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The activity of nitrate reductase, glutamine synthetase and phosphoenolpyruvate carboxylase in leaf blades of developing Zea mays
Biochem. Physiol. Pflanzen 179, 445- 454 (1984)
The Activity of Nitrate Reductase, Glutamine Synthetase and Phosphoenolpyruvate Carboxylase in Leaf Blades of Developing Zea mays GISELA MACK, RUDOLF TISCHNER and HARALD LORENZEN Pflanzenphysiologisches I nstitut der UnivcTsitiit Go ttingen, BRD K (' Y T er mIn d ex: glutamine synthetase, nitrate reductase, phosphoenolpyruvate carboxylase, enzyme distribution in leaves, development ; Z ea mays
Summary Th e spedfic activities of nitrate reductase, glutamine synthetase and phosphoenolpyruvate carboxylase in the leaf canopy of 2-, 3-, 5-, and 8-week-old Zea mays (Forla) were recorded from early vegetative growth to anthesis. A reg lJiation of the ratio between th e activity of the N assimilating enzymes and that of phosphornolpyruvate carboxylase has been found in the leaf blades as follows: a) A t emporal separation of the max imum of the N assimilating enzymes (3rd week after sowing) and that of phosphoenolpyruvate I:arboxylase (2nd and 8th week after sowing); b) a spatial separation of the maximum of the N assimilating ('nzymes (uppermost leaves of the plant) and that of phosphoenolpyruvate carboxylase (middle leaves); and c) a spatial separation of the maximum of the N assimilating enzy mes (in the base and inner sections of the leaf blade) and that of phosphoenolpyruvate carboxylas e (in the midsections of the leaf blade). The activity of the N assimilating enzymes was closely related to th e protein and chlorophyll content, but negatively correlated to the nitrate and ammonium content of the leaf blade. PhosphoenolpYIllvate carboxylase activity was associated with enlargement of the leaf blade and with stem clonga tion.
Introduction
In natural environments nitrogen may commonly limit photosynthesis. Thus, field crops with the C4 pathway of CO 2 assimilation have an important advantage, as they utilize nitrogen more efficiently than do C3 species (BROWN 1978; BOLTON and BROWN 1980) This difference appears related to the higher CO 2 fixation capacity and the reduced amount of RuBP carboxylase (Ku et al. 1979). These advantages are due to "Kranz" leaf anatomy which results in spatial separation of metabolic pathways between the mesophyll and bundle sheath cells (RAY and BLACK 1979). Besides photosynthesis, nitrogen assimilation also is compartmentalized in the C4 plants and therefore the competition for electrons and ATP between N03 - and CO:! reduction is reduced (VENKATARAl\{ANA and DAS 1982). Abbreviations: GS, glutamine synthetase; GDH, glutamate dehydrogenase ; GHA, y-glutamylhydroxamat e; NR, nitrate reductase; PEP, phosphoenolpyruvate; prot., protein; RuBP, ribulosebisphosphate 29
Biochcm. Physiol.PiJanzen.Bd.179
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G. }IACK, R. TISCHNER and H. LORENZEN
In this paper we present data for a regulation between the activity of the N assimilating enzymes and that of phosphoenolpyruvate carboxylase in the leaf blades dependent on plant age. These enzyme activities were also examined dependent on leaf position, leaf age and tissue differentiation. Material and Methods Plant growth
Soaked caryopses of Zea mays (Forla) were germinated in vermiculite. The seven-day-old seedlings were separated into containers with Fruhstorfer Erde (type FRUSTOSOIL N, by Industrie-ErdenWerk Erich Archut, Lauterbach) and grown in the green-house during spring and summer. Only in spring natural light was supplemented by 400 Watt lamps (OSRAM, HPL- W, 170.u Einstein/m2/s) to provide 16 hid of light. The greenhouse temperature ranged from 20-24°C and humidity was about 70 %. The plants were watered with rain water (pH 5). Plant age
At the 2nd (early seedling stage), 3rd, 5th, and 8th (anthesis) week after sowing each leaf blade of 3 to 5 equally grown plants was analyzed for its content of protein and chlorophyll, nitrate and ammonium, and for the specific activity of NR, GS, and PEP carboxylase. Leaf age, position, and division
The 5th leaf blade (serial numbers from the bottom to the top of the plant) of the 2-, 3-, and 5week-old plant, the 8th leaf blade of the 5-week-old plant, the 10th leaf blade of the 5- and 8-week-old plant, and the 16th leaf blade of the 8-week-old plant were harvested (3-5 replications) for the same analyses as mentioned above (except nitrate and ammonium content). The leaf blades were divided into 10 sections according to Fig. 1, each of it being examined separately. Enzyme extraction and chlorophyll determination
Samples were taken always 4 h after onset of illumination. After removal of the midrib the whole leaves and also the leaf strips were frozen with liquid nitrogen and ground in a mortar with potassium phosphate buffer (0.1 M, pH 7.4, 1 mM EDTA, 5 mM L-cystein). The ratio of fresh weight (g): buffer (ml) was 1: 5. An aliquot of that homogenate was retained for chlorophyll determination in acetone (ARNON 1949), the rest was centrifuged for 10 min at 4°C and 45,000· g. The supernatant was used directly for the enzyme assays. Enzyme assays and protein determination
NR activity was evaluated at 31°C by the procedure of SCHLESIER (1977). GS activity was determined at 37°C using a biosynthetic assay with hydroxylamine as substrate instead of ammonium (STEWART and RHODES 1977). PEP carboxylase activity was measured at room temperature by following NADH oxidation at 340 nm due to oxaloacetate reduction via malate dehydrogenase (HATCH and OLIVER 1978). Protein content was determined as described by LOWRY et al. (1951). Nitrate and ammonium determination
Fresh leaf blades were cut into small pieces and boiled for 15 min in 10-60 ml distilled water. After filtration the clear extract was used for the determination of the nitrate and ammonium content by a modified method of MERCK (1963). In a Zn/HCI solution nitrate is reduced completely to ammonium which is estimated with NESSLER'S reagent at 376 nm. Calculations
All data shown are means of 3 to 5 replications. Standard deviation was about 3-5 % throughout.
