Properties of maize root mitochondria from plants grown on different nitrogen sources

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Properties of Maize Root Mitochondria from Plants Grown on Different Nitrogen Sources VESNA HAoZI-TASKOVH: SUKALOVIC

and

MIRJANA VULETIC

Laboratory of Plant Physiology, Maize Research Institute Zemun Polje, P.O. Box 89,11081 Beograd-Zemun, Yugoslavia Received January T7, 1997 . Accepted September 26, 1997

Summary The role of maize (Zea mays L. inbred line VA35) root mitochondria in nitrogen assimilation was investigated. Maize plants were grown for 2 weeks on a modified Knopp solution containing different forms of nitrogen: 10.9 mM nitrate or 10.9 mM nitrate + 7.2 mM ammonium. Mitochondria were isolated from root tissue and purified on a PercoU gradient. Glutamate dehydrogenase (GDH; EC 1.4.1.2), alanine aminotransferase (GPT; EC 2.6.1.2), NAD+ -isocitrate dehydrogenase (NAD+ -ICDH; EC 1.1.1.41), succinate dehydrogenase (SDH; EC 1.3.99.1) and malate dehydrogenase (MDH; EC 1.1.1.37) all showed increased specific activities in the case of mitochondria isolated from plants grown in the presence of ammonia. These results indicate the involvement of such mitochondria in ammonia assimilation by synthesizing glutamate in a reductive amination reaction catalyzed by glutamate dehydrogenase and enhanced TCA metabolism. Increased oxygen consumption rates of mitochondria isolated from ammonia grown plants was demonstrated in our experiments, supponing the occurrence of such a metabolic shifr. The possibility that higher oxygen consumption rates of both respiratory pathways (phosphorylative and non-phosphorylative) with malate as substrate for oxidation result from stimulation of biosynthetic and catabolic functions of mitochondria from plants grown in the presence of ammonia is discussed.

Key words: Zea mays L., enzyme, mitochondria, nitrogen form, respiration, root. Abbreviations: GDH = glutamate dehydrogenase; GPT = alanine aminotransferase; NAD+ -ICDH = NAD+ -isocitrate dehydrogenase; MDH = malate dehydrogenase; ME = malic enzyme; PVP = polyvinylpyrrolidone; SDH = succinate dehydrogenase; SHAM = salicylhydroxamic acid; TPP = thiamine pyrophosphate. Introduction Nitrogen, a major constituent of plant macromolecules, is available in the soil as nitrate (N0 3 -) or ammonium (NH 4 +) ions or as reduced nitrogen derived from the degradation of plant and animal remains. It was shown that both inorganic nitrogen forms are required for maximal growth of maize plants (Schrader et al., 1972). Nitrogen assimilation involves the uptake of available nitrogen from soil by the root system. If the source of nitrogen is nitrate, it is either reduced in the root or transferred to the shoot (via the xylem) and then reduced and incorporated into biochemical pathways. In optimal environmental conditions the proportion of nitrate reduced in maize roots was shown to be 37 % of the total nitrate uptake (van Bensichem et al., 1989). Ammonium, on

J Plant Physiol Wll. 153. pp. 67-73 (1998)

the other hand, is totally and rapidly metabolized in the roots (Lewis, 1986). If ammonium is the dominant form of nitrogen supplied to the plant, it is incorporated into amide nitrogen of glutamine and/or asparagine in the root, and these are transponed to the other parts of the plant. Our previously published results (HadZi-TaSkovic Sukalovic, 1994) demonstrated an increase in the activity of the mitochondrial enzyme GDH in maize root tissue grown on mixed nitrogen compared with nitrate-grown plants, suggesting an altered involvement of root mitochondria in nitrogen metabolism. Plant mitochondria were shown to tolerate high ammonium concentrations otherwise considered to be toxic for plant tissues (Yamaya and Matsumoto, 1985). The same-authors (Yamaya et al., 1986) demonstrated that GDH localized

68

VESNA HAnzI-TAAKOVIC 5UKAWVIC and MIRJANA VULETIC

in pea and corn shoot mitochondria can mediate the assimilation of either exogenously fed NH4 + or NH4 + released internally from glycine in a synthetic reaction (amination of 2-oxoglutarate), and that transamination, rather than deamination by GDH, was involved in the oxidation of glutamate. Besides this in vitro effect on mitochondria, the results of Nauen and Hartmann (1980), and Zheng-Qiang et al. (1992) demonstrated that root mitochondria are involved in ammonia assimilation when a high concentration of ammonium was supplied to the plant. Since plant mitochondria are the site of synthesis of important biosynthetic precursors for amino acid synthesis, such as 2-oxoglutarate and oxaloacetate, increased activity of tricarboxylic acid cycle enzymes and mitochondrial respiration could also be expected at high lev-

ration characteristics of the Percoll gradient were improved by including Pvp, which helped to narrow the distribution of the mitochondria. PVP was added to gradient layers of decreasing concentration in 1 % steps, from 6% (w/v) to 1 % from the bottom to the top of the gradient. All Percoll/PVP solutions contained 0.35 M mannitol, 50 mM Tes buffer (pH 7.4), 1 mM EDTA, 1 mM MgCh and 0.1 % (w/v) BSA. Centrifugation was performed at 13,000 & for 30 min in a Sorvall SS-34 angle rotor (Schwittguebel and Siegenthaler, 1984). The band of mitochondria at the 27/45 % interface was collected with a Pasteur pipette and diluted approximately 10 times with the washing medium. Purified mitochondria were concentrated by centrifugation at 12,000& for 15 min. Possible contaminations of mitochondria with microbodies and plastids were determined by analyzing activities of marker enzymes, catalase (EC 1.11.1.6) and phosphogluconate dehydrogenase (EC 1.1.1.44), respectively.

elsofN~+.

