Matsumoto, Hideaki

Ohno, Seishi

Yamaya, Tomoyuki

Okayama University Scientific Achievement Repository

Ber. Ohara 1nst. landw. Biol.， Okayama Univ. 18: 115-123 (1983) CHANGES IN SOME ENZYME ACTIVITIES OF RICE AND CHINESE CABBAGE DUE TO NITROGEN ST ATUS Hideaki MATSUMOTO， Seishi OHNO* and Tomoyuki Y AMA Y A It is generally accepted that a plant species has a preferred nitrogen form， i.e.， nitrate-nitrogen (N) or ammonium-nitrogen (N). Although many plants can utilize either nitrate or ammonium salts， nitrate is the preferable nitrogen source for various crops幻 Manyworkers have in-vestigated the relative values of nitrate and ammonium salts as nitrogen sources5.8.9). Several environmental factors concerning uptake and utili-zation of these nitrogen sources for plant growth have been investigated in agricultural studies directed toward establishing ferti1izer practice. The external pH and oxygen supply in the culture solution are important factors that affect the uptake and uti1ization of nitrogen3.B). Besides these factors， metabolic capabi1ities of plants toward di百erentni trogen sources could be reflected in the differen t preferences to ni trogen forms1. 7.10.1). In this study， some characteristic behaviors of nitrate reductase (NR)， glutamine synthetase (GS) and glutamate dehydrogenase (GDH) under different nitrogen status were investigated using rice plants which tend to prefer ammonium-N and chinese cabbage which strict1y prefer nitrate8). MA TER1ALS AND METHODS Plant materials一一一Seedsof rice (Oryza sativa cv. Akebono) and chinese cabbage (Brassica rapa cv. Kashin-hakusai) were allowed to ger-minate approximately 10 days after sowing， the seedlings were cultured in a standard culture solution containing the following minerals: 1 mM NaH2PO.， 1 mM MgSO.， 2 mM CaCb， 4 mM KNOa， 1 mM NaN03， 0.5 ppm B， O.5 ppm Mn， 0.05 ppm Zn， 0.02 ppm Cu， 0.01 ppm Mo and 1 ppm Fe. The solution was adjusted to pH 5.5. The culture solution diluted 4 fold was given during the first week. Rice was given an additional 2 mM H2Si03・ Theplants in the greenhouse were aerated throughout the experiment and the culture solution was renewed weekly. After approximately 1 month of culture， the samples were used for the ex-periment. Received April 20， 1982. 牟 Presentaddress: Bunsen Company Limited， Shingu 116 H. Matsumoto， S.Ohno and T. Yamaya Enzyme preparation and assays一一一Enzymepreparation was carried out at 2-60C. Samples were weighed， washed by distilled water， and then homogenized with three times their volume of 0.1 M Tris-HCl buffer (pH 7.5) containing 10 mM cysteine in a Waring blender for 2 min. Polyvinylpyrolidone corresponding to 1/5 the sample weight was added during homogenization. The homogenate was passed through 4 layeres of cheese cloth and centrifuged at 15，000 x g for 20 min. The resulting supernatant was used as the crude enzyme. The reaction components and assay conditions for NADH-nitrate reductase (NR)， glutamine synthetase (GS) and glutamate dehydroge-FIG. 1. Rice and chinese cabbage fed with either 5 mM ammonium-N or nitrate-N for 5 days. Note that chinese cabbage， especially roots， was injured byammonium-N. Chinese cab-bage (lower): treated with ammonium-N (right) and nitrate-N (left). Rice (upper): treated with ammonium-N(left) and nitrate-N (right). Enzyme Activities of Rice and Chinese Cabbage due to N Status 117 nase (GDH) were the same as those reported previously2.6.l2). was determined by the method of Lowry et al.o Protein RESUL TS AND DlSCUSSION Characteristic Growth of Rice and Chinese Nitrate or Ammonium Salts Cabbage Cultured by Either As shown in Fig. 1， rice plants grew normally on either nitrate or ammonium-N， whi1e chinese cabbage was severely injured by ammonium-N. This supports the previous finding that nitrate-N is the strict1y preferred nitrogen source for the growth of chinese cabbage8】.Nitrate Reductase Activity from Rice and Chinese Cabbage NR activity from the leaves of rice and chinese cabbage which were grown with nitrate medium showed near1y the same activity on a pro-tein basis (Fig. 2). The reaction progressed linearly up to 300μg pro-tein in both enzymes. It can be said that the level of NR activity might not be an important factor determining the preference of nitrogen form of rice and chinese cabbage. This implies that other factors， 1) NR regulating system1への thecapacity of absorptionjaccumulation of nitrate and 3) the tolerance to ammonium-N， may be responsible for the different response of rice and chinese cabbage to each nitrogen form. 0.04 ~ 0.03 0 、τ， E 。