447
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Fig. 2. Changes in leaf blade N R, GS, and PEP carboxylase specific activity (A) and in N Oa- and NH4+ content (B) from the seedling stage until anthesis of Z ea mays. NR, NO a- ; +-+, GS, NH4+; 0 - 0, PEP carboxylase. NR: 1 reI. unit = 14 nmoles N0 2 -1
*- *,
mg prot./h; GS: 1 reI. unit = 300 nmol GHA/mg prot./h; P EP carboxylase: 1 rel.unit = 520 nmol. NADHj mg prot./h ; NOs -: 1 reI. unit. = 6.7 nmoles/ mg freshw eight; NH4+: 1 rl.unit = 3.5 nmolesjmg fresh weight.
Results
Changes in the specific activities of NR,
as, and PEP carboxylase with plant age
The 3rd week after sowing seemed to be most important for leaf blade nitrate reduction and for ammonium assimilation via GS (Fig. 2A). Thereafter the specific activity of GS declined to its minimum value at anthesis (8th week), whereas minimum specific activity of NR was obtained in the 5th week followed by a slight increase until anthesis. The sharp rise in both activities between the 2nd and 3rd week was accompanied by maximum increase in leaf blade protein and chlorophyll content (data not shown). 29*
448
G.
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and H.
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The time course of PEP carboxylase specific activity (Fig. 2A) exhibited 2 maxima: the first at the early seedling stage of the plant (2nd week) and the second at anthesis (8th week). Minimum activity coincided with maximum activity of the N assimilating enzymes (3rd week). Increasing PEP carboxylase activity was associated with maximum enlargement of the leaf blade and with maximum stem elongation (data not shown). Thus, an inverse time course of the specific activity of PEP carboxylase compared to that of NR and GS could be noted during vegetative growth of Zea mays.
Changes in nitrate and ammonium content with plant age Leaf blade nitrate and ammonium content showed an inverse time course with respect to NR and GS specific activity during the vegetative growth of the plant (Fig. 2A and B). Minimum and maximum of the nitrate and ammonium content coincided. Their first maximum in the 2nd week appeared 1 week before maximum increase in leaf blade area began. The second maximum in the 5th week appeared concomitantly with the beginning of maximum stem elongation (data not shown). Maximum ratio of nitrate content: NR (specific activity) was obtained in the seedling (2nd week), whereas maximum ratio of ammonium content: GS (specific activity) was observed at anthesis (8th week) (Table 1). Table 1. Changes in the ratios of leaf blade NO a- conteut: NR spedfic activity and of NH4+ content:
as specific activity dependent on plant age. Details as for Fig. 2 Plant age
NO a-: NR
2 weeks 3 weeks
1.06 0.05
Plant age 6.0 2.0
5 weeks 8 weeks
0.93 0.39
12.5
14.5
NR, as, and PEP carboxylase specific activity in all leaves of the 2-, 3-, 5-, and 8-week-old plant The highest values of the specific activities of NR and GS were obtained in the uppermost leaf blades of the developing plant (Fig. 3A and B), although these leaves were partially enclosed in the whorl and therefore had less chlorophyll content than the older ones which were fully exposed to the light (data not shown). On the contrary, PEP carboxylase specific activity was low in the uppermost leaves but reached its maximum value in the middle and upper leaf blades (Fig. 3C) of the plant at each sampling date. The low specific activity of all 3 enzymes in leaf blades no. 4-7 of the 5-week-old plant was associated with elongation of the connected leaf sheaths. The high PEP carboxylase activity of the 8-week-old plant in the leaf blades above the 10th leaf may be due to the fact that at its node the ear within the husk began to grow and develop rapidly. In this plant no NR activity was detectable in leaf blades no. 8-13 (leaves no. 1-7 are lost or wilted), although nitrate was available.
Nitrate Reductase in Leaves of Developing Zea mays
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Fig. 3. Distribution of NR (Aj, GS (Nj, and PEP carboxylase (Cj specific activity within the leaf canopy of developing Zea mays. 2-week-old plant; +-+, 3-week-old plant; 0-0, 5-week-old plant; x-x, 8-week-old plant.
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Fig. 4. Localization of NR (A, B j, a8 (C, D j, and PEP carboxylase (E, F j specific activity along the length of leaf blade no. 5. 2- (*-*); 3- (+-+); and 5-week-old (0-0) plant. A, C, E, outer sections (odd numbers); B, D, F, inner sections (even numbers) of the leaf blade, as illustrated in Fig. 1.
