Changes in the Activity of Enzymes Involved in Nitrogen Metabolism in Maize Seedlings Dependent on Different Nitrogen Sources

Biochem. Physiol. Pflanzen 177, 567-576 (1982)

Changes in the Activity of Enzymes Involved in Nitrogen Metabolism in Maize Seedlings Dependent on Different Nitrogen Sources PETER-CHRISTIAN QUETZ1) , RUDOLF TISCHNER and HARALD LORENZEN Pflanzenphysiologisches Institut der Universitat Gottingen, F. R. G. l)Institut fiir Landeskultur und Pilanzenokologie der Universitat Hohenheim, Stuttgart, F. R. G. Key Term Index: nitrate reductase, glutamine synthetase, glutamate dehydrogenase, nitrogen starvation; Zea mays

Summary 1. Changes in the specific activities of nitrate reductase, glutamine synthetase and NAD + NADP glutamate dehydrogenase were estimated in seedlings of Zea mays for 20 d after germination. 2. N-starvation resulted in a lengthening of the roots, while the increase in shoot lenght was reduced. 3. The activities of the enzymes observed were higher in the roots than in the shoots. Probably a major role of the root in N-metaboism can be interpreted from this observation. 4. Recovery from N-starvation first increased the activities of enzymes investigated in the roots. With different delays their activities increased also in the shoots. The capacity to abolish N-starvation is only slightly reduced in both roots and shoots during starvation. 5. The nitrate reductase activity in the roots was limited by the NOa- supply. If the NOa- supply was enhanced, nitrate reductase activity increased via de novo synthesis.

Introduction

Nitrogen can be consumed by plants both as cations (NH4+) and as anions (N0 3 -). NH4 + is assimilated directly via the aminoacid pathways, whereas N0 3 - at first must be reduced by an energy-consuming process. Moreover the reduction of N0 3 - catalyzed by NR is believed to be rate-limiting. NR activity varies with N-source, plant part, species and age (BEEVERS and HAGEMAN 1969) and may therefore serve as index of the N-status of a plant (SRIVASTAVA 1980). The literature on the adaptive development of NR in response to N0 3 - addition is abundant especially for Zea mays (GASRARIKOVA et al. 1976; OAKS et al. 1980). The effect of NH4+ on NR activity has been examined by several authors, but their results are contradictory. An inhibition of NR synthesis (SMITH and THOMSON 1971) has been observedas wellas a stimulation (INGLE et al. 1966; METHA and SRIVASTAVA 1980). Two pathways of NH4 - assimilation are possible: GDH catalyzes the direct reaction of NH3 - with oxoglutarate and NAD(P)H to glutamate, whereas via GS NH3 - reacts with glutamate and ATP to glutamine (MIFLIN and LEA 1976). Information of the influAbbreviations: GDH, glutamate dehydrogenase; GS, glutamine synthetase; NR, nitrate reductase

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ence of exogenously supplied nitrogen on GS and GDH is rare and mostly dealing with the influence of NH 4+-rather than with that of NOa--feeding (MIFLIN and LEA 1977). In the present study the variaions of NR, GS and NAD(P)-GDH activities during the first 20 d in the life of Maize seedlings were investigated. Additionally the effect of recovery from N-starvation was observed. The enzyme activites in the shoot were compared with those in the roots. Material and Methods Plant growth Caryopsis of Zea mays were germinated for 4 d on moist filter paper at 24 "C, After germination the seedlings were placed in a nutrient solution (6 mM N0 3 -) according to BERGMANN (1958). In nitrogen free medium the ionic streugth was adjusted by the addition of KGI and NaG!. The plants were illuminated 14 hId (9 W/m 2 obtained with Phillips WTL 40 W: TTL 40 W = 3: 3) at 24 "O.

Experimental procedure Samples were taken every day from the fifth day after germination always two hours after onset of the illumination. At a younger age the changes in enzyme activities were low and are therefore not presented. Plants were cultivated in N-free nutrient and N0 3 --medium respectively. Additionally, plants were transfered from N-free medium to N0 3 -- or NH~ +-medium (6 mM each) 8 or 12 dafter germination.

Preparation of the enzyme extracts Shoots and roots were separated; 1.0-1.8 g fresh weight were ground in a mortar with potassium phosphate buffer (0.1 M, pH 7.4,1 mM EDTA, 5 mM cysteine) at 4 -o. After centrifugation (10 min, 20,000' g) the supernatant was used for the enzyme assays.

