0032-0889/79/63/0956/07/$00.50/0. Low Root Temperature Effects on Soybean Nitrogen Metabolism and Photosynthesis'. Received for publication October 17, ...
Plant Physiol. (1979) 63, 956-962 0032-0889/79/63/0956/07/$00.50/0
Low Root Temperature Effects on Soybean Nitrogen Metabolism and Photosynthesis' Received for publication October 17, 1978 and in revised form January 2, 1979
STANLEY H. DuKE, LARRY E. SCHRADER, CYNTHIA A. HENSON, JEROME C. SERVAITES', ROBERT D. VOGELZANG, AND JOHN W. PENDLETON Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706 recent studies have shown that a limiting temperature for a biochemical event may limit a physiological process in such
ABSTRACT The influences of low root temperature on soybeans (Glycine max IL.1 Merr. cv. Wells) were studied by germinating and maintaining plants at root temperatures of 13 and 20 C through maturity. At 42 days from the beginning of imbibition, 13 and 20 C plants were switched to 20 and 13 C, respectively. Plants were harvested after 63 days. Control plants (13 C) did not nodulate, whereas those switched to 20 C did and at harvest had C2H2 reduction rates of 0.2 micromoles per minute per plant. Rates of C2H2 reduction decreased rapidly in plants switched from 20 to 13 C; however, after 2 days, rates recovered to original levels (0.8 micromoles per minute per plant) and then began a slow decline until harvest. Arrhenius plots of C2H2 reduction by whole plants indicated a large increase in the energy of activation below the inflection at 15 C. Highest C2H2 reduction rates (1.6 micromoles per minute per plant) were at 58 days for the 20 C control. Root respiration rates followed much the same pattern as C2H2 reduction in the 20 C control and transferred plants. At harvest, roots from 13 C-treated plants had the highest activities for malate dehydrogenase, glutamate oxaloacetate transaminase, and phosphoenolpyruvate carboxylase. Roots from transferred plants had intermediate activities and those from the 20 C treatment the lowest activities. Newly formed nodules from plants switched from 13 to 20 C had much higher glutamate dehydrogenase than glutamine synthetase activity. Photosynthetic rates on a leaf area basis were about three times as high in the 20 C control as compared to 13 C control plants. Photosynthetic rates of plants switched from 20 to 13 C decreased to less than half the original rate within 2 days. Photosynthetic rates of plants switched from 13 to 20 C recovered to rates near those of the 20 C control plants within 2 weeks. All leaf enzymes assayed at harvest, with the exception of nitrate reductase, were highest in activity in the 20 C control plants.
chilling-sensitive plants (13, 25). Also, there have been few studies in which root temperatures and other environmental parameters were rigidly controlled under otherwise near optimal conditions from germination to maturity. Most studies on temperature effects on legume C2H2 reduction have been conducted with detached nodules, root-nodule sections, or root-soil bores (7, 17, 31, 32). In this study we measured whole plant C2H2 reduction, root respiration rates, and leaf photosynthetic and transpiration rates while controlling root and shoot temperatures of soybeans from germination to maturity under a high irradiance photoperiod. Our data show hitherto unknown low root temperature responses and relationships in soybeans. MATERIALS AND METHODS Plant Material. Soybean (Glycine max [L.] Merr. cv. Wells) seeds were imbibed and germinated at 13 and 20 C as previously described (13). At 15 and 19 days (20 and 13 C treatments, respectively), seedlings were transferred to pots (Fig. 1) containing nutrient solution (1.4 mm CaCl2, 1.0 mM MgSO4, 0.5 mm KH2PO4, 125 AM K2SO4, 22.7 t.M H3BO3, 0.91 ftM MnCl2, 0.208 ytM (NH4)6Mo7024, 0.20 uM ZnSO4, 0.787 tLM CuSO4, 0.017 ytM CoSO4, 0.05 g liter-' sodium ferric diethylenetriamine pentaacetate (about 87.5 ylM Fe, Sequestrene 330 Fe, Ciba-Geigy Corp., Ardsley, N.Y.). Nutrient solution was titrated to pH 6.5 with KOH. Fresh nutrient solution was used to replace nutrient solution in pots that had risen above pH 7.5. Pots were inoculated with 0.5 g (about 50