451
Nitrate Reductase in Leaves of Developing Zea mays
Localization of NR, as, and PEP carboxylase specific activity in the sections of the leaf blade no. 5 of the 2-, 3-, and 5-week-old plant The 5th leaf of the 2-week-old plant was the youngest leaf of that plant. It was just 3 d old (since being visible in the whorl) and so small that it was cut into only 5 segments (a-e) according to Fig. 1. Therefore the distribution pattern of the enzymes was identical in both the outer and inner sections (Fig. 4). 4 d later the collar of the leaf was visible and the leaf had reached its final size on the 3-week-old plant. Now it could be cut into 10 sections according to Fig. 1. On the 8-week-old plant it was no more functional. ~R and GS specific activity was located predominantly in the young (base) tissue of the leaf blade (Fig. 4A-D), whereas PEP carboxylase activity dominated in the midsections (Fig. 4E and F). A higher specific activity of NR (17 %) and of GS (11 %) was obtained in the inner than in the outer sections. For PEP carboxylase activity no similar distribution was obs('fved. But the ratio of PEP carboxylase: NR (specific activity) was about 1.5 times, that of PEP carboxylase: GS about 1.3 times, higher in the outer than in the inner sections. This distribution pattern did not vary with leaf position nor with leaf age, because in leaf blades no. 8, 10, and 16 of the 5- and 8-week-old plant respectively the enzymes' distribution pattern was similar to that described for leaf blade no. 5.
Distribution of protein and chlorophyll contents within the leaf blades The protein content (Table 2A) increased 3-fold and the chlorophyll content (Table 2 B) increased 2-fold from the base to the top of the leaf blade. The inner sections contained about 12 % more protein than the outer ones. On the contrary 11 % more chlorophyll was found in the outer than in the inner sections of the leaf blade. Table 2. Distribution of protein ( A) and chlorophyll (R) contents (1lYlmg fresh weight ) within the lea f blades A. Leaf number
5
8
10
16
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Discussion
Plant age During leaf ontogeny of barley and wheat maximum NR activity has been demonstrated to appear prior to full activity of the photosynthetic apparatus (KOHL etal.1975). In the present study a similar temporal separation of canopy N assimilation and CO 2 fixation was observed dependent on plant age of Zea mays. The development of both processes was inverse during vegetative growth.
452
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At anthesis the increased PEP carboxylase activity and the simultaneously decl'€ased activity of NR and GS indicate that C-supply is adequate but newly reduced K is not sufficient for ear development of maize as being reported by BELOW et al. (1981). Although GS specific activity decreased continuously between the 3rd and 8th week after sowing, the ammonium content did not change markedly. Therefore a maximum ratio of leaf blade ammonium content: GS specific activity was obtained at anthesis. This indicates that GS activity was inhibited by high ammonium concentrations (RHODES et al. 1976) and that with increasing plant age other N assimilating enzymes than GS were favoured. That view is supported by the results of MIFLIN and LEA (1976) who suggest a switch from the GSjGOGAT to the GDH pathway at high ammonium and low energy levels. Evidence for low energy levels at anthesis of Zea mays with respect to N assimilation is given by the fact that canopy photosynthesis (BELOW et al. 1981) and a maximum of PEP carboxylase activity, as shown in this paper, now required the bulk of energy. A former maximum of this enzyme activity was observed with the 2-week-old seedling presumably in order to provide C-skeletons for increasing N assimilation. A maximum ratio of leaf blade nitrate content: NR specific activity and as well of nitrate: ammonium content indicates that the seedling accumulated nitrate in its leaves and preferentially utilized ammonium (SPRATT and GASSER 1970). The accumulated nitrate then induced (VENNESLAND and GUERRERO 1979) maximum NR activity at the 3rd week. Increasing N assimilation was associated with maximum increase in leaf blade pro~ tein and chlorophyll content; increasing PEP carboxylase activity was correlated to a maximum enlargement of the leaf blade and maximum stem elongation. This agrees with the results of RAPER and PEEDIN (1978) who demonstrated that an increase in net C assimilation is predominantly determined by an increase in total photosynthetic leaf area.
Leaf position A spatial separation of maximum N assimilation and CO 2 fixation was observed along the shoot axis of developing maize. Maximum N assimilation was found in the uppermost leaf blades that partially had been enclosed in the whorl, whereas PEP carboxylase specific activity dominated in the middle leaf blades which had been fully exposed to light. In the uppermost leaves energy for N assimilation presumably is supplied by the oxidation of carbohydrates (KOHL et al. 1975), whereas PEP carboxylase specific activity seems to be dependent on photosynthesis.
Leaf blade sections From our results evidence was given for an additional spatial separation of Nand C assimilation: a separation within the leaf blade itself. The specific activity of the N assimilating enzymes predominated in the base (near collar) and inner (near midrib) sections of the leaf blade, whereas PEP carboxylase specific activity was found mainly in the midsections and thus being associated with a high chlorophyll content. We conclude that PEP carboxylase activity is more dependent on tissue differentiation and photo-
Nitrate Reductase in Leaves of Developing Zea mays
453
synthetic capacity than is N assimilation. Similar results were obtained by PERCH OROWICZ and GIBBS (1980) who found the C4 pathway to be functioning only in the middle and top sections of the (total) leaf accompanied by increasing tissue differentiation and chlorophyll content. The localization of maximum NR and GS activity may be due to the fact that nitrate and ammonium are absorbed by the roots and transported to the leaf blade in the midrib. NEYRA and HAGEMAN (1976) demonstrated that high ambient CO 2 concentrations decrease the influx of nitrate to the leaf blade. A distribution pattern of the activity of PEP carboxylase and the N assimilating enzymes, as presented by us, therefore could protect the inner sections from too high CO 2 concentrations in order to minimize inhibition of nitrate influx. • To sum up, the separations between Nand C assimilation seem to be necessary for the plant in order to avoid competition for ATP and electrons. It remains to be examined whether this is typical only for Zea mays.