Enzyme assays Nitrate reductase assay was as reported by SCHLESIER (1977) for both, in vivo and in vitro assay. The glutamine synthetase activity was estimated according to STEWART and RHODES (1977). The assay of glutamate dehydrogenase was the same as reported by DOHERTY (1970). Protein content was determined according to LOWRY et al. (1951).

Nitrate reductase assay (in vivo) The aim of this experiment was to demonstrate, that the NR activity obtained in vivo is limited by the N03 - supply. Thus the assay was not performed in a N2-atmosphere as usually, but in air under different N0 3 - supply and in the presence of different concentrations of cycloheximide. Slices of roots (1-2 mm of thickness, 1-2 g fro wt.) were incubated in the assay mixture at 32 "C, The reaction was started by the addition of N0 3 -. After different times samples were taken and the N0 2 - concentration was estimated. Of course, the change in the volume caused by evaporation was corrected.

Calculation The data presented show the average of 4-7 different experiments. Mostly the standard deviations are noted in the figures by vertical bars.

Results

Morphological development at different nitrogen-sources The shoots of N-starved plants were only half as long as that of NOa--grown plants (Table 1). A transfer to NOa- or NH 4+ containing medium after 8 d of starvation in-

569

Enzymes of Nitrogen Metabolism in Maize

Table 1. Length and fresh weight of shoots and roots of Zea mays seedlings at the 20th day of germination as percentage of the control (N 0 3 - grown seedlings). The N-starved maize seedlings were transferred into N0 3-- or NH 4 +-medium after 8 d of N-starvation fresh weight of

length of

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102

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creased shoot growth immediately. After 20 d nearly 70 % of the shoot length of NOa- grown plants was reached with both, NOa--and NH4+-medium. The roots of N-starved seedlings (9th-14th d of starvation) were longer than those of NOa- grown plants at the same stage. After the 12th d no increase in root lenght was detected (data not shown). With both, NOa- and NH4+ a complete recovery was obtained 20 d after germination.

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Fig. 2. 111 vil:o assay of NR activity in slices of roots from NOa- -grown plants as dependent on NOa- and cycloheximide cOllcentrations. Rates in OD 540 nm. 0.2 ~{ NOa - ( e), 0.1 M NOa- (0),0.02 M NOa- (~ 0.1 ],I NOa-, plu s 0.1 ml\I cycloheximide (L:~ ) 0.1 :M NOa- , pr eincubation in 0.1 m~I cycloheximide for 10 min (.) 0.1 }[ NOa- , preincubatio n in 0.1 ml\[ cycloheximide for 20 min (0 ) 0.1 M NOa- , pr eincub ation in 0.1 mM cycloheximide for 30 min ( x),

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Changes in NR-activity In th e shoots of NOa- grown plants the spedific activity of NR had a maximum around the 9th d after germination (F ig. lA). Later th e activity decreased until th e end of t he observation. In the shoots of N-starve d plant s NR act ivity remained at a low level throughout the experiment. NR activit y increased 10- or 8-fold after a transfer to NOa--medium after 8 or 12 d of starvat ion . Aft er th e maximum activity at th e 10th or 15th d respecti vely a decrease simular to t hat of the reference was observed. The maximum value of acti vity was 30 %---40 % higher than in the reference. After th e transfer of N-sta rved plants into NH 4 +-nutrient NR activity increased only 3-fold. The maximum activity was 30 % of that in NOa--grown plants.

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In the roots of N03 --grown plants (Fig. 1 B) the maximum of NR activity was again around the 9th d after germination. Later the specific activity decreased until the end of our observation. However the values were always higher than in the shoots. In the roots of N-starved plants a only very little NR activity was present. After the transfer to N0 3--medium a drastic increase was observed. The maximum activity was two days after the transfer. The same time course of NR activity was obtained during the recovery (N0 3-) after 8 d or 12 d of starvation. The transfer to NH 4 +-mediumresulted in a 4-fold increase; the maximum activity was also here reached after 2 d. Similar time courses of NR activity were obtained, if slices of leaves and roots were incubated with the assay mixture (data not shown). However, values of the specific activities achieved in this way were only 1/3of those with cell-free extracts. With slices of roots from N0 3 --grown seedlings the amount of N0 2- produced during the assay increased linearly with time for 2-3 h (Fig. 2). Later an exponential increase was observed. The rate of this enhancement was correlated to the N0 3 - concentration. Cycloheximide (0.1 mM) reduced this increase, especially if the slices were incubated with the poison before the assay. This effect of long-term incubation was never obtained with leaf slices.

