of the growth light conditions used in the present study was made in a previous screening using Nrdependent plants and was based on the fact that the rate of apparent N2 fixation in plants grown at 120 ",E was similar to the rate in plants at 60 ",E (Fig. 1). Under these light intensities, the rate of leaf net photosynthesis was 3fold greater for the plants at the higher irradiance (Fig. 2). Thus, under such conditions, neither the current photosynthesis nor the partitioning of photosynthate were limiting symbiotic nitrogen fixation. The addition of relatively low levels of combined nitrogen such as 1.75 and 3.5 mM N0 3 - decreased C 2H 2 reduction and CO 2 uptake by plants in each irradiance treatment (Figs. 1 and 2). The sensitivity of nitrogen fixation to nitrate has been related to a competition between nitrogenase and nitrate reductase for photosynthate (Oghoghorie and Pate, 1971), to a depressed nodule and bacteroid respiration (Mahon, 1977; Trinchant and Rigaud, 1981), to a direct effect of nitrite on nitrogenase activity (Kennedy et al., 1975; Trinchant and Rigaud, 1980) and to an effect on the loading of amino acids or other nitrogen compounds into the xylem (Streeter, 1985). The finding that low levels of nitrate diminished net photosynthesis seems to suggest that nitrogenase activity was inhibited through an impaired carboxylation process rather than a limitation in the availability of photosynthate. This effect may be attributed to the competition for electrons and ATP between the CO 2 and N0 2- reduction processes in the chloroplast. Such inhibitory effects have been shown in nitrogen-deficient cells of Chiarella (Thomas et aI., 1976) and in Anabaena and Radospirillum growing in the presence of NH4 + under low light conditions (Yoch and Gotto, 1982). Short exposures of 24 to 48 h duration to combined nitrogen inhibited nitrogen fixation activity of nodules maintained in hydroponic conditions at high (500 - 600 ",E) levels of irradiation (Rigaud, 1976; Gibson and Pagan, 1977). However, our results showed that the addition of 7 or 14 mM N0 3 - to alfalfa plants did not affect nitrogenase activity in plants at 120 ",E, while the rate of nitrogen fixation in plants at 60 ",E was lower than that of the control plants (Fig. 1). These nitrate con-
J. Plant. Physiol.
Vol. 128. pp. 467-472 (1987)
470
3
u.
--
MIGUEL
A.
HERRERA, EULOGIO
J. BEDMAR, and JosE OLIVARES
0.4
CJI
.c N
0
u
0.3
-0 E E
.
.!!!
1/1
£
0.2
c >-
1/1
0
'0 .c
0-
a; z
0.1
o i
1.75
i
i
i
i
3.5
7
14
28
i
56
Nitrate concentration (mM)
Fig. 2: Effect of N03 - concentration on the net photosynthesis of alfalfa plants under 120 (0) and 60 (.) fLE· m - 2. S -1 of light intensity. Data are given ± SE (n = 12).
centrations increased the photosynthetic CO 2 uptake in plants at both growth light conditions (Fig. 2). Most N03 - in alfalfa is reduced in the leaves (Vance and Heichel, 1981). Then, it is possible that after 48 h of growth the reduction of nitrate may have resulted in an increased intracellular metabolic pool which enhanced plant net photosynthesis. Improved rates of CO2 assimilation counteracted the inhibition of N2 fixation by N03 - in plants at 120/lE, but it was not sufficient to abolish the depressive effect on acetylene reduction activity in plants at 60/lE (Fig. 1). Partial relief of the inhibition of N2 fixation by N03 - with increasing growth light intensities has been previously reponed in pea plants (Bethlenfalvay and Phillips, 1977). Since increases in net photosynthesis were not followed by increases in nitrogenase activity, the results indicate that either part of the photosynthate was being retained in the shoots rather than being partitioned to the roots and nodules or that the photosynthate was not capable of supporting simultaneously both nitrogen fixation and nitrate reduction. These results sustain the case presented by Oghoghorie and Pate (1971) that nitrate reduction competes with nitrogenase for products of photosynthesis.
