Diurnal Variation in the Functioning of Cowpea Nodules ' Received

Nitrogenase (EC 1.7.99.2) activity of nodules of cowpea ( Vigna aguiculata IL.1 Walp), maintained under conditions of a 12-hour day at 30°C and 800 to 1,000 microelastelns per square meter per second (photosyntheticaHly active radiation) and a 12-hour night at 20°C, showed a marked diurnal variation with the total electron flux through the enzyme at night being 60% of that in the photoperiod. This diurnal pattern was, however, due to changes in hydrogen evolution. The rate of nitrogen fixation, measured by short-term 15N2 assimilation or estimated from the difference in hydrogen evolution in air or Ar02 (8&0, v/v), showed no diurnal variation. Carbon dioxide released from nodules showed a diurnal variation synchronized with that of nitrogenase functioning and, as a consequence, the apparent 'respiratory cost' of nitrogen fixation in the photoperiod was almost double that at night (9.74 ± 0.38 versus 5.70 ± 0.90 moles CO2 evolved per mole N2 fixed). Separate carbon and nitrogen balances constructed for nodules during the photoperilod and dark period showed that, at night, nodule functioning required up to 40% less carbohydrate to achieve the same level of nitrogen fixation as during the photoperiod (2.4 versus 1A moles hexose per mole N2 fixed). Stored reserves of nonstructural carbohydrate of the nodule only partly satisfied the requirement for carbon at night, and fixation was depndent on continued import of translocated assimilates at all times. Measurements of the soluble nitrogen pools of the nodule together with 1"N studies indicated that, both during the day and night, nitrogenous products of fixation were effectively translocated to all organs of the host plant despite low rates of transpiration at night. Reduced fluxes of water through the plant at night were apparently counteracted by increased concentration of nitrogen, especially as ureides, in the xylem stream. In addition to showing long-term changes in the economy of nodule functioning throughout development (7, 15), many legume symbioses exhibit pronounced diurnal fluctuations in nitrogenase activity as assayed by the reduction of acetylene (2, 6, 11, 16, 19). These fluctuations have been interpreted in relation to changes in the respiration of the nodulated root (7, 11), the carbohydrate status of nodules (5, 11, 19), and the accumulation and export of nitrogenous solutes by the nodules (11). Conversely, where significant diurnal variations in acetylene reduction were not observed, the symbioses studied were regarded not to be currently limited by availability of carbohydrate from the host plant (6, 21). Although acetylene reduction assays may well offer a reliable measure of overall nitrogenase activity, and thus reflect the availability and rate of utilization of oxidizable substrate in the nodule, ' Supported by funds from a University of Western Australia General Development Grant and from the Wheat Industry Research Council of Australia. ' Present address: Central Research and Development Department, Experimental Station, Dupont, Wilmington, DE 19898. they fail to provide information on how electron flow through the enzyme complex in vivo is partitioned between nitrogen and proton reduction (17). Marked diurnal fluctuation in this partitioning could well be a significant factor in the daily economy of the nodule, particularly were times of low availability of carbohydrate to coincide with periods of more intense nitrogen fixation relative to H2 evolution. This paper examines such a possibility in a symbiosis (Vigna unguiculata:Rhizobium strain 176A27) lacking an uptake hydrogenase. The experimental procedure used involved measurements of diurnal fluctuation in H2 evolution in air or Ar/02 and of '5N2 fixation in air to estimate separate rates of nitrogen and proton reduction, and diurnal variations in nitrogenase function were then related to overall daily operation of the plant's nodules under a regime of fluctuating temperature and illumination. MATERIALS AND METHODS Plant Material. Surface-sterilized seed of cowpea ( Vigna unguiculata [L.] Walp. cv Caloona) was inoculated with Rhizobium strain 176A27 (Nitragin Co.). Ten d after sowing in sand, groups of five seedlings were transplanted to 3.5-L containers of N-free liquid culture (10) maintained in a naturally lit glasshouse. Fifteen d after establishment, the liquid cultures were transferred to a controlled environment cabinet with a 12-h day at 30'C and 800 to 1,000 liE/m2.s (PAR) and a 12-h night at 20°C. After 5 to 10 d growth in the cabinet, the lids of the culture containers were sealed at the edge and around the stem of each of the five plants