million rhizobia) Rhizobium japonicum inoculant (Nitragen "S" inoculant, Nitragen Co., Milwaukee, Wis.). KNO3 was added (20 ,umol per addition) to pots when plants appeared chlorotic (days
There have been few investigations on low temperature effects on roots. However, in legumes, low soil or root temperature decreases both nodulation and rates of N2 fixation (7, 17, 19, 26, 32). Many adverse effects of low soil temperature on chillingsensitive plants can be attributed to low-temperature-induced membrane phase transitions which slow protoplasmic streaming, increase permeability, and decrease the activities of membranebound enzymes by increasing Eas3 (13, 14, 25). Furthermore, these ' Research supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, the National Soybean Crop Improvement Council, USDA-CSRS Grant 616-15-72, and the American Soybean Association Research Grant 75-ASARF-208-3. 2 Present address: Light and Plant Growth Laboratory, Plant Physiology Institute, SEA, USDA, Beltsville, Maryland 20705. 3 Abbreviations: E.: energy of activation; 1,: diffusive resistance; NR: nitrate reductase (EC 1.6.6.1); GDH: glutamate dehydrogenase (EC
27, 45, 50, and 63 for the 23 C treatment; days 41 and 63 for the 13 C treatment). All plants (12 per temperature treatment) were grown in a plant growth room at the University of Wisconsin Biotron in thermostatically controlled vats at their respective germination temperatures. Vat solutions (H20 and NaClO) were just below the tops of pots and maintained pot temperatures at ± I C. Pots were aerated (2.3 liters min-) for 15 out of every 30 min through an aeration tube (Fig. 1). Each tank was placed under an identical bank of lamps (18 l00-w incandescent, 30 200w fluorescent (Sylvania cool-white F 96T12-VHO), and four 400w metal halide (Sylvania Metalarc GTE MS 400/C/HOR) lamps) which yielded a maximal flux density of 679 ± 25 ,tE m-2 s-1 (400-700 nm) at pot top levels for the 24 pots. The light-dark cycle (14:10 h) was phased at the beginning and end of the photoperiod 1.4.1.3); MDH: malate dehydrogenase (EC 1.1.1.37); GS: glutamine synthetase (EC 6.3.1.2); GOGAT: glutamate synthase (EC 2.6.1.53); GOT: glutamate oxaloacetate transaminase (EC 2.6.1.1); PEPC: phosphoenolpyruvate carboxylase (EC 4.1.1.31); GAO: glycolic acid oxidase (EC 1.1.3.1); RuBPC: ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)
956
957 Vol.Vl.63 Plat hyio. 179 LOW ROOT TEMPERATURE EFFECTS IN SOYBEANS95 Plant 63, 1979 Physiol. to approximate natural spectral and intensity changes. Day-night shaft. The air was circulated through the pot at 333 ml min-' with temperature regimen for the shoots was 28:21 C. RH was main- an electromagnetic piston pump (Reciprotor Co., Copenhagen). tained at 55 to 60%1. At 42 days from imbibition two pots from Before air entered the pump a fraction was bled into a Beckman each treatment (13 and 20 C) were switched. Switched plants and 865 IR gas analyzer at 140 ml min-'. The IR gas analyzer outlet two control plants from each treatment were harvested at 63 days was introduced back into the system near the pump outlet. After from imbibition. Other control plants were monitored until ma- flushing the system for several minutes with compressed air, assays were initiated by disconnecting the compressed air line and sealing turity. Acetylene Reduction Assays. Nutrient solution (about 1,500 ml) all open ports. At this time the system was completely closed and was removed from each pot through a tube connected to the had a volume of 3.6 liters. Rates Of CO2 flUX (U CO2 min-' siphon shaft (Fig. 1). This left about 3.1 liters of gas space in each plant-') were calculated from the time necessary to produce 100 pot. Air lines were then removed and all ports were sealed. Air PI liter-' CO2 over the initial concentration Of CO2 in the system. (120 ml) was replaced with an equal volume of purified acetylene Rates were linear over this period. with 60-ml syringes. This yielded a 3.8 to 4.0%7 (v/v) acetylene Photosynthesis Assays. The middle leaflet of the third (13 C) concentration in air. Acetylene was mixed in pots by the pumping or sixth (20 C) trifoliolate leaf of each plant was used in all action of a 60-ml syringe for about 30 s. Samples (three 1-ml photosynthetic assays. Leaflets were clamped into an air-tight syringes) were immediately collected for a zero time reading and Plexiglas cuvette which exposed a leaf area of either 2 or 10 CM2. again at 15, 30, and 60 min. Arrhenius plots were derived from Major veins were avoided and leaflets were not measured until acetylene reduction data collected when 20-C-grown plants were they had a width of >2 cm. Compressed air (325 ± 10 tdl liter-' transferred to 13 C. The drop in temperature from 20 to 13 C took C02) was introduced into the system (Fig. 2) and through the