J. Plant. Physiol.
Vol. 128. pp. 467-472 {1987}
Nitrate and irradiance effects on alfalfa
471
Finally, rates of apparent nitrogen fixation drastically decreased when plants under both light intensities were provided with 28 or 56 mM N0 3 - (Fig. 1). Nitrate also depressed net CO2 uptake in plants at 60 ",E, but did not affect photosynthesis of plants under 160 ",E (Fig. 2). The inhibition of nitrogenase activity was probably due to any of the mechanisms that repress N2 fixation mentioned before. Ammonia accumulation within the roots has been shown to uncouple A TP synthesis (Hewitt, 1975) and therefore can be responsible for the inhibition of the nitrogenase enzyme. Ammonia also has a toxic effect on leaves when present in excess of its assimilation. CO 2 uptake by N0 3 - -treated plants was depressed in plants at 60/LE. However, this effect was not observed in plants under 120/LE, indicating that products of photosynthesis were available to prevent ammonia accumulation in leaves and the decay in CO2 assimilation. The fact that intermediate levels of N0 3 - (7 and 14 mM) did not decrease acetylene reduction activity in alfalfa root nodules is of interest because many agricultural plants grow in mixed crops where the nitrogen fixation process can be impaired by conditions of partial shade or the presence of nitrate in the soil solution. Acknowledgements The authors thank Prof. J. Lopez-Gorje for useful advice during 14C determinations. This work was supported by the Comision Asesora de Investigacion Ciendfica y Tecnica (Spain) grant No. 1764/82.
References BEDMAR, E. J. and J. OLIVARES: Effect of chemical inhibitors of photorespiration on nitrogenase activity in nodulated alfalfa plants. Planta 150, 299-302 (1980). BETHLENFALVAY, G. J. and D. A. PHILLIPS: Ontogenetic interactions between photosynthesis and symbiotic nitrogen fixation in legumes. Plant Physio!. 60, 419-421 (1977). - Interactions between symbiotic nitrogen fixation, combined-N-application and photosynthesis in Pisum sativum. Physio!. Plant. 42, 119-123 (1978). GIBSON, A. H.: Recovery and compensation by nodulated legumes to environmental stress. In: NUTMAN, P. S. {ed.}: Symbiotic Nitrogen Fixation in Plants. Internat. Bio!. Programme Vo!' 7. pp. 385-403. Cambridge University Press. Cambridge {1976}. GIBSON, A. H. andJ. D. PAGAN: Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium. Planta 134, 17 -22 {1977}. HAGEMAN, R. H.: Integration of nitrogen assimilation in relation to yield. In: HEWITT, E. 1. and C. V. CUTTING {eds.}: Nitrogen Assimilation of Plants. Proc. VI Long Ashton Symposium. pp. 591-611. Academic Press, New York {1979}. HARDY, R. W. F. and U. D. HAVELKA: Photosynthate as a major factor limiting nitrogen fixation by field-grown legumes with emphasis on soybeans. In: NUTMAN, P. S. {ed.}: Symbiotic Nitrogen Fixation in Plants. Internat. Bio!. Programme Vo!' 7. pp. 421-439. Cambridge University Press. Cambridge (1976). HEWITT, E. J.: Assimilatory nitrate-nitrite reduction. Annu. Rev. Plant Physio!. 26, 73 -100 (1975). KENNEDY, J. R., J. RIGAUD, and J. R. TRINCHANT: Nitrate reductase from bacteroids of Rhizo· bium japonicum: enzyme characteristics and possible interaction with nitrogen fixation. Biochim. Biophys. Acta 397, 24-35 {1975}. LOPEz-GORJE, J., O. LASTRA, A. CHUECA, and M. LACHICA: Use of photosynthetic parameters for the diagnosis of copper deficiency in Pinus radiata seedlings. Physiol. Plant. 65, 508-512 (1985).
J Plant. Physiol. Vol.
128. pp. 467-472 {1987}
472
MIGUEL A. HERRERA, EULOGIO J. BEDMAR, and JosE OLIVARES
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j. Plant. Physiol. Vol. 128. pp. 467-472 (1987)