In this article we present the results of our study on the role of maize root mitochondria in nitrogen assimilation, by analyzing the respiration rates and the activity of some key enzymes of mitochondria isolated from plants grown in media containing different nitrogen forms.

Materials and Methods Plant rnatmal Maize (Zea mays L.) inbred line VA35 was used for the experi-

ments. The seed, germinated on water, was transferred after 3 days to plastic pots containing Knopp solution, modified in nitrogen content. The first 7 days, plants were grown on ~ strength nutrient solution and during the following 4 days on full strength solution, both being supplemented with different forms of nitrogen. Nitrogen was supplied as KN03, Ca(N03h and (N~hS04 in two treatments, the concentration of N03- and NH4 + in full strength solution being 10.9: 0 and 10.9: 7.2 (in mM), respectively, i.e. the concentration of nitrogen in the form of nitrate was kept constant, while the presence of ammonium varied. The initial pH of the solution was adjusted to 5.6. Plants were kept in a growth chamber under a 12 h light/dark regime at 22/18 ·C, with light intensity of 40Wm- 2 and a relative humidity of70%.

Isolation ofmitochondria Mitochondria were prepared by a modified procedure described by Schwittguebel and Siegenthaler (1984). Roots were cut in 5 volumes of medium containing 0.4 M mannitol, 50 mM TES buffer (pH 7.5), 4 mM cysteine, 1 mM EDTA, 1 mM MgC12' 0.1 % (w/v) BSA and 1 % (w/v) soluble polyvinylpyrrolidone (PVP) 25,000, and ground with a mortar and pestle. The homogenate was filtered through 4 layers of muslin, centrifuged at 4,000 & for 5 min, and the obtained supernatant centrifuged at 39,000 & for 5 min. The resulting pellet was resuspended in a washing medium containing 50 mM TES buffer (pH 7.5), 0.4 M mannitol, 1 mM EDTA, 1 mM MgCl2 and 0.1 % (w/v) BSA. This suspension was centrifuged at 10,000& for 15 min, yielding a pellet of washed mitochondria.

Purification ofmitochondria Washed mitochondria were suspended in 3 -4 mL of the washing medium and layered on the top of a discontinuous Percoll gradient made of six layers prepared with 5 mL 60 % (v/v), 5 mL 50 %, 10mL45%, 5mL 27%, 5mL 20% and 5mL 13.5% Percoll. Sepa-

Analysis ofproteins Protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. The content of BSA in the washing medium was subtracted from the total protein content in the case of mitochondrial protein calculation.

Enzymt activitits Enzyme activities were assayed spectrophotometrically at 30·C using mitochondria solubilized in 0.04 % (v/v) Triton X-lOO. The activity of SDH was measured by the decrease of absorbance at 600 nm caused by reduction of 2,6-dichlorophenol-indophenol according to King (1967). GDH was assayed by monitoring the oxidation of NADH (aminating activity), or the reduction of NAD+ (deaminating activity) at 340 nm (Bulen, 1956). NAD+ -ICDH activity was determined according to Rasmusson and M0ller (1990) by monitoring the increase of absorbance at 340 nm due to NAD+ reduction. GPT activity was determined using the colorimetric Thonazy method (Bergmeyer and Bernt, 1974). MDH was determined in both directions, oxaloacetate reduction and malate oxidation, resulting in decrease and increase of NADH absorbance at 340 nm, respectively, as described by Hayes et al. (1991). Catalase was assayed by measuring the loss of H 20 2 (Aebi, 1974). Phosphogluconate dehydrogenase activity was determined by increase of absorbance due to NADP+ reduction Oournet and Douce, 1985). Succinate: Cyt c oxidoreductase activity was assayed by measuring reduction of Cyt c, absorbing at 550nm, according to Douce et al. (1m).

Mtasurtmtnt ofmitochondrial rtSpiration ratt Oxygen consumption by mitochondria was measured at 25 ·C using a Clark-type polarographic electrode (Hansatech Ltd., England). Mitochondria (0.2-0.4 mg of protein) were added to 1 mL of the medium containing 10 mM KCI, 5 mM MgC12' 10 mM KH2P04, 0.4 M mannitol, 1 mglmL BSA and 10 mM MOPS buffer, adjusted to pH 7.5 with KOH. Oxygen consumption was measured with a cocktail of substrates containing malate, glutamate and succinate in the presence of thiamine pyrophosphate (TPP) to ensure maximum electron transpott rates (Lambers et al., 1983) or with malate alone. State 3 respiration was triggered by the addition of 0.1 mM ADP. In each assay two state 3 to state 4 transitions were performed, and respiratory control and ADP/O values were calculated according to Estabrook (1967). Salicylhydroxarnic acid (SHAM) was added from a stock solution of 2 M in (100 % v/v) dimethyl sulfoxide. Other details are provided in the legends to figure and tables.