about 70 min with samples taken every 3 to 7 min. Air-acetylene were monitored continuously over the course of experimentation. Pots were refilled with the original nutrient solution after air-acetylene temperatures had equilibrated for 30 min. Acetylene and ethylene concentrations were determined with a Hewlett Packard 7620 A gas chromatograph with a 2.4-in column (0.5 cm i.d.) containing Porapak type N (100 mesh). Injection temperature was 100 C and column temperature 80 C. All acetylene reduction values are expressed as nmol C2H2 reduced miiiin' plant-' or g-1 fresh weight of nodules. Root Respiration Assays. Nutrient solution was removed from pots as for acetylene reduction. A compressed air line (325 Al liter'1 C02) was then connected to the siphon shaft and an exhaust air line to a gassing and sampling port (Fig. 1) opposite the siphon
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962
13 to 20 C root temperatures displayed a marked increase in the activities of leaf GS and GDH over the 13 C control plants. GDH is known to be induced by NH4' (1 1). Differences in NR activity with the various treatments reflect the small amounts of N03added to plants at 13 C in the N03- strongly induces NR activity (10). Photosynthesis. In plants with 20 C root temperatures we found a high correlation between low leaf Xrs and high photosynthetic rates (Figs. 5 and 6), as is normally found (21). This negative correlation was also true to some extent in the 13 C control plants after 50 days. Mrs for plants switched from 13 to 20 C and 20 to 13 C root temperatures increased at first, and then decreased (Fig. 6). It should be noted that leaves which were partially folded away from incident radiation may have lowered their Xrs by this type of orientation. Transpiration rate is also negatively correlated with leaf Er (21). In such species as Zea mays it appears that photosynthesis, Er, and transpiration rate are controlled by leaf water potential, which is drastically lowered by low soil temperature (1). Our data would suggest that in soybeans this relationship may be much more complicated (Figs. 5 and 6) and further studies are needed. As with the leaf enzymes of nitrogen metabolism, all leaf enzymes assayed which are related to photosynthesis (RuBPC, PEPC and GAO) had higher activities in the 20 C control plants (Table II). However, unlike the enzymes of nitrogen metabolism, when 20 C control plants were transferred to 13 C root temperatures, these leaf enzymes were diminished in activity to rates below those of the 13 C control plants. In contrast, the respiratory enzyme MDH did not decrease nearly as much. This indicates that soybean leaf photosynthetic enzymes are severely affected by low root temperatures during the middle of the growing season. LITERATURE CITED 1. BARLOW EWR, L BOERSMA, IL YOUNG 1977 Photosynthesis, transpiration, and leaf elongation in corn seedlings at suboptimal soil temperatures. Agron J 69: 95-100 2. BETHLENFALVAY GJ, DA PHILIPS 1977 Ontogenetic interactions between photosynthesis and symbiotic nitrogen fixation in legumes. Plant Physiol 60: 419-421 3. CATSKY J, J JANAC, PG JARVIS 1971 General principles of using IRGA for measuring CO2 exchange rate. In Z Sestak, J Catsky, PG Jarvis. eds. Plant Photosynthetic Production: Manual of Methods. Dr W Junk, The Hague, pp 161-166 4. CHRISTELLER JT, WA LAING, WD SurrON 1977 Carbon dioxide fixation by lupin root nodules. 1. Characterization, association with phosphoenolpyruvate carboxylase. and correlation with nitrogen fixation during nodule development. Plant Physiol 60: 47-50 5. COOPER AJ 1973 Root temperature and plant growth. Res Rev No 4, Commonwealth Bureau of Horticulture and Plantation Crops, East Malling Kent, England 6. CRISWELL JG, RWF HARDY, UD HAVELKA 1976 Nitrogen fixation in soybeans: measurement techniques and examples of applications. World Soybean Res, Sept 1976. pp 108-124 7. DART PJ, JM DAY 1971 Effects of incubation temperature and oxygen tension on nitrogenase