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In the shoots of N0 3 - grown plants the specific activity of GS was first slowly reduced (Fig. 3A). A minimum occurred at the 10th d and then the activity increased again.

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Enzymes of Nitrogen Metabolism in Maize

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In the shoots of N-starved plants GS activity slowly increased until the 12th day and then decreased slightly. In the roots of NOa- grown plants the maximum GS activity was around the 9th d after germination (Fig. 3B). Later it decreased until the 12th d after which it increased till the end of the experiments. In the roots of N-starved plants the GS activity rose between day 5 and day 12 and then it was reduced till the end of the experiments. A transfer on day 8 of N-starved plants to NOa- and NH 4 + medium, respectively resulted in a more rapid increase of GS activity than in the leaves. The maximum activity was higher and came later with NOathan with NH 4 +. The time course of GS activity was similar for both, recovery after 8 and 12 d of N-starvation.

Changes in NADP-GDH activity The activity of this enzyme in the shoots of NOa- grown plants decreased between the 5th and the 8th d after germination (Fig. 4). Then it increased till the 15th d and was reduced until the 18th d. In the shoots of N-starved plants this enzyme activity was continuously somewhat enhanced. If N-starved (8 d) plants were transferred to NOa-- or to NH 4 "-medium, a drastic increase (although somewhat delayed for NH 4 +) in NADP-GDH activity was detected in the shoots. The maximum activity was 3-4-fold of that in N-starved plants and about twice that of control plants. A transfer after 12 d of starvation resulted in the same general effect; the reaction towards NH 4 + was obtained even quicker than with NOa-. The ability to increase NADP-GDH activity was thus not reduced during the time of starvation. As compared with that in the shoots the specific activity of NADP-GDH in the roots (Fig. 4B) was 5-fold in N-starved plants and 7-fold in NOa- frown plants. A transfer after 8 or 12 d of starvation into NOa- or NH 4 +-medium caused an increase in NADPGDH activity. The reaction upon NH 4 + again occurred with a delay after 8 d of starvation and was quicker than with NOa-. The grade of recovery in shoots and roots was almost the same.

Changes in NAD-GDH activity In the shoots of NOa- grown plants the specific activity of NAD-GDH was continously enhanced until the 15th d after germination (Fig. 5A). Then it slowly decreased until the end of our observations. In N-starved plants the NAD-GDH activity showed small variations; a minimum was observed after 10 d of starvation. After the transfer to NOa- or NH 4+ medium an increase in NAD-GDH activity occurred. After transfer, both at day 8 and day 12 the activity rose more quickly and to higher values in NOamedium than in NH 4 + medium. In the roots a higher NAD-GDH activity was present. The specific activity was reduced in both NOa- grown and N-starved plants (Fig. 5 B). Lowest values were obtained around the 10th d in both cases. Then the activity increased in NOa- grown plants while it varied slightly in N-starved plants.

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After transfer into NOa- as well as into NH 4+ medium the activity was rapidly enha nced. If the r ecovery sta rte d after 12 d the values of the activity obtained were lower than in plants transferred after 8 d. The recovery reacti on occurred mor e quickly in t he roots than in the shoots. Discussion