‘-~ O.OZ 2 3 h 苦. ・: 0.01 0: z 。。lT' す涼噌O.Z 0.3 0.4 Protein (mg) 0.5 FIG. 2. NR activity in leaves of rice and chinese cabbage as a function of enzyme vol・ume. Samples were cul-tured with nitrate-N added for approximately 1 month. H. Mjltsumoto， S.Ohno and T. Yamaya FIG. 3. The rate of induction of NR in excised leaves of rice and chinese cabbage. Rice and chinese cabbage were grown with a standard cul-ture solution for 1 month followed by nitrogen starva-tion for 2 weeks. Nitrogen starved leaves were cut into small segments(approx-imately 5 mm squares) and fioated on O. 1 M KNO. in O. 1 M potassium phosphate buffer (pH 7.5) under 30，0∞ lux light at 300C for 0， 3 and 6 hr. The values are expressed by % increase in the activity of the non-induced sample. ???????? ? ?3 6 20 ?《????? ? ??? ? ??????????? ? ?5 118 Chinese cabbas・100 90 80 き32・70喜S 360 • gTime (hr) 100 S .~ 70 E H = 360 a -g 90 80 • ? ?• 50 50 40 01. 36  3 Tim・(hr) Tin咽 (hr)FIG. 4. Effect of ammonium-N on in vivo stability of NR of rice and chinese cabbage leaves. Ric怠 andchinese cabb也gewere grown normally for 1 month followed by nitrogen starvation for 2 weeks. Then the samples were given 5 mM KNO. for 48 hr to induce NR. The leaves were cut int刀 smallsegments and fioated on either 1∞mM NH，Cl or HzO. NR activity was measured and the value at 0 hr of fioating was calculated as 1∞弘i 6 40 Enzyme Activities of Rice and Chinese Cabbage due to N Status 119 It is well known that NR is an inducible enzyme. The rate of in-duction within a short period was compared in the following experiment. Rice and chinese cabbage were grown normally for one month and then in a nitrogen deficient condition for another two weeks. Then the leaves of nitrogen starved plants were cut into small segments and floated on nitrate solution to induce NR. The rate of NR induction of rice after 3 and 6 hr was 1. 8 and 2.5 fold， and that of chinese cabbage 9.4 and 18.7 fo1d， respective1y (Fig. 3). However， the NR activity of nitrogen-starved rice and chinese cabbage was O. 62 and O. 04 p.mo1 N02 formed per mg protein for 10 min， respective1y. These results suggest that chinese cabbage effective1y responds to the nitrate supplied concerning NR induction. Effect 01 Ammonium-N on in vivo Stability 01 NR As shown in Fig. 4， NR activity in both 1eaf segments decreased after they were floated on water， and the 10ss was slightly bigger in chinese cabbage than in rice p1ants. Furthermore， the 10ss was promoted by the floating of 1eaf segments on the solution containing ammonium-N. Near1y 67 % of the activity of rice NR remained after 6 hr， but on1y 45 % remained in chinese cabbage 1eaves. This suggests that the NR in chinese cabbage becomes 1ess stab1e by coexisting ammonium-N than NR of rice does. This may be connected to the fact that ammonium-N represses the induction of NR in chinese cabbage as wi1 be shown 1ater. In the following e玄periments，the effects of ammonium-N on NR， GS and GDH were investigated further. Effect 01 Ammonium-N on the Activities 01 NR， GS and GDH GS and GDH p1ay an important ro1e in the assimi1ation of ammonia. NR is the key enzyme in the process of reducing nitrate to ammonia. Thus the effects of ammonium treatment on the activities of GS and TABLE 1. NR activity of rice and chinese cabbage fed with different nitrogen forms N-form NR specific activity inleaf* NO.-N 5 mM NO.-N 2.5mMl NH，-N 2. 5 mMJ NH，-N 5 mM Rice 13 x 10-'(1∞%) 10x 10・I(76.9) 3 x 10-'( 23. 1) Chinese cabbage 13 x 10-1(1∞%) 9 X1O-'( 69.2) O. 6 X 1O-1( 4. 