with Terostat VII (Teroson Gmbh, Heidelberg, F. R. G.), and a moisturized stream of either C02-free air or C02-free Ar:02 (80%:20%, v/v) was passed through the enclosed gas space above the liquid culture at a flow rate of 50 to 100 cm3/min. Plants were in mid vegetative growth and were fixing N at near maximum rates (9) when used. Measurement of Hydrogen Evolution and Estimation of Nitrogen Fixation. Three sealed water cultures (total of 15 plants) gassed with C02-free air and three similar cultures gassed with C02-free Ar:O were connected to an automated analysis system which sequentially sampled the effluent gas stream from each over a 6-min period. H2 concentration of the gas stream was measured using a gas liquid chromatograph equipped with a 2-m column of molecular sieve 5A (100-120 mesh; Waters Associates) and a thermal conductivity detector. The chromatograph incorporated a motorized gas sampling valve which was controlled to sample 1.4 cm3 at times coinciding with the sequential switching of the effluent gas streams from the six cultures. Measurement of H2 evolution from each of the six cultures was thus made every 36 min for a period of 33 to 69 h. Values were integrated on a 3-h basis. In the absence of hydrogenase reactions which utilize H2, the rate of H2 evolution into Ar:02 may be regarded as a measure of the total flow of electrons to nitrogenase functioning (17). In the present study, H2 evolution in air and in Ar:02 was therefore used to estimate the rate of N2 fixation for each 3-h period of the day and night. In air, the decrease in H2 evolution was presumed to be 308 www.plantphysiol.org on October 15, 2017 Published by Downloaded from Copyright © 1983 American Society of Plant Biologists. All rights reserved. DIURNAL FUNCTIONING OF NODULES due to electron flow to N2 reduction, and, assuming that reduction of N2 required three electron pairs compared to one for H2 production, the following relationship was used to estimate the rate of N2 fixation. N2fixation = H2 evolution in Ar:02H2 evolution in air 3 (1) Measurement of CO2 Evolution. The nodulated zone on the primary root of intact plants cultured as above was enclosed in a small (volume 10 cm3) plastic cuvette as detailed previously (10), and the CO2 evolved into a C02-free air or C02-free Ar:02 (80%:20%o, v/v) stream was measured using an IR gas analyzer. The effluent stream from six such cuvettes was sampled sequentially as described above for H2 assay. The total CO2 evolved into the gas stream flowing through the cuvettes was due to respiration of both nodules and the segment of supporting root which was also enclosed. The separate respiratory contribution of the nodules in each time interval of the study was estimated by subtracting from the total CO2 efflux the respiration rate of the root segment measured immediately following detachment of the nodules. As described previously (10), the small cuvettes enclosed all the nodules on the root system of a plant which was nodulated only on the primary root. Plant Harvest and Analysis. Plants cultivated and maintained under the same conditions as those used for measurement of gas exchange were harvested at 4to 5-h intervals during the study period for assay of ureides (8), soluble amino nitrogen (23), and nonstructural carbohydrate (3) in nodules. Dry weight and total N, measured by Kjeldahl digestion, were determined for nodules and for whole plants at the beginning and end (after 33 or 69 h) of periods of measurement of gas exchange. Transpiration. The rate of water loss from liquid cultures identical to those used for measurement ofgas exchange was estimated gravimetrically or as the change in volume of the culture solution at varying periods (3-6 h) throughout the photoperiod and dark period of diurnal studies. Xylem Sap Collection. Five to 10 plants were decapitated at 3h intervals throughout the study period, and root bleeding (xylem) sap was collected for assay of ureide (8) and total amino acid content (1). Measurement of 1"N2 Fixation. Cuvettes were attached to the nodulated root regions of two plants as described above and connected in series to a closed gas exchange system incorporating the two cuvettes, a pump, and a small soda lime CO2 absorber followed by a water bubbler. The gas within the system (260 cm3) was cycled at a flow of 80 cm3/min and, following a period of equilibration, 50 cm3 was removed and, simultaneously, 50 cm3 '5N2 (95 atom % excess 15N) was added. After 2 h, plants were separated into component organs and dried. Samples of the dry, finely milled material were taken for '5N assay by MS following Kjeldahl digestion and oxidation of the resultant ammonia by the Rittenberg procedure (4).