Thetim e course ofN R activity in the shoots of NOa- grown plan ts rea ched a maximum around the 10th d aft er germination. In younger leaves th ere is a higher level of NR act ivity (WALLACE 1973) than in older ones (HARPER et aI. 1972). It may be that around t he 10th d the ratio between young and old leaves is relatively high and that the following development of each new leaf cannot compensate the low activit y in older tissues. Thus the specific activity of the whole shoot decreased. A similar timecourse for the development of NR activity was detected in the root s of NOa- grown plants. The decrease after the maximum around the 10th d may be due t o t he ratio between youn ger and older tissues as discussed abov e. The highest NR acti vity was demonstrated in root t ips by GASPARIKOVA et aI. (1978) and OAKS et aI. (1980). The relati on of NR act ivity between shoots and roots of NOa- grown plan ts is opposit e t o that reported by WALLACE (1973) and PSENAKOVA et aI. (1976). This discrepan cy may he based on the fact , t hat seedlings were used in our experiments. In th e early stages of growth or at low levels of NOa- most of it will be reduced in the roots. In t he presence of high levels or in older plants NOa- is t ransporte d to t he shoot and NR is indu ced in the a erial ti ssues (BI.;EVERS and HAGEMAN 1969). A basic activity of NR is always present in shoots and roots od N-starve d plan ts (cf. L E E and STEWART 1978). After the tra nsfer of such plants into NOa- medium a rap id increase of NR activity occurred in the shoots . The tra nsfer after 8th d of sta rvation resulted in a maximum activity 2 d later. If th e recovery was starte d after 12 d of starvat ion th e maximum act ivit y was reached three days later. Thus there is only a small decrease in the capa city for forming NR furing N-starvati on. In both cases of recovery the maximum activity of NR was even higher than in NOa- grown plants. Such a behaviour was described for Lemna by J OY (1969). It is possibly due to the high cont ent of carb ohydrates in N-straved plants supplying energy and C-cha ins for the synthesis of ami noacids. The maximum recovery of NR activity was reached one day earlier in the roots afte r transfer at day 12 than in the shoots. The tra nsfer of N-starve d seedlings int o NH 4+ medium result ed in a small but significant increase of NR activity in the shoot, t ransfer after 8 or 12 d. Similar result s were reported by GASPARIKOVA et aI. (1976) and METHA and SRIVASTAVA (1980). The tra nsfer t o NH4+ medium probably increases the synthesis of aminoacids thus effecting protein synt hesis in general (compare the result s of VIJAYARAGHAVAN et al, 1979). NR activity increased if root segments from NOa- grown plan t s were supplied with a sufficient amount of nitrat e. Probably the transport of ions such as NOa- is dominant compared with it s reducti on. The incubation with sufficient NOa- concentrations

Enz ymes of Nitro gen Metabolism in Marze

575

resul ted in a de novo synthesis of NR at 80S ribosomesas shown by the action of cycloheximide. Similar results were reported by ~1ETHA and SRIVISTAVA (1980). Thu s the a ctivity of NR seems to be regulated via th e available NOa- in the cells. This availability depends on NOa- uptake, NOa- storage in the va cuoles and NOa- transport. From our result s it seems as if th e latter is of str ongest evidence. This is confirmed by the r esults of FERRARI et al. (1973), NOa- just taken up was availabel for reduction rather than NOa- present in the vacuoles although the plants were always grown in NOa- medium, ju st as our plants were. After th e transfer of N-starved plants into NOa- or NH4 +-medium the activity of both, GS and NADP-GDH increased immediately in the roots and lat er (ca. 3-4 d for maximum activity) also in th e shoots. This lag phase is due to the time taken for transport of NOa- and NH 4 + from th e roots to the shoots. A similar result was obtained for NR activity but the delay was only one day. Thus it seems as if the transfer into NOa- medium first enhanced NR activity in th e roots. This enhancement is possibly due to de novo synthesis. Later the NR activity in th e leaves increased. The activities of GS and NADP-GDH in the roots increase parallel with NR activity. Thr ee to four days later th ese enzyme activities also increased in th e shoots. This findin g indi cated the reduced role of th e shoot in N-metabolism compared with that of t he root s in young maize seedlings. This interpretation is confirm ed by th e higher amoun ts of thi s enzyme activiti es in the roots than in th e shoots : (GS 2- 3-fold, NADP-GDH 5 - 7-fold and NAD-GDH 4-5-fold).