Acknowledgements We thank Prof. NORBERT ELSNER for the use of his computer and its devices and DipI.- BioI. EBERHARD SCHMITT for programming. The work was supported by a grant from Deutsche Forschungsgemeinschaft to R T. and H.L.
References ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenol oxydase in Beta bulgaris. Plant Physioi. 24, 1-15 (1949). BELOW, F. E., CHRISTENSEN, L. E., REED, A. J., and HAGEMAN, R. H.: Availability of reduced nitrogen and carbohydrates for ear development of maize. Plant Physioi. 68, 1186-1190 (1981). BOLTON, J. K., and BROWN, R H.: Photosynthesis of grass species differing in carbon dioxide fixation pathways. Plant Physiol. 66, 97-100 (1980). BROWN, R H.: A difference in N use efficiency in C3 and C4 plants and its implications in adaptation and evolution. Crop Science 18, 93-98 (1978). HATCH, M. D., and OLIVER, J. R: Activation and inactivation of phosphoenolpyruvate carboxylase in leaf extracts of C4 species. Aust. J. Plant Physioi. 5, 571-580 (1978). KOHL, J.-G., APEL, M., and DUDEL, G.: Enzymatische Charakteristik der Entwicklungsphasen des Getreide-Primarblattes im Hinblick auf funktionelle Beziehungen zwischen Nitratreduktion und anderen Stoffwechselwegen. Biochem. Physioi. Pflanzen 167, 501-512 (1975). Ku, M. S.B., SCHMITT, M. R, and EDWARDS, G. E.: Quantitative determination of RuBP carboxylase - oxygenase in leaves of several C3 and C4 plants. J. Exp. Bot. 30, 79-98 (1979). LOWRY, O. W., ROSENBROUGII, M. J., FARR, A. L., nd RANDALL, R S.: 1951. Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193, 265-275 (1951). MERCK, E.: Die Untersuchung von Wasser. Eine Auswahl chemischer Methoden fiir die Praxis. 7. unveriinderte Ausgabe. Darmstadt. 115 S. (1962). lIrFLIN, B. J., and LEA, P. J.: The pathway of nitrogen assimilation in plants. Phytochemistry I;'), 873-885 (1976). )iEYRA, C. A., and HAGEMAN, R H.: Relationships between carbon dioxide, malate, and nitrate accumulation and reduction in corn (Zea mays) seedlings. Plant Physioi. 58, 726-730 (1976).
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PERCHOWICZ, J. T., and GIBBS, M.: Carbon dioxide fixation and related properties in sections of the developing green maize leaf. Plant Physiol. 65, 802-809 (1980). RAPER, C. D., Jr., and PEEDIN, G. F.: Photosynthetic rate during steady-state growth as influenced by carbon dioxide concentration. Bot. Gaz. 139, 147-149 (1978). RHODES, D., RENDON, G. A., and STEWART, G. R.: The regulation of ammonia assimilating enzymes in Lemna minor. Planta 128, 203-210 (1976). SCHLESIER, G.: Methodische Verbesserungen zur Bestimmung der Nitratreduktase-Aktivitat in Hiiheren Pflanzen. Biochem. Physiol. Pflanzen 171,503-510 (1977). SPRATT, E. D., and GASSER, J. K. R.: The effect of ammonium sulphate treated with a nitrification inhibitor, and calcium nitrate, on growth and N-uptake of spring wheat, rye-grass and kale. J. Agr. Sc., Camb. 74, 111-117 (1970). STEWART, G. R., and RHODES, D.: Comparison of characteristics of glutamine synthetase and glutamate dehydrogenase from Lemna minor. New Phytol. 79, 257-268 (1977). VENKATARAMANA, S., and DAS, V. S. R.: Distribution of nitrogen assimilating enzymes in relation to photosynthesis in certain C4 ' grasses. Z. Pflanzenphysiol. lOa, 289-296 (1982). VENNESLAND, B., and GUERRERO, M. G.: Reduction of nitrate and nitrite. In: "Photosynthesis II" (GIBBS, M., LATZKO, E., Eds.), Encyclopedia of Plant Physiology, New Series, Vol. 6, pp. 425-444, Springer-Verlag, Berlin-Heidelberg-New York 1979.
Received December 22, 1983; accepted February 1, 1984 Authors' address: GISELA MXCK, RUDOLF TISCHNER, HARALD LORENZEN, Pflanzenphysiologisches Institut der Universitat Giittingen, Untere Karspiile 2, D - 3400 Giittingen.
The Activity of Nitrate Reductase, Glutamine Synthetase and Phosphoenolpyruvate Carboxylase in Leaf Blades of Developing Zea mays GISELA MACK, RUDOLF TISCHNER and HARALD LORENZEN Pflanzenphysiologisches I nstitut der UnivcTsitiit Go ttingen, BRD K (' Y T er mIn d ex: glutamine synthetase, nitrate reductase, phosphoenolpyruvate carboxylase, enzyme distribution in leaves, development ; Z ea mays
Summary Th e spedfic activities of nitrate reductase, glutamine synthetase and phosphoenolpyruvate carboxylase in the leaf canopy of 2-, 3-, 5-, and 8-week-old Zea mays (Forla) were recorded from early vegetative growth to anthesis. A reg lJiation of the ratio between th e activity of the N assimilating enzymes and that of phosphornolpyruvate carboxylase has been found in the leaf blades as follows: a) A t emporal separation of the max imum of the N assimilating enzymes (3rd week after sowing) and that of phosphoenolpyruvate I:arboxylase (2nd and 8th week after sowing); b) a spatial separation of the maximum of the N assimilating ('nzymes (uppermost leaves of the plant) and that of phosphoenolpyruvate carboxylase (middle leaves); and c) a spatial separation of the maximum of the N assimilating enzy mes (in the base and inner sections of the leaf blade) and that of phosphoenolpyruvate carboxylas e (in the midsections of the leaf blade). The activity of the N assimilating enzymes was closely related to th e protein and chlorophyll content, but negatively correlated to the nitrate and ammonium content of the leaf blade. PhosphoenolpYIllvate carboxylase activity was associated with enlargement of the leaf blade and with stem clonga tion.
Introduction
In natural environments nitrogen may commonly limit photosynthesis. Thus, field crops with the C4 pathway of CO 2 assimilation have an important advantage, as they utilize nitrogen more efficiently than do C3 species (BROWN 1978; BOLTON and BROWN 1980) This difference appears related to the higher CO 2 fixation capacity and the reduced amount of RuBP carboxylase (Ku et al. 1979). These advantages are due to "Kranz" leaf anatomy which results in spatial separation of metabolic pathways between the mesophyll and bundle sheath cells (RAY and BLACK 1979). Besides photosynthesis, nitrogen assimilation also is compartmentalized in the C4 plants and therefore the competition for electrons and ATP between N03 - and CO:! reduction is reduced (VENKATARAl\{ANA and DAS 1982). Abbreviations: GS, glutamine synthetase; GDH, glutamate dehydrogenase ; GHA, y-glutamylhydroxamat e; NR, nitrate reductase; PEP, phosphoenolpyruvate; prot., protein; RuBP, ribulosebisphosphate 29
Biochcm. Physiol.PiJanzen.Bd.179
446
G. }IACK, R. TISCHNER and H. LORENZEN
In this paper we present data for a regulation between the activity of the N assimilating enzymes and that of phosphoenolpyruvate carboxylase in the leaf blades dependent on plant age. These enzyme activities were also examined dependent on leaf position, leaf age and tissue differentiation. Material and Methods Plant growth
Soaked caryopses of Zea mays (Forla) were germinated in vermiculite. The seven-day-old seedlings were separated into containers with Fruhstorfer Erde (type FRUSTOSOIL N, by Industrie-ErdenWerk Erich Archut, Lauterbach) and grown in the green-house during spring and summer. Only in spring natural light was supplemented by 400 Watt lamps (OSRAM, HPL- W, 170.u Einstein/m2/s) to provide 16 hid of light. The greenhouse temperature ranged from 20-24°C and humidity was about 70 %. The plants were watered with rain water (pH 5). Plant age
At the 2nd (early seedling stage), 3rd, 5th, and 8th (anthesis) week after sowing each leaf blade of 3 to 5 equally grown plants was analyzed for its content of protein and chlorophyll, nitrate and ammonium, and for the specific activity of NR, GS, and PEP carboxylase. Leaf age, position, and division
The 5th leaf blade (serial numbers from the bottom to the top of the plant) of the 2-, 3-, and 5week-old plant, the 8th leaf blade of the 5-week-old plant, the 10th leaf blade of the 5- and 8-week-old plant, and the 16th leaf blade of the 8-week-old plant were harvested (3-5 replications) for the same analyses as mentioned above (except nitrate and ammonium content). The leaf blades were divided into 10 sections according to Fig. 1, each of it being examined separately. Enzyme extraction and chlorophyll determination
Samples were taken always 4 h after onset of illumination. After removal of the midrib the whole leaves and also the leaf strips were frozen with liquid nitrogen and ground in a mortar with potassium phosphate buffer (0.1 M, pH 7.4, 1 mM EDTA, 5 mM L-cystein). The ratio of fresh weight (g): buffer (ml) was 1: 5. An aliquot of that homogenate was retained for chlorophyll determination in acetone (ARNON 1949), the rest was centrifuged for 10 min at 4°C and 45,000· g. The supernatant was used directly for the enzyme assays. Enzyme assays and protein determination
NR activity was evaluated at 31°C by the procedure of SCHLESIER (1977). GS activity was determined at 37°C using a biosynthetic assay with hydroxylamine as substrate instead of ammonium (STEWART and RHODES 1977). PEP carboxylase activity was measured at room temperature by following NADH oxidation at 340 nm due to oxaloacetate reduction via malate dehydrogenase (HATCH and OLIVER 1978). Protein content was determined as described by LOWRY et al. (1951). Nitrate and ammonium determination
Fresh leaf blades were cut into small pieces and boiled for 15 min in 10-60 ml distilled water. After filtration the clear extract was used for the determination of the nitrate and ammonium content by a modified method of MERCK (1963). In a Zn/HCI solution nitrate is reduced completely to ammonium which is estimated with NESSLER'S reagent at 376 nm. Calculations
All data shown are means of 3 to 5 replications. Standard deviation was about 3-5 % throughout.
447
Nitrate Reductase in Leaves of Developing Zea mays
A 6
2
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2
3
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PLANT AGE (WEEKS) en 5
....
~ ~
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. 3
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6
6
5
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PLANT AGE (WEEKS)
2 Fig. 1. Division of the leaf blade into sequential outer (odd nu mber) and inner (even numbers) sections. 1
Fig. 2. Changes in leaf blade N R, GS, and PEP carboxylase specific activity (A) and in N Oa- and NH4+ content (B) from the seedling stage until anthesis of Z ea mays. NR, NO a- ; +-+, GS, NH4+; 0 - 0, PEP carboxylase. NR: 1 reI. unit = 14 nmoles N0 2 -1
*- *,
mg prot./h; GS: 1 reI. unit = 300 nmol GHA/mg prot./h; P EP carboxylase: 1 rel.unit = 520 nmol. NADHj mg prot./h ; NOs -: 1 reI. unit. = 6.7 nmoles/ mg freshw eight; NH4+: 1 rl.unit = 3.5 nmolesjmg fresh weight.
Results
Changes in the specific activities of NR,
as, and PEP carboxylase with plant age
The 3rd week after sowing seemed to be most important for leaf blade nitrate reduction and for ammonium assimilation via GS (Fig. 2A). Thereafter the specific activity of GS declined to its minimum value at anthesis (8th week), whereas minimum specific activity of NR was obtained in the 5th week followed by a slight increase until anthesis. The sharp rise in both activities between the 2nd and 3rd week was accompanied by maximum increase in leaf blade protein and chlorophyll content (data not shown). 29*
448
G.
MACK,
R.
TrscllNER
and H.
LORENZEN
The time course of PEP carboxylase specific activity (Fig. 2A) exhibited 2 maxima: the first at the early seedling stage of the plant (2nd week) and the second at anthesis (8th week). Minimum activity coincided with maximum activity of the N assimilating enzymes (3rd week). Increasing PEP carboxylase activity was associated with maximum enlargement of the leaf blade and with maximum stem elongation (data not shown). Thus, an inverse time course of the specific activity of PEP carboxylase compared to that of NR and GS could be noted during vegetative growth of Zea mays.
Changes in nitrate and ammonium content with plant age Leaf blade nitrate and ammonium content showed an inverse time course with respect to NR and GS specific activity during the vegetative growth of the plant (Fig. 2A and B). Minimum and maximum of the nitrate and ammonium content coincided. Their first maximum in the 2nd week appeared 1 week before maximum increase in leaf blade area began. The second maximum in the 5th week appeared concomitantly with the beginning of maximum stem elongation (data not shown). Maximum ratio of nitrate content: NR (specific activity) was obtained in the seedling (2nd week), whereas maximum ratio of ammonium content: GS (specific activity) was observed at anthesis (8th week) (Table 1). Table 1. Changes in the ratios of leaf blade NO a- conteut: NR spedfic activity and of NH4+ content:
as specific activity dependent on plant age. Details as for Fig. 2 Plant age
NO a-: NR
2 weeks 3 weeks
1.06 0.05
Plant age 6.0 2.0
5 weeks 8 weeks
0.93 0.39
12.5
14.5
NR, as, and PEP carboxylase specific activity in all leaves of the 2-, 3-, 5-, and 8-week-old plant The highest values of the specific activities of NR and GS were obtained in the uppermost leaf blades of the developing plant (Fig. 3A and B), although these leaves were partially enclosed in the whorl and therefore had less chlorophyll content than the older ones which were fully exposed to the light (data not shown). On the contrary, PEP carboxylase specific activity was low in the uppermost leaves but reached its maximum value in the middle and upper leaf blades (Fig. 3C) of the plant at each sampling date. The low specific activity of all 3 enzymes in leaf blades no. 4-7 of the 5-week-old plant was associated with elongation of the connected leaf sheaths. The high PEP carboxylase activity of the 8-week-old plant in the leaf blades above the 10th leaf may be due to the fact that at its node the ear within the husk began to grow and develop rapidly. In this plant no NR activity was detectable in leaf blades no. 8-13 (leaves no. 1-7 are lost or wilted), although nitrate was available.
Nitrate Reductase in Leaves of Developing Zea mays
449
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Fig. 3. Distribution of NR (Aj, GS (Nj, and PEP carboxylase (Cj specific activity within the leaf canopy of developing Zea mays. 2-week-old plant; +-+, 3-week-old plant; 0-0, 5-week-old plant; x-x, 8-week-old plant.
*-*,
450
G.
MXCK,
R.
TISCHNER
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Fig. 4. Localization of NR (A, B j, a8 (C, D j, and PEP carboxylase (E, F j specific activity along the length of leaf blade no. 5. 2- (*-*); 3- (+-+); and 5-week-old (0-0) plant. A, C, E, outer sections (odd numbers); B, D, F, inner sections (even numbers) of the leaf blade, as illustrated in Fig. 1.
451
Nitrate Reductase in Leaves of Developing Zea mays
Localization of NR, as, and PEP carboxylase specific activity in the sections of the leaf blade no. 5 of the 2-, 3-, and 5-week-old plant The 5th leaf of the 2-week-old plant was the youngest leaf of that plant. It was just 3 d old (since being visible in the whorl) and so small that it was cut into only 5 segments (a-e) according to Fig. 1. Therefore the distribution pattern of the enzymes was identical in both the outer and inner sections (Fig. 4). 4 d later the collar of the leaf was visible and the leaf had reached its final size on the 3-week-old plant. Now it could be cut into 10 sections according to Fig. 1. On the 8-week-old plant it was no more functional. ~R and GS specific activity was located predominantly in the young (base) tissue of the leaf blade (Fig. 4A-D), whereas PEP carboxylase activity dominated in the midsections (Fig. 4E and F). A higher specific activity of NR (17 %) and of GS (11 %) was obtained in the inner than in the outer sections. For PEP carboxylase activity no similar distribution was obs('fved. But the ratio of PEP carboxylase: NR (specific activity) was about 1.5 times, that of PEP carboxylase: GS about 1.3 times, higher in the outer than in the inner sections. This distribution pattern did not vary with leaf position nor with leaf age, because in leaf blades no. 8, 10, and 16 of the 5- and 8-week-old plant respectively the enzymes' distribution pattern was similar to that described for leaf blade no. 5.
Distribution of protein and chlorophyll contents within the leaf blades The protein content (Table 2A) increased 3-fold and the chlorophyll content (Table 2 B) increased 2-fold from the base to the top of the leaf blade. The inner sections contained about 12 % more protein than the outer ones. On the contrary 11 % more chlorophyll was found in the outer than in the inner sections of the leaf blade. Table 2. Distribution of protein ( A) and chlorophyll (R) contents (1lYlmg fresh weight ) within the lea f blades A. Leaf number
5
8
10
16
B. Leaf number
Outer sl'ctions Inu€'f sections Top: base
42 47 3.2
45 54 3.3
30 35 3.1
48 48 2.1
Outer sections Inner sections Top:basc
5 3.0
-
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2.0
8
10
16
2.4 2.2 3.0
1.3 1.1
1.9 1.8 1.5
2.7
Discussion
Plant age During leaf ontogeny of barley and wheat maximum NR activity has been demonstrated to appear prior to full activity of the photosynthetic apparatus (KOHL etal.1975). In the present study a similar temporal separation of canopy N assimilation and CO 2 fixation was observed dependent on plant age of Zea mays. The development of both processes was inverse during vegetative growth.
452
G.
MXCK,
R.
TISCHNEI<
and H.
LOHENZEN
At anthesis the increased PEP carboxylase activity and the simultaneously decl'€ased activity of NR and GS indicate that C-supply is adequate but newly reduced K is not sufficient for ear development of maize as being reported by BELOW et al. (1981). Although GS specific activity decreased continuously between the 3rd and 8th week after sowing, the ammonium content did not change markedly. Therefore a maximum ratio of leaf blade ammonium content: GS specific activity was obtained at anthesis. This indicates that GS activity was inhibited by high ammonium concentrations (RHODES et al. 1976) and that with increasing plant age other N assimilating enzymes than GS were favoured. That view is supported by the results of MIFLIN and LEA (1976) who suggest a switch from the GSjGOGAT to the GDH pathway at high ammonium and low energy levels. Evidence for low energy levels at anthesis of Zea mays with respect to N assimilation is given by the fact that canopy photosynthesis (BELOW et al. 1981) and a maximum of PEP carboxylase activity, as shown in this paper, now required the bulk of energy. A former maximum of this enzyme activity was observed with the 2-week-old seedling presumably in order to provide C-skeletons for increasing N assimilation. A maximum ratio of leaf blade nitrate content: NR specific activity and as well of nitrate: ammonium content indicates that the seedling accumulated nitrate in its leaves and preferentially utilized ammonium (SPRATT and GASSER 1970). The accumulated nitrate then induced (VENNESLAND and GUERRERO 1979) maximum NR activity at the 3rd week. Increasing N assimilation was associated with maximum increase in leaf blade pro~ tein and chlorophyll content; increasing PEP carboxylase activity was correlated to a maximum enlargement of the leaf blade and maximum stem elongation. This agrees with the results of RAPER and PEEDIN (1978) who demonstrated that an increase in net C assimilation is predominantly determined by an increase in total photosynthetic leaf area.
Leaf position A spatial separation of maximum N assimilation and CO 2 fixation was observed along the shoot axis of developing maize. Maximum N assimilation was found in the uppermost leaf blades that partially had been enclosed in the whorl, whereas PEP carboxylase specific activity dominated in the middle leaf blades which had been fully exposed to light. In the uppermost leaves energy for N assimilation presumably is supplied by the oxidation of carbohydrates (KOHL et al. 1975), whereas PEP carboxylase specific activity seems to be dependent on photosynthesis.
