Fertilizer-N scheduling based on ontogenetic characteristics of nitrate reductase in rice genotypes

R.K. Dutta and M. Badruddin

Crop Physiology Division, Bangladesh Institute of Nuclear Agriculture, PO Box 4; Mymensingh, Bangladesh

INTRODUCTION

Rice crops are totally dependent on soil or fertilizer-N for their nitrogen nutrition. In soils, nitrogen may be present as organic or inorganic nitrogen in the form of nitrate or ammonium. In acidic soils nitrate may be present in ionic form and may be leached (Mohanty and Patnaik, 1975) but ammonium may be fixed (Ponnamperuma, 1972). In alkaline soil with a high pH, nitrate may be present in free form whereas ammonium may be volatilized (Mohanty, Dash and Patnaik, 1978; Patrick and Mahapatra, 1968). Fertilizer-N in the form of urea rapidly converts to ammonium (Collings, 1955) and thereafter may be converted to nitrate after nitrification (Patrick and Mahapatra, 1968). Although rice has a preference for ammonium uptake, it can also take up nitrate. In acidic soil, the most abundant form of nitrogen is ammonium, whereas in alkaline soil, nitrate is most abundant (Sahrawat, 1980; Ponnamperuma, 1972). Thus, most upland crops take up nitrate whereas rice under submerged conditions takes up ammonium as its principal source (Ponnamperuma, 1965; 1975). However, autumn rice cultivation in Bangladesh is rainfed, sometimes under submerged and sometimes under dry conditions, depending on the rainfall. Plants utilize the nitrate reduction system for nitrate assimilation or, whenever ammonium is taken up, it is reduced to glutamine by glutamine synthetase (Lee, Blackwell and Jony, 1992). It is widely known that the main nitrogen source for rice is ammonium. However, the importance or effectiveness of nitrate reduction has not been comprehensively evaluated in rice, unlike in wheat and barley. It has been demonstrated that rice genotypes have a wide variability and considerable potential for nitrate reduction, and the relationship of nitrate reduction capacity with biomass productivity and grain yield has not been evaluated. This study was undertaken to investigate the ontogenetic potential of nitrate reduction in some élite varieties/mutants of rice, while determining the relationship of this potential with rice grain yield and evaluating the nitrate reduction pathway in rice.

Two experiments were conducted, one under field conditions and the other in pot culture in sand. The field experiment was used 11 rice genotypes/mutants with a high nitrate reductase activity, as measured in the earlier experiment (Dutta, Badruddin and Lahiri, 1996). The genotypes used were Binasail, Nizersail, BR11, Nizersail mutants, viz. NS3, NS11, NS12, NS14, NS15, NS18, and BR3 somaclones, viz. P7 and P9. The experiment was conducted in split plots, with fertilizer-N rates observed in the main plots and varieties/mutants in subplots in three replicates. The rice seedlings were planted in 3-m rows and spacing was 20 x 15 cm. Thirty-day-old seedlings were transplanted. Phosphorus and potash were applied at 80 kg P₂O₅ and 60 kg K₂O ha^-1 during land preparation. Nitrogen was applied once 28 days after transplanting at 10 and 100 kg N ha^-1 in the form of potassium nitrate. Appropriate cultural practices, including irrigation and weeding, were carried out when necessary. Nitrate reductase activity was determined 48 hours after the nitrogen application and then every four days up to 64 days. The ontogenetic stages were determined as active tillering (30 days after tillering - DAT), panicle initiation (50 DAT) and the booting stage (65 DAT). Data on yield and yield components were determined from ten hills at maturity.

A pot experiment was conducted in sand culture with five rice varieties/mutants, viz. Binasail, Nizersail, BR11, NS14 and NS18. A complete nutrient solution (-N) was prepared, containing 4 mM KNO₃; 4 mM Ca(NO₃); 1.5 mM MgSO₄; 7H₂O; 1.3 mM NaHPO₄; 0.1 mM NaCl; 0.007 mM NH₄MO₇O₂5H₂O and 0.05 mM H₃BO₃. Half-strength working solution was taken from the stock solution and applied twice a week up to the age of 30 days while nitrogen treatments were applied as 5 mM NO₃, 2.5 mM NO₃ + 2.5 mM NH₄⁺. The experiment was completely random and comprised four replicates. Nitrate reductase activity was measured 48 hours after the application of the nitrogen.