6) Samples cultured with a standard culture solution for 1 month were treated with nitrogen starvation for 2 weeks. Then the samples were fed with diferent nitrogen forms for 72 hr. * Values meanμmol NO， formedjmg proteinjlO min. 120 H. Matsumoto， S.Ohno and T. Yamaya GDH together with NR were investigated. The samples treated for nitrogen deficiency were fed either with nitrate-N or with ammonium-N， or a mixture of both N sources for 72 hr. As shown in Table 1， am・monium-N strongly affected the level of NR in the leaves of rice and chinese cabbage， especially in the latter. The NR activity of chinese cabbage fed with ammonium-N was suppressed to only 4.6% of that of the nitrate-fed plants. The eoexistence of equal concentrations of ammonium-and nitrate-N reduced the NR activity by approximately 70 % of that of the nitrate-fed plants. On the other hand， rice plants were much less affected by ammonium-N. The NR activity was sup-pressed by ammonium-N to 77 % of the NR activity of nitrate-fed plants. These results show that ammonium-N a旺ectsthe level of NR more prominent1y in chinese cabbage than in rice as shown in Fig. 4. The effects of ammonium-N on GS activity in rice and chinese cab-bage were also c1ear1y different (Table 2). GS activities in both leaves TABLE 2. GS activity of rice and chinese cabbage fed with different nitrogen forms N-form NO，-N 5mM NO.-N 2.5 mMl NH..N 2.5mMf NH.-N 5mM 夏Iee貰叩t0.30 (1∞%) 0.88 (293) 1.02 (340) GS speclftc関tivity・chinese晶 bbage百五r- Root Leat 1.01 (1∞%) 0.67 (l∞%) 2.10 (l∞%) 0.98 ( 97) o.ω( 90) 1. 84 ( 8) 1. 40 (139) O. 10 ( 15) 1. 21 ( 58) Samples cultured with a standard culture solution for 1 month were treated with nitrogen starvation for 2 weeks. Then the samples were treated with diferent nitrogen forms for 72 hr. • Values meanμmol小glutamylhydroxamateformedjmg proteinj10 min. and roots of rice were increased by ammonium treatment， whi1e those of chinese cabbage were strongly suppressed. The increase in GS activity was especially prominent in the roots of rice， GS activity being 340 % of that of the sample without ammonium treatment. Also the presence of ammonium-N in rice roots caused an increase in GS activity， which was 2. 9 fold that of the nitrate-fed plants. These results coincide with those obtained on the induction of GS in rice roots by increasing amounts of ammonia2>. Unlike in rice plants， GS activity of chinese cabbage was strongly suppressed as was the case for pea roots10) and Osmunda regalis1l); the rate of inhibition was 42 % in leaves and 85 % in roots of chinese cabbage. The GDH activity of rice as affected by ammonium treatment was also somewhat different from that of chinese cabbage. 1n leaves of rice Enzyme Activities of Rice and Chinese Cabbage due to N Status 121 TABLE 3. GDH activity of rice and chinese cabbage fed with different nitrogen forms N-form 買両主軍属GDO .pedtc actirity. Chinese団 bb唱eE扇軍一一- Root Leaf NO.-N 5 mM 8.1 x 10-1(1∞%) 1. 7 x 10-1(1∞%) 24.0 x 10-1(1∞%) 0.8 x 10-1(1∞%) N01-N 2.5 mMl NÌÍ:~N :  ~M} 8.1 x 10-1(1∞) 1. 6 x 10叱 94) 15.9 x 10寸(飴) 1.1 x 10吋138)NH，-N 5 mM 13.5 x 10-5(167) 2.9 x 10・‘(171) 15.9 x 1O-5( 6) 1. 8 x 10-1(225) Samp1es cultured with a standard culture solution for 1 month were treated for nitrogen starvation for 2 weeks. Then the samples were given diferent nitrogen forms for 72 hr. • Va1ues mean nmol NADH oxidizedjmg proteinjmin. and chinese cabbage and in roots of rice， GDH activity increased due to ammonium treatment (Table 3). These r白 ultsare supported by the findings that GDH activity is induced by ammonium-N in several plant species7•m. However， a c1ear suppression of GDH activity was observed in the roots of chinese cabbage treated with ammonium-N. This is ap-parent1y in contrast to the roots of rice which show an increase in GDH activity by 67 % of the nitrate-fed plants. The above results suggest that NR， GS and GDH activities in chinese cabbage which strict1y prefers nitrate-N are inhibited more by the treatment with ammonium-N than rice plants are. So far many investigators have discussed why the nitrate-N is generally accepted to be the preferred from for optimal growth. Much attention has been paid to the conditions of the culture solution such as changes in pH and the amount of 0玄ygenavai1able"5.I'. The pr回entwork was designed to investigate the effect of ammonium-and nitrate-N on the growth of two plants whose preferred nitrogen forms are different. Particular emphasis was placed on the changes in enzyme activities concerned in the assimilation of nitrogen. It can be conc1uded that the strong preference for nitrate of chinese cabbage does not depend on the strong activity of NR (Fig.2)， but on its susceptibi1ity to ammonium-N which suppressed the activities of NR， GS and GDH variously (Fig. 4， Table 1，2 and 3). On the other hand， rice NR showing a similar activity to chinese cabbage NR is less suppressed by ammonium-N， and the level of GS and GDH in rice plants is even increased by ammonium treatment. Although such di百erentbehaviors of nitrogen assimi1ating enzymes upon the given treatments are reflected in di旺erentgrowth rate due to am-monium-and nitrate-N， there is as yet no evidence that these changes can be the direct cause of the different preference for nitrogen of rice and chinese cabbage. 