Nitrogen form Influence on Root Mitochondria

Results

Nutrient and general plant characteristics In order to investigate the effect of different nitrogen forms on root mitochondria, we used nutrient solutions containing 10.9 mM N0 3- as a sole source of nitrogen or 7.2 mM NHt + in the presence of 10.9 mM N03 -. A high concentration ofNH 4+ applied together with N0 3- was not toxic for plants and enabled differentiation of the effects of different nitrogen forms on mitochondrial activity. Although this treatment led to an increase in total nitrogen supply, our previous investigations demonstrated that increasing concentration ofN03- as the sole nitrogen source in the range from 10.9 to 18 mM exerted no effect on the investigated enzyme activities (data not presented). When N0 3- was the only nitrogen source, maize produced larger roots with more developed lateral roots compared with those of plants grown in a mixture of both nitrogen forms, root fresh weight and length being higher (data not presented). However, soluble protein content per g fresh weight of roots was decreased by approximately 40 % in N0 3- - grown plants. During plant growth pH of the unbuffered nutrient solution changed as a consquence of different ion uptake from 5.6 to 6.2 or 4.3 for N03- and mixed nitrogen grown plants, respecitively.

Mitochondria quality By testing the bands collected from the Percoll gradient, using mitochondrial marker enzyme activities, succinate: Cyt c oxidoreductase (Table 1) succinate dehydrogenase (SDH) (Table 2), and respiratory activity (Table 3), we demonstrated that the fraction isolated at the 27/45 % interface was enriched in mitochondria. This fraction also exhibited the highest outer membrane integrity. The protein quantity in the 27/45 % interface of isolated mitochondria from roots grown in the presence of ammonia was 15 % greater (Table 1). Mitochondria of greater density appeared in the 45/50 % &actions only in the case of plants grown in the presence of ammonium. Taking into account this &action, the total mitochondrial protein content was approximately 30 % greater than that of nitrate grown plants. Lower density fractions appearing in the gradient consisted of broken mitochondria or did not show the activities of mitochondrial marker

69

enzymes (data not presented). We compared the activities of the 27/45 % interface mitochondria. Mitochondria from plants grown on different nitrogen sources were of similar quality with respect to outer membrane integrity (Table 1). This was tested by succinate: Cyt c oxidoreductase activity and was approximately 80 % for both mitochondrial samples. In order to rule out the possible effect of contamination on enzyme and respiration activities of isolated mitochondria we tested for the presence of microbodies and plastids. An equal or even lower level of contamination with microbodies and plastids in mitochondria from N03 --fed plants was demonstrated by similar specific activities of catalase (marker enzyme for microbodies) in both samples, and even a doubling in specific activity of phosphogluconate dehydrogenase (marker enzyme for plastids) in mitochondria isolated from mixed nitrogen-fed plants compared with N0 3 - -grown plants (Table 1).

Mitochondrial enzyme activities The effect of nitrogen forms in the nutrient solution on activities of several key matrix enzymes involved in carbon metabolism, NAD+-ICDH, MDH and SDH, and enzymes of nitrogen metabolism, GDH and GPT, was examined. Mitochondria isolated from maize grown on mixed nitrogen showed higher specific activities of the investigated enzymes when compared with those obtained from N0 3 --grown plants. Glutamate dehydrogenase, localized in mitochondria, exhibited about 5 times higher activity in plants grown in the presence of ammonia (Table 2). This enzyme catalyzes both the synthetic (amination of 2-oxoglutarate) and catabolic (deamination of glutamate) reaction. The K.n value of GDH for NH4 + in the aminating reaction, determined in the presence of 10 roM 2-oxoglutarate, was 31.4 ± 1.8 roM in mixed nitrogen-grown plants, compared with a K.n of 52.1 ± 5 roM in nitrate-grown maize. This result demonstrated different GDH affinities for NH4 +, depending on growth conditions. The activity of the second enzyme involved in amino acid synthesis, alanine aminotransferase (GPT), catalyzing glutamate oxidation through glutamate-pyruvate transamination, was increased to a similar extent in the presence of NH4 + (Table 2). Also, specific activities of investigated TCA-cycle enzymes; NAD+ -ICDH, MDH and SDH, were increased when NH4 +

Table 1: The quality and purity of mitochondria isolated from maize roots and purified on a PercolUPVP density gradient. The values of enzyme activities are the mean of at least two or three independent experiments (±SE).

Source of nitrogen

Parameter

10.9 mM N03 - + 7.2 mM N~ + Integrity of outer membrane (%) Succinate: