Diurnal variations in acetylene reduction is shown in nitro gen-fixing faba bean.
The highest values for ARA occurred during the light period, while the lowest values
were recorded during the dark periodo However, the glutamate synthase cycle enzymes
(GS and NADH-GOGAT) and NADH-GDH show rhythmic behaviour on a cycle of 16
h, with maximum and minimum activities both being observed during the photoperiod
as well the dark periodoLa reducción de acetileno en plantas de haba fijadoras de nitrógeno, mostró
diferencias entre el día y la noche. Los mayores valores de ARA se detectaron durante
el periodo de luz, mientras que en periodo de oscuridad los valores fueron inferiores.
Por otra parte, las enzimas del ciclo de la glutamato sintasa (GS y NADH-GOGAT) y
la NADH-GDH mostraron un comportamiento cíclico de 16 horas, observándose actividades
máximas y mínimas tanto en el periodo de luz como en el de oscuridad

CORDOVILLA, M.P.

LIGERO, F.

LLUCH, C.

OCAÑA, A

Spanish

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Diurnal variation in nodule nitrogen metabolism in faba bean (Vicia faba L.) Variaciones del metabolismo del nitrógeno en nódulos radicales de habas (Vicia faba L.) a lo largo del fotoperiodo. CORDOVILLA, M. P., OCAÑA, A., LIGERO, F. and LLUCH, C. Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Granada, E-1807 l Granada, Spain ABSTRACT Diurnal variations in acetylene reduction is shown in nitro gen-fixing faba bean. The highest values for ARA occurred during the light period, while the lowest values were recorded during the dark periodo However, the glutamate synthase cycle enzymes (GS and NADH-GOGAT) and NADH-GDH show rhythmic behaviour on a cycle of 16 h, with maximum and minimum activities both being observed during the photoperiod as well the dark periodo Key words: arnmonium assimilation, GS, NADH-GOGAT, nitrogen fixation, Vicia faba. RESUMEN La reducción de acetileno en plantas de haba fijadoras de nitrógeno, mostró diferencias entre el día y la noche. Los mayores valores de ARA se detectaron durante el periodo de luz, mientras que en periodo de oscuridad los valores fueron inferiores. Por otra parte, las enzimas del ciclo de la glutamato sintasa (GS y NADH-GOGAT) y la NADH-GDH mostraron un comportamiento cíclico de 16 horas, observándose acti­vidades máximas y mínimas tanto en el periodo de luz como en el de oscuridad. Palabras clave: asimilación de amonio, fijación de nitrógeno, GS, NADH-GOGAT, Vicia faba. AbbreviatioDs: ARA, acetylene reduction activity; y-GH, y-glutarnyl-hydroxamate; GS, glutamine synthetase; NADH-GOGAT, NADH-dependent glutamate synthase; NADH-GDH, NADH­dependent glutamate dehydrogenase. Recibido: 18-10-1997 Aceptado: 9-12-1997 BIDLID [0004-2927(1997) 38:4; 365-373] Ars Pharmaceutica, 38:4; 365-373, 1997 366 CORDOVILLA. M. P .• OCAÑA. A .• LIGERO, F. and LLUCH, C. Introduction Biological N2 fixation is the major means by which organic N is added to soils. The legume-Rhizobium :Bradyrhizobium symbiosis is the most signifiant source of N for agriculture. In addition, legumes yield nutritious fodder, protein-rich seeds and fruits. Faba bean (Vicia faba L.) is widely used as a grain legume crop in south Australia and Spain, China and Egypt (1,2) but there are few reports of N2 fixation by this species (3, 4). Sorne herbaceous symbioses show pronounced diurnal fluctuations in nitrogenase activity detected as acetylene reduction acitivity (ARA) (S, 6, 7). In field studies of tropical nitrogen-fixing tree symbioses, diurnal patterns in ARA have been indicated (8, 9, 10). In sorne studies a midday peak in ARA was observed (8, 9). The ARA assay has been widely used as a simple and rapid method of estimating rates of N2 fixation by legumes grown in both greenhouse and field experiments (11, 12). According to Herdina and Silsbury (3), and Minchin el al. (12) an ARA assay in a closed system was satisfactory for measuring the N2 fixation of faba bean plants in the vegetative growth stage. During nodulation, both glutamine synthetase (GS) and NADH-dependenl glutamate synthase (NADH-GOGAT) have been shown to increase its activitieE in parallel with nitrogenase and other enzymes in the nitro gen assirnilatoI) pathway (13, 14). In vitro enzymatic studies indicate that GS and GOGAT rathel than glutamate dehydrogenase (GDH) are involved in the assimilation of NH to produce glutamate and glutamine. The resulting glutamate and glutamim are utilized to produce a variety of amino acids. Ohyama and Kumazawa (IS: and Fujihara and Yamaguchi (16), using the 15N tracer technique, reported tha GS/GOGA T played a predominant role in the assimilation of N in soybean This study shows data on diurnal variation in ARA, ammonium assimilatin! enzyme activities and soluble protein content in nitrogen-fixing symbioses o faba bean plants in the vegetative phase. Materials and methods Plant material and growth conditions Commercial cultivar Alborea of Vicia faba L. var. minor was bought fron Semillas Pacífico S.A. , Sevilla, Spain. Seeds were surface-sterilized in 96� (v/v) ethanol for 3 min, then washed with sterile water and germinated a 26°C in moist autoclaved vermiculite. Seedlings, selected for uniformit) were planted (2 per jar) in sterile 1-liter Leonard jars with vermiculite and nutrient solution (17) containing 2 mM KN03, which stimulated plant growt Ars Pharmaceutica, 38:4; 365-373, 1997 DIURNAL VARIATION IN NODULE NITROGEN METABOLlSM IN FABA BEAN (VICIA FABA L.) 367 but did not inhibit nodule growth and activity (18). Each seedling was given 1 mi of a suspension of R. leguminosarum biovar viciae strain GRA19 (19) from a 3-day-old Allen 79 solid medium culture (ca 109 cells mi"). The seedlings were then covered with moist vermiculite and a layer of dry, sterile perlite. The jars were arranged randomly in a growth chamber with a 16-8 h light-dark cycle, 23-16°C day-night temperature, relative humidity 55-75% and photosynthetic photon flux density (400-700 nm) of 450 imol m·2 S" supplied by Sylvania Cool-White Lifeline fluorescent lamps (F96T12-CW­VHO, Sylvania Ltd, Quebec, Canada) and incandescent lamps (30% fluorescent wattage). The plants were harvested 23 days after transplanting. Nitrogen fixation assays Nitrogenase (EC l.7.99.2) activity was determined by acetylene reduction on the entire root systems of six plants, as well as on small nodulated portions of root from remaining plants, according to the recommendations of Herdina and Silsbury (3). Nodulated root portions (1 g root plus nodules) and root systems were placed in vials and sealed with serum caps. Ten percent of the internal atmosphere was replaced with acetylene, and 0.2 mi aliquots were taken after 5, 10 and 15 min of incubation and analyzed for ethylene in a Perkin Elmer 8600 gas chromatograph equipped with a Porapak R colurnn (20). Preparation of cel/-free extracts Nodule homogenates for determining GS, NADH-GOGAT, NADH-GDH and protein were prepared according to a slightly modified version of the technique of Groat and Vanee (21). Samples (1 g fresh weight) were homogenized on ice with acid-washed quartz sand and 12 mi (ice-cold) of an extraction medium containing 100 mM maleic acid-KOH, pH 6.8, 100 mM sucrose, 2% (vol/vol) 2-mercaptoethanol, and 15% (vol/vol) ethylene glycol, plus 0.5 g polyvinyl polypyrrolidone. The homogenate was filtered through 4 layers of cheesec10th and the nodule debris removed; the filtrate was centrifuged at 3500 g at 2°C for 8 mino The resulting supernatant was centrifuged once more at 30000 g for 20 min, which produced a c1ear solution of host cell cytoplasm and its organelles; this solution was used for enzyme assay. Enzyme assays Glutamine synthetase (EC 6.3. l . 2) was determined by the hydroxamate synthetase assay, adapted from Farnden and Robertson (22) and Kaiser and Ars Pharmaceutica, 38:4; 365-373, 1997 368 CORDOVILLA, M. P., OCAÑA, A., LIGERO, F. and LLUCH, C. Lewis (23). Assays were optimized for the amount of enzyme to give a linear reaction within at least 30 min. Two controls, one without enzyme and one without L-glutamate, were also analyzed. NADH-glutamate synthase (EC 1.4.1.14) and NADH-glutamate dehydrogenase (EC 1.4.1.2) activities were assayed spectrophotometrically at 30°C by monitoring the oxidation of NADH at 340 nm, essentially as indicated by Groat and Vance (21) and Singh and Srivastava (24), always within 2 h of extraction. Two controls (without a-ketoglutarate and without glutamine in the case of GOGAT, without NH and without a-ketoglutarate in the case of GDH) were used to correct for endogenous NADH oxidation. The de crease in absorbance (linear for at least 10 min) was recorded for 8 min in a Beckman DU-70 spectrophotometer. Protein determination Soluble proteins in tissue extracts were determined by the Bradford method (25), with bovine serum albumin (Merck, fraction V) as the standard. Statistical design and analysis The experimental layout was a randomized block design replicated 6 times. The results were subjected to an one-way analysis of variance with the least significant difference (LSD) test between means. Results and discussion Diurnal variations in ARA were found in V. faba (Fig. 1), as was al so found for tropical and subtropical nitrogen-fixing tree symbioses (26). In V. faba, the highest values for ARA (on average, 105 imol C2H4 h-' g DW-') were recorded during the light period (between 4 and 7 h), while the lowest values were recorded during the dark periodo The maximum ARA value during exposure to light was 1.6 times higher than the minimum detected during periods of darkness. In sorne field studies of tropical tree species a midday peak in ARA was observed (8, 9). This may be due to stomatal closure following excessive water loss under high light intensities, as was discussed by Roskoski and van Kessel ( lO). Midday peaks could not be observed in this study. Acetylene reduction acitivity showed maximum value for several hours during the light period, which had the same light intensity during the day, as was also found for Casuarina obesa ARA (26). In Acacia albida, Leucaena Ars Pharmaceutica, 38:4; 365-373, 1997 DIURNAL VARIATION IN NODULE NITROGEN METABOLISM IN FABA BEAN (VICIA FABA L.) 369 105 LSD (0.05) 95 .. . -� e Q 85 -.. . � .. :I: 75 N o 'O E 65 ::L light darkness Iight darkness o 5 10 15 20 25 30 35 40 45 50 Hours Fig. l .-Diurnal variation on nodule ARA (imol C2H4 h·1 g.1 nodule) for V. faba plants inoculated with R. leguminosarum GRA 19. leucocephala, Prosopis chilensis and Casuarina glauca ARA increased throughout the light period (26), and in soybean the ARA remained constant (27). The enzyme activity involved in the glutamate synthase cycle is 2: 1, that is, GS activity is double that of GOGAT, to ensure assimilation of aH the ammonia that the nodules are able to produce (28). However, recent results obtained with other genotypes and other strains of R. leguminosarum (4) indicate that this relationship may depend on the genotype of the plant and the strain of Rhizobium. The GS/GOGAT ratio found in the present study indicates that GS activity does not limit NH3 assimilation. The arnmonium assimilation enzymes showed a rhythmic behaviour on a 14 h cycle, maximum and minimum activities both occurring during the light as weH as the dark period (Figs. 2, 3). The maximum activity values of GS, NADH-GOGAT and NADH-GDH were 1.3 times higher than the minimums. In accord with these results, Femández-Conde el al. (29), studying sunflower leaves, describe the rhythmic behaviour for Fd-GOGAT not controHed by light/darkness conditions but atlributable to a biological clock. However, these authors (29) described an evolution of the GS completely dependent upon light. Other authors have reported differences in enzymatic behaviour Ars Pharmaceutica, 38:4; 365-373, 1997 370 CORDOVILLA. M. P .. OCAÑA, A., LIGERO. F. and LLUCH. C. 300 LSD (0.05) -. i 280 u. a ---. � J: 260 (!) (5 E :::1. 