122 H. Matsumoto. S. Ohno and T. Yamaya SUMMARY Comparison of the growth and nitrogen assimilating enzymes was made between rice and chinese cabbage which responded di旺erentlyto nitrate-and ammonium-N. Rice grew normally on both nitrogen forms， whi1e chinese cabbage was severly injured by ammonium-N. NR activi-ties in leaves of rice and chinese cabbage were alike， when the plants were grown on nitrate-N. The rate of induction of NR in excised chinese cabbage leaves was strikingly higher than that of rice. The in vivo stabi1ity of NR in the presence of ammonium-N was smaller in chinese cabbage than in rice. The treatment with ammonium-N reduced the level of NR more strongly in chinese cabbage than in rice. GS activity in chinese cabbage and rice was inversely a任ectedby ammonium treat-ment， and the activity in the chinese cabbage being suppressed. 1n addition， GDH activity in leaves and roots of rice was increased by ammonium treatment， while it was lowered in the roots of chinese cab-bage. The results were discussed wi th respect to the di鉦erencein the preferred nitrogen form of chinese cabbage and rice. Acknowledgement We wish to thank Prof. T. Kawasaki of our institute for his kind reading of the manuscript. We are indebted to Prof. T. Kawasaki and Dr. M. Moritsugu of our institute for their kind advice on the culture of plants. REFERENCES 1. Bayley. J. M.. King. J. and Gamborg. O. L. 1972. The effect of the source of inorganic nitrogen on growth and enzymes of nitrogen assimilation in soybean and wheat cells in suspension cultures. Planta 105: 15-24. 2. Kanamori. T. and Matsumoto. H. 1972. Glutamine synthetase from rice plant roots. Arch. Biochem. Biophys. 152: 404-412. 3. Lee. R. B. 1978. lnorganic nitrogen metabolism in barley roots under poor1y aerated conditions. J. Exp. Bot. 29 : 69~708. 4. Lowry. O. H.. Rosebrough. N. J.， Farr. A. L. and Randall. R. J. 1951. Protein measure. ment with the Folin phen01 reagent. J. Biol. Chem. 193: 265ー275.5. Matsumoto. H. and Tamura， K. 1981. Respiratory stress in cucumber roots treated with ammonium or nitrate nitrogen. Plant Soil 60: 195-204. 6. Mat剖 moto，H.. Tanaka. T.. Matoh. T.. Hashizume. K. and Takahashi. E. 1979. ln-hibition of NADH.nitrate reductase activity in cucumber leaves due to NADH oxidation. P1ant & Cell Physi01. 20: 573-582. 7. Mohanty. B. and F1etcher. J. S. 1980. Ammonium infiuence on nitrogen assimilating enzymes and protein accumulation in suspension cultures of Paul's Scarlet rose. Physio1. Plant. 48 : 453-459. 8. Moritsugu. M吋 Suzuki.T. and Kawasaki. T. 19肌 Effectof nitrogen sources upon plant growth and mineral uptake. 1. Comparison between constant pH and con. ventional culture method. J. Sci. Soi1 Manure. Jpn. 51 : 447-456 (in Japanese). 9. Moritsugu. M. and Kawasaki. T. 1982. Effect of solution pH on growth and mineral uptake in plants under constant pH condition. Ber. Ohara Inst. landw. Bi01句Okayama Univ. 18: 77-92. Enzyme Activities of Rice and Chinese Cabbage due to N Status 123 10. Sahulka， J.and Lisa， L.1979. Regulation of glutamine synthetase level in isolated pea roots. 1. Di宜erentialeffects of ammonium salts in sugar-supplied roots. Biochem. Physio1. Pftanzen 174: 646-652. ]1. Taylor， A. A. and Stewart， G. R. 1980. The effect of ammonia and light/dark tran-sitions on the leve1 of glutamine synthetase activity in Osmunda regaliJ. Plant Sci. Letters 20 : 125-131. 12. Wakiuchi， N吋 Matsumoto，H. and Takahashi， E. 1971. Changes of some enzyme activities of cucumber during ammonium toxicity. Physio1. Pl'ant. 24: 24ふ253.13. Yamaya， T. and Ohira， K. 1976. Nitrate reductase inactivating factor from rice cells in suspension culture. Plant & Cell Physio1. 17: 日~1.

CHANGES IN SOME ENZYME ACTIVITIES OF RICE AND CHINESE CABBAGE DUE TO NITROGEN STATUS

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