Plant Physiol. Vol. 63, 1979
activity of legume root nodules. Plant Soil (Special Vol): 167-184 8. DAVID KAV, SK APTE, J THOMAS 1978 Stimulation of nitrogenase by acetylene: fresh synthesis or conformational change? Biochem Biophys Res Commun 82: 39-45 9. DAVID KAV, P FAY 1977 Effects of long-term treatment with acetylene on nitrogen-fixing organisms. Appl Environ Microbiol 34: 640-646 10. DuKE SH, JW FRIEDRICH, LE SCHRADER, WL KOuKKARI 1978 Oscillations in the activities of enzymes of nitrate reduction and ammonia assimilation in Glycine max and Zea mays. Physiol Plant 42: 269-276 11. DUKE SH, GE HAM 1976 The effect of nitrogen addition on N2 fixation and glutamate dehydrogenase and glutamate synthase activities in nodules and roots of soybeans inoculated with various strains of Rhizobium japonicum. Plant Cell Physiol 17: 1037-1044 12. DUKE SH, WL KOUKKARI, TK SOULEN 1975 Glutamate dehydrogenase activity in roots: distribution in a seedling and a storage root, and the effects of red and far-red illuminations. Physiol Plant 34: 8-13 13. DUKE SH, LE SCHRADER, MG MILLER 1977 Low temperature effects on soybean (Glvcine max [L.] Merr. cv. Wells) mitochondrial respiration and several dehydrogenases during imbibition and germination. Plant Physiol 60: 716-722 14. DUKE SH, LE SCHRADER, MG MILLER, RL NIECE 1978 Low temperature effects on soybean (Glycine max [L.] Merr. cv. Wells) free amino acid pools during germination. Plant Physiol 62: 642-647 15. FISHBECK K. HJ EVANS, LL BOERSMA 1973 Measurement of nitrogenase activity of intact legume symbionts in situ using the acetylene reduction assay. Agron J 65: 429-433 16. HARDY RWF, RC BURNS, RD HOLSTEN 1973 Application of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol Biochem 5: 47-81 17. HARDY RWF, RD HOLSTEN, EK JACKSON, RC BURNS 1968 The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol 43: 1185-1207 18. HAVELKA WD, RWF HARDY 1976 Legume N2 fixation as a problem of carbon nutrition. In WE Newton, CJ Nyman, eds, International Symposium of N2 fixation, Vol 2. Washington State Univ Press, Pullman, pp 456-474 19. JONES FR, WB TtSDALE 1921 Effect of soil temperature upon the development of nodules on the roots of certain legumes. J Agric Res 22: 17-31 20. KLUCAS RV 1974 Studies on soybean nodule senescence. Plant Physiol 54: 612-616 21. KRIEDEMANN DE 1971 Photosynthesis and transpiration as a function of gaseous diffusive resistances in orange leaves. Physiol Plant 24: 218-225 22. MAGUE TH, RH BURRtS 1972 Reduction of acetylene and nitrogen by field grown soybeans. New Phytol 71: 275-286 23. MAHON ID 1977 Root and nodule respiration in relation to acetylene reduction in intact nodulated peas. Plant Physiol 60: 812-816 24. QUEBEDEAUX B, UD HAVELKA, KL LtVAK, RWF HARDY 1975 Effect of altered pO2 in the aerial part of soybean on symbiotic N2 fixation. Plant Physiol 56: 761-764 25. JK, EA CHAPMAN 1976 Membrane phase changes in chilling-sensitive Vigna radiala and their significance to growth. Aust J Plant Physiol 3: 291-299 26. ROUGHLEY RJ. PJ DART 1970 Growth of Trifolium subierraneum L. selected for sparse and abundant nodulation as affected by root temperature and Rhizobium strain. J Exp Bot 21: 776-786 27. SCHRADER LE, DA CATALDO, DM PETERSON 1974 Use of protein in extraction and stabilization of nitrate reductase. Plant Physiol 53: 688-690 JC. LE SCHRADER, GE EDWARDS 1979 Glycolate synthesis in C 4 C and interme28. diate photosynthetic plant types. Plant Cell Physiol 19(8). In press 29. StNCLAtR AG 1973 Nondestructive acetylene reduction assay of nitrogen fixation applied to white clover plants growing in soil. NZ J Agric Res 16: 263-270 30. MM, ME MCCULLY 1977 Mild temperature "stress" and callose synthesis. Planta 136: 65-70 31. TRtNtCK MJ, MJ DtLWORTH, M GROUNDS 1976 Factors affecting the reduction of acetylene by root nodules of Lupinus species. New Phytol 77: 359-370 32. WAUGHMAN GJ 1977 The effect of temperature on nitrogenase activity. J Exp Bot 28: 949-960
RAtSON
SERVAtTES SMtTH