240 light darkness light darkness O 5 10 15 20 25 30 35 40 45 50 Hours 160 LSD (0.05) B -. � 1 50 u. CI --o;- 140 � ,,¡ o J: e 130 « z o E :::1. 120 light darkness light darkness O 5 10 15 20 25 30 35 40 45 50 Hours Fig. 2.-Diurnal variation on GS (A) (imol g-GH h" g FW") and GOGAT (B) (imol NADHo, 11" g FW") in nodules of V. Jaba plants inoculated by R. leguminosarwn GRAI9. Ars Pharmaceutica, 38:4; 365-373,1997 DIURNAL VARIATION IN NODULE NITROGEN METABOLlSM IN FABA BEAN (VICIA FABA L.) 371 38 I LSD (0.05) A -I i � 33 Q -":' � ,¿ o 28 x e < z o E 23 � light darkness light darkness O 5 10 15 20 25 30 35 40 45 50 Hours 20 8 I LSD (0.05) 1 5  ":' --3: � a 1 0  --a E 5 light darkness light darkness O O 5 10 15 20 25 30 35 40 45 50 Hours Fig. 3.-Diurnal variatioo 00 GDH (A) (imol NADHox h·1 g FW·I) aod soluble proteio cooceotratioo (B) (rng g FW·I) io oodules of V. faba plaots iooculated by R. leguminosarum GRAI9. Ars Pharmaceutica, 38:4; 365-373, 1997 372 CORDOVILLA, M. P., OCAÑA, A., LIGERO, F. and LLUCH, C. for different plant organs (30). Legume root nodules are morphologically and biochemically unique organs which have enzymes (nodulines) not expressed in other parts of the plants; therefore GS conceivably behaves differently in different plant organs. The soluble protein content in the nodule did not show cyclic behaviour, having similar values for light and darkness. Conclusion Acetylene reduction acitivity increased over the light period and decreased in darkness, while the ammonia assimilation enzymes in the nodules showed a 16 h cyclic behaviour independent of the light/darkness conditions. The soluble pro te in content remained constant, regardless of light and darkness. Acknowledgements Financial support was obtained by grant No. PB95-1159from the DGICYT, and by grant No ERBICI8-CT97-0081 CE-INCOo References ( 1) BUTTERY, B. R., PARK., S. J., HUME, D. J.: "Potential for increasing nitrogen fixation in grain legumes". Can. J. Plant Sci. ( 1992), 72:323-349. (2) HILL-COTTINGHAM, D. G., LLOYD-JONES, C. D.: "The influence of nitrate supply on nitrogen fixation during growth of field bean, Vicia faba, in sand". Physiol. Plant. ( 1980), 48:1 16-120. (3) HERDINA, J. A., SILSBURY, J. H.: "Estimating nitro gen ase activity of faba bean (Viciafaba) by acetilene reduction (AR) assay. Aust. J. Plant Physiol. ( 1990), 17: 489-502. (4) CABA, J. M., LLUCH, C., LIGERO, F.: "Genotypic differences in nitrogen assimilation in Vicia faba: Effect of nitrate". Plant Soil ( 1993) 151: 167-174. (5) A YANABANA, A., LA WSON, T. L.: "Diurnal changes in acetylene reduction in field -grown cowpeas and soybeans". Soil Bio/. Biochem. ( 1977), 9: 125- 129. (6) MINCHIN, F. R., PATE, J. S.: "Diurnal functioning of the legume root nodule". J. Exp. Bot. ( 1974), 25:295-308. (7) RAINBIRD, R. M., ATKINS, C. A., PATE, J. S.: "Diurnal variations in the functioning of cowpea nodules". Plant Physiol. ( 1983), 72, 308-3 12. (8) LANGKAMP, P. J., SWINDE, L. B., DALLING, M. J.: "Nitro gen fixation (acetylene reduction) by Acacia pellita on areas resto red after mining at Groote Eylant, Northern Territory. Aust. J. Bot. ( 1979) 30:57-106. (9) HÓGBERG, P., KV ARNSTRÓM, M.: "Nitro gen fixation by the woody legume Leucaena eucocephala in Tanzania". Plant Soil ( 1982), 66: 2 1-28. ( lO) ROSKOSKI, J. P., VAN KESSEL, C.: "Annual, seasonal and diel variation in nitrogen fixation activity by Ingajinicuil, a tropical leguminous tree". Oikos (1985), 44:306-3 12. Ars Pharmaceutica, 38:4; 365-373, 1997 

Variaciones del metabolismo del nitrógeno en nódulos radicales de habas (Vicia faba L.) a lo largo del fotoperiodo.

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Variaciones del metabolismo del nitrógeno en nódulos radicales de habas (Vicia faba L.) a lo largo del fotoperiodo.

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