References lJEEVERS, L., and HAGEMAN, R H.: Nit rate redu ction in higher plan ts. Ann. Rev. Plant Ph ysiol. 20, 495-522 (1969). ll E R G ~[ A N N, W.: Methoden zur Ermittlung mineralischer Bediirfni sse der Pflanz en. In: " Die mineralische Emahrung der Pflanz en" (RUHLAND, W., Ed s.), Handbuch der Pflanz enphy siologie, Vol. 4 pp . 37-89. Springer, Berlin-H eidelberg-New York 1958. ]) OHERTY, D.: Glutamate Dehydr ogenase. In: "Methods of Enz ymology" (TABOR, H., TABO R, C. W. Ed s.) Vol. 17, pp. 850- 856. Academic Press, New York 1970. FERRARI, T. E., YODER, O. C., and FILNER, P.: Anaerobic nitrite production by plant cells and tissu es. Evidence for two nitrate pools. Plant Physiol. 51, 423-431 (1973). GASPARIKOVA, 0., PSENAKOVA, T., and NIZNANSKA, A.: Influence of various nitrogen sources on the activity of nitrate and nitrite redu ctases and glutamate dehy drogenases in Zea mays roots Biologia (Bratislava) 31, 527-535 (1976). - - - Location of nitrate redu ctase and gluta mat e dehycrogenase in the Zea mays root. Biologia (Bratislava) 33, 35-42 (1978). H ARPER, J. E., NIC HOLAS, J. C., and HAG E}[AN, R H.: Seasonal and canopy variation in nitr ate reductase act ivit y of soybean (Glycine max L.) vari eties. Crop. Sci. 12, 382-386 (1972). I NGLE, J. , J OY, K. W., and H AGE}[ AN, R H.: The regulat ion of activity of t he enzymes involv ed in the assimilation of nit ra t e by higher plants. Biochem. J. 100, 577-588 (1966). J OY, K. W.: Nitr ogen metab olism of Lemna minor. II. Enz ymes of nitr at e assimilation and some aspects of their regulati on. Plant Ph ysioJ. 44, 849-853 (1969) . ] ,EE, J. A., and STEWART, G. R: Ecological aspects of nitro gen assimilation. Adv. Bot. Res. 6, 1- 43 (H178).

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LOWRY, O. W., ROSEBROUGH, M. J., FARlt, A. 1., and RANDALL, R S.: Protein measurement with the folin phenol reagent. J. BioI. Chern. 193, 265-275 (1951). METIIA, P., and SRIVASTAVA, H. S.: Comparative stability of ammonium- and nitrate-induced nitrate reductase activity in maize leaves. Phytochem. 19, 2527-2530 (1980). MIFLIN, B. J., and LEA, P. J.: The pathway of nitrogen assimilation in plants. Phytochem. Hi, 873-885

(1976). MIFLIN, B. J., and LEA, P. J.: Aminoacid metabolism. Ann. Rev. Plant Physiol. 28, 299-329 (1977). OAKS, A., STULEN, I., JONES, K., WINSPEAH, M. J., MIRA, S., and BOESEL, I. L.: Enzymes of nitrogen assimilation in maize roots. Planta 148, 477-484 (1980). PSENAKOVA, T., GASPARIKOVA, 0., and NIZNANSKA, A.: Nitrate reductase, nitrite reductase and glutamate dehydrogenase levels in roots and leaves of maize seedlings. BioI. Plant. 18, 283-288

(1976). SCHLESIER, G.: Methodische Verbesserungen zur Bestimmung der Nitratreduktase Aktivitiit in hoheren Pflanzen. Biochem. Physiol. Pflanzen 171, 503-510 (1977). SMITH, F. W., and THOMPSON, J. F.: Regulation of nitrate reductase in excised barley roots. Plant Physiol. 48, 219-223 (1971). SRIVASTAVA, H. S.: Regulation of nitrate reductase activity in higher plants. Phytochem. 19, 725-733

(1980). STEWART, G. R., and RHODES, D.: Comparison of Characteristics of glutamine synthetase and glutamate dehydrogenase from Lemna minor. New Phytol. 79,257-268 (1977). VlJAYARAGHAV.tN, S. J., SOPORY, S. K., and GUHu-MuKKERJEE, S.: Ammonium stimulation of nitrate reductase induction in excised leaves of wheat (Triticum aesticum). Z. Pflanzenphysiol.

93, 395-402 (1979). WALLACE, W.: The destribution and characteristics of nitrate reductase and glutamate dehydrogenase in the maize seedlings. Plant Physiol. 52, 191-196 (1973).

Received March 23, 1982 Authors' addresses: PETER-CHRISTIAN QUETZ, Institut fiir Landeskultur und Pflanzenokologie der Universitat Hohenheim, SchloB-Mittelbau (West), D - 7000 Stuttgart 70; RUDOLF TISCHNER and HARALD LORENZEN, Pflanzenphysiologisches Institut der Universitat Gottingen, Untere Karspiile 2, D - 3400 Gottingen,