Leaf blade sections From our results evidence was given for an additional spatial separation of Nand C assimilation: a separation within the leaf blade itself. The specific activity of the N assimilating enzymes predominated in the base (near collar) and inner (near midrib) sections of the leaf blade, whereas PEP carboxylase specific activity was found mainly in the midsections and thus being associated with a high chlorophyll content. We conclude that PEP carboxylase activity is more dependent on tissue differentiation and photo-
Nitrate Reductase in Leaves of Developing Zea mays
453
synthetic capacity than is N assimilation. Similar results were obtained by PERCH OROWICZ and GIBBS (1980) who found the C4 pathway to be functioning only in the middle and top sections of the (total) leaf accompanied by increasing tissue differentiation and chlorophyll content. The localization of maximum NR and GS activity may be due to the fact that nitrate and ammonium are absorbed by the roots and transported to the leaf blade in the midrib. NEYRA and HAGEMAN (1976) demonstrated that high ambient CO 2 concentrations decrease the influx of nitrate to the leaf blade. A distribution pattern of the activity of PEP carboxylase and the N assimilating enzymes, as presented by us, therefore could protect the inner sections from too high CO 2 concentrations in order to minimize inhibition of nitrate influx. • To sum up, the separations between Nand C assimilation seem to be necessary for the plant in order to avoid competition for ATP and electrons. It remains to be examined whether this is typical only for Zea mays.
Acknowledgements We thank Prof. NORBERT ELSNER for the use of his computer and its devices and DipI.- BioI. EBERHARD SCHMITT for programming. The work was supported by a grant from Deutsche Forschungsgemeinschaft to R T. and H.L.
References ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenol oxydase in Beta bulgaris. Plant Physioi. 24, 1-15 (1949). BELOW, F. E., CHRISTENSEN, L. E., REED, A. J., and HAGEMAN, R. H.: Availability of reduced nitrogen and carbohydrates for ear development of maize. Plant Physioi. 68, 1186-1190 (1981). BOLTON, J. K., and BROWN, R H.: Photosynthesis of grass species differing in carbon dioxide fixation pathways. Plant Physiol. 66, 97-100 (1980). BROWN, R H.: A difference in N use efficiency in C3 and C4 plants and its implications in adaptation and evolution. Crop Science 18, 93-98 (1978). HATCH, M. D., and OLIVER, J. R: Activation and inactivation of phosphoenolpyruvate carboxylase in leaf extracts of C4 species. Aust. J. Plant Physioi. 5, 571-580 (1978). KOHL, J.-G., APEL, M., and DUDEL, G.: Enzymatische Charakteristik der Entwicklungsphasen des Getreide-Primarblattes im Hinblick auf funktionelle Beziehungen zwischen Nitratreduktion und anderen Stoffwechselwegen. Biochem. Physioi. Pflanzen 167, 501-512 (1975). Ku, M. S.B., SCHMITT, M. R, and EDWARDS, G. E.: Quantitative determination of RuBP carboxylase - oxygenase in leaves of several C3 and C4 plants. J. Exp. Bot. 30, 79-98 (1979). LOWRY, O. W., ROSENBROUGII, M. J., FARR, A. L., nd RANDALL, R S.: 1951. Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193, 265-275 (1951). MERCK, E.: Die Untersuchung von Wasser. Eine Auswahl chemischer Methoden fiir die Praxis. 7. unveriinderte Ausgabe. Darmstadt. 115 S. (1962). lIrFLIN, B. J., and LEA, P. J.: The pathway of nitrogen assimilation in plants. Phytochemistry I;'), 873-885 (1976). )iEYRA, C. A., and HAGEMAN, R H.: Relationships between carbon dioxide, malate, and nitrate accumulation and reduction in corn (Zea mays) seedlings. Plant Physioi. 58, 726-730 (1976).
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G. MXCK, R. TISCHNER and H. LORENZEN, Nitrate Reductase in Leaves etc.
PERCHOWICZ, J. T., and GIBBS, M.: Carbon dioxide fixation and related properties in sections of the developing green maize leaf. Plant Physiol. 65, 802-809 (1980). RAPER, C. D., Jr., and PEEDIN, G. F.: Photosynthetic rate during steady-state growth as influenced by carbon dioxide concentration. Bot. Gaz. 139, 147-149 (1978). RHODES, D., RENDON, G. A., and STEWART, G. R.: The regulation of ammonia assimilating enzymes in Lemna minor. Planta 128, 203-210 (1976). SCHLESIER, G.: Methodische Verbesserungen zur Bestimmung der Nitratreduktase-Aktivitat in Hiiheren Pflanzen. Biochem. Physiol. Pflanzen 171,503-510 (1977). SPRATT, E. D., and GASSER, J. K. R.: The effect of ammonium sulphate treated with a nitrification inhibitor, and calcium nitrate, on growth and N-uptake of spring wheat, rye-grass and kale. J. Agr. Sc., Camb. 74, 111-117 (1970). STEWART, G. R., and RHODES, D.: Comparison of characteristics of glutamine synthetase and glutamate dehydrogenase from Lemna minor. New Phytol. 79, 257-268 (1977). VENKATARAMANA, S., and DAS, V. S. R.: Distribution of nitrogen assimilating enzymes in relation to photosynthesis in certain C4 ' grasses. Z. Pflanzenphysiol. lOa, 289-296 (1982). VENNESLAND, B., and GUERRERO, M. G.: Reduction of nitrate and nitrite. In: "Photosynthesis II" (GIBBS, M., LATZKO, E., Eds.), Encyclopedia of Plant Physiology, New Series, Vol. 6, pp. 425-444, Springer-Verlag, Berlin-Heidelberg-New York 1979.
Received December 22, 1983; accepted February 1, 1984 Authors' address: GISELA MXCK, RUDOLF TISCHNER, HARALD LORENZEN, Pflanzenphysiologisches Institut der Universitat Giittingen, Untere Karspiile 2, D - 3400 Giittingen.