Nitrate reductase assay. The nitrate reductase assay was done following the method of Orebamjo and Stewart (1979). The assay solution contained 0.1 mM phosphate buffer, pH 7.5; 1.5% (w/w) KNO₃; 1.5 % (v/v) N-Propanol and 0.01% Neutronix. The colour reagent contained 1% sulphanylic acid and 0.02% NEED. A 50 mg sectioned leaf was placed in 5 ml of assay solution in scintillation vials. The leaf sections were vacuum-infiltrated for five minutes until the leaf section settled on the bottom. The vials with leaf sections were incubated in darkness in a water bath at 25°C for one hour; 1.0 ml of assay mixture was extracted to mix with 2 ml of colour reagents in the ratio of 1:1; the colour was measured at 540 nm and nitrate reductase was expressed as m mol NO2 g^-1 fresh weight hour^-1.

FIGURE: Ontogenetic induction of nitrate reductase activity in rice

RESULTS AND CONCLUSIONS

Results presented in the Figure show three major peaks of activity of nitrate reductase in all the genotypes, mainly at active tillering (30 DAT), panicle initiation (50 DAT) and the booting stage (64 DAT). However, there were genotypic differences in the characteristics of their peaks at different stages. Binasail, Nizersail, BR11, NS3, NS14, NS15, P7 and P9 showed well-defined characteristics of three peaks at different stages. NS11, NS12, NS18 showed sharp peaks of nitrate reductase at the active tillering stage whereas, at other phases, nitrate reductase peaks were not prominent. The different peaks of nitrate reductase resulting from high and low nitrogen treatments were mostly prominent at the active tillering stage, especially in those varieties/mutants with less nitrate reductase activity in later stages. The rice growth curve may demonstrate the significance of such activity peaks at different stages. Rice shows active leaf area and biomass development up to the flowering stage (Baset et al., 1994).

Table 1 shows the significant difference of grain yield and yield components among varieties/mutants and owing to high and low nitrogen treatments. The ontogenetic average of nitrate reductase showed higher activity with a high level of nitrogen and a positive correlation with effective tillers/hill-¹, filled grain/panicle-¹, TDM/hill-¹, grain yield/hill-¹ and harvest index, but it showed a negative correlation with 1 000 grain weight and a weak correlation with panicle length.

The flag leaf nitrate reductase, measured after unfolding of the leaf, showed a higher level of activity owing to its low level of nitrogen. The ontogenetic average value of nitrate reductase was highest in Nizersail. Many authors obtained a significant positive correlation of yield and yield components in many crops, for example dry bean (Neyra, Sales and Pollack, 1980); wheat (Abrol et al., 1978; Nair and Abrol, 1982), barley and wheat (Sairam and Singh, 1989; Stepanova, 1989) and wheat (Harper and Paulsen, 1967). In this experiment, potassium nitrate was used as the sole source of nitrogen, although some nitrogen may have been taken up in the form of ammonium (NH₄⁺). Thus, the correlation with yield and yield components with nitrate reductase was obtained.

Results of the pot experiment presented in Table 3 show that nitrate reductase activity was highest with 5 mM nitrate treatment and lower with ammonium treatment, especially in the case of Nizersail. The presence of 2.5 mM NH₄⁺ + 2.5 mM NO₃ produced a medium level of activity. The results indicated that nitrate reductase is not completely repressed in the presence of NH₄⁺. Ammonium has been conclusively determined to be a negative effector of nitrate reductase in Lemna minor (Stewart and Rhodes, 1976). However, in many crops the presence of NH₄⁺ in addition to nitrate may regulate the activity of nitrate reductase in a different way. In some crops, nitrate reductase could be induced in the presence of NH₄⁺ (Dutta et al., 1994). In rice, nitrate reductase may be regulated metabolically in the presence of nitrate or ammonium.

The results suggested that fertilizer nitrogen application in three splits at the active tillering, panicle initiation and booting stages may be the appropriate method for increasing rice productivity by minimizing leaching and volatilization loss of nitrogen. Rao and Murty (1975) obtained maximal rice productivity with three splits of nitrogen application over two splits. In some varieties with a high level of nitrate reductase, 10 kg N/ha did not produce much difference in nitrate reductase activity compared with 100 kg N/ha, except at the active tillering stage (see Figure). Thus, in rice lines with high nitrate reductase, a minimum level of nitrogen may be sufficient for maximum rice productivity. Khamis and Lamage (1990) obtained maximal productivity of maize with a minimum nitrate concentration in the leaf tissue. The flag leaf showed a higher value of nitrate reductase at 10 kg N/ha. This might be because of the rapid decay of nitrate reductase activity with 100 kg N after reaching its peak. The experimental results also suggested that nitrate reductase may not be completely repressed by NH₄⁺. Thus, its high positive of correlation with grain yield and yield components suggests that NO₃^- assimilation may be an alternative or complementary route of nitrogen metabolism in rice crops.

REFERENCES

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Harper, J.E. & Paulsen, G.M. 1967. Changes in reduction and assimilation in nitrogen during the growth cycle of winter wheat. Crop. Sci., 7: 205-209.

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Lee, P.J., Blackwell, R.D. & Jony, K.W. 1992. Ammonia assimilation in higher plants. In K. Mengel & D.J. Pilbeam, eds. Nitrogen metabolism in plants, p. 153-186. Oxford, UK, Clarendon Press.

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Neyra, C.A., Sales, B.A. & Pollack, B.L. 1980. Characterization of nitrate reductase in field grown beans (Phaseolus vulgaris L.). Plant Physiol., S-65: 54.

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Patrick, W.H. Jr & Mahapatra, A.K. 1968. Transformation and availability of nitrogen and phosphorus in water-logged soils. Adv. Agron., 20: 323-359.

Ponnamperuma, F.N. 1965. Dynamic aspects of flooded soils. In Proceedings of the Symposium on the Mineral Nutrition of the Rice Plant, p. 295-328. February 1964, IRRI, Los Baños, the Philippines. Baltimore, MD, USA, The Johns Hopkins University Press.

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Calendrier des engrais azotés sur la base des caractéristiques ontogénétiques
de la nitrate réductase dans les génotypes du riz

Les expériences menées sur 11 génotypes d'élite dans les conditions du terrain avec deux taux de fertilisation azotés (10 kg et 100 kg/ha^-1) épandus 28 jours après le repiquage ont fait apparaître trois grands sommets dans l'activité de la nitrate réductase dans tous les génotypes, principalement aux stades du tallage (30 DAT), de l'initiation florale (50 DAT) et de la montaison (64 DAT). Toutefois, on constate des différences génotypiques dans les caractéristiques des points culminants atteints. Binasail, Nizersail, BR11, NS3, NS14, NS15, P7 et P9 ont atteint trois points culminants distincts à différentes étapes. Mais NS11, NS12 et NS13 montrent que les pics de nitrate réductase dus à des traitements forts en azote (100 kg/ha^-1) ou faibles en azote (10 kg/ha^-1) sont surtout marqués aux stades du tallage actif, plus particulièrement dans les lignées qui ont moins d'activité de nitrate réductase aux stades ultérieurs. L'activité de NR moyenne ontogénétique fait apparaître une corrélation positive et significative avec la talle fertile^-1, la panicule à grain rempli^-1, TDM^-1, le rendement du grain^-1, et l'indice de récolte tandis qu'on constate une corrélation négative avec le poids de 1 000 grains. Ces résultats indiquent que les plants de riz sont capables d'utiliser le chemin de l'assimilation de l'azote de façon efficace et le calendrier de fertilisation à l'azote des différents cultivars peut être conçu en fonction des caractéristiques ontogénétiques de la nitrate réductase. Il est apparu également que la présence de NH₄⁺ ne réprime peut-être pas complètement l'activité de la nitrate réductase.

Fertilizantes: programación del nitrógeno sobre la base de las características
ontogenéticas de la nitrato reductasa en genotipos de arroz

En experimentos realizados sobre el terreno con genotipos selectos de arroz a los que se aplicaron dos tasas distintas de fertilizantes nitrogenados (10 kg/ha y 100 kg/ha) 28 días después del trasplante se observaron sistemáticamente tres crestas principales en la actividad de la nitrato reductasa en todos los genotipos, principalmente en las etapas de formación de hijuelos fértiles (30 días después del trasplante), formación de la panícula (50 días después del transplante) e hinchamiento (64 días después del trasplante). Sin embargo, hubo diferencias genotípicas en las características de las crestas. Las variedades Binasail, Nizersail, BR 11, NS3, NS14, NS15, P7 y P9 alcanzaron tres crestas distintas en diferentes etapas. Pero NS11, NS12 y NS13 mostraron crestas acusadas de nitrato reductasa como consecuencia de tratamientos con dosis de nitrógeno altas (100 kg/ha) o bajas (10 kg/ha), especialmente destacadas en la etapa de formación de hijuelos fértiles y en las líneas que tenían menos actividad de nitrato-reductasa en etapas posteriores. La actividad ontogenética media de nitrato reductasa mostró una correlación positiva y significativa de hijuelos fértiles/macolla, grano relleno/panícula, MST/macolla, rendimiento en grano y grano/paja, y una correlación negativa con el peso de 1 000 granos. Los resultados indicaron que las plantas de arroz tienen la capacidad de utilizar efectivamente la vía de la asimilación de nitrógeno, por lo que podrían formularse calendarios de aplicación de fertilizantes nitrogenados a diferentes cultivares de acuerdo con las características ontogenéticas de la nitrato-reductasa. También se puso de manifiesto que es posible que la presencia de NH₄⁺ no inhiba por completo la actividad de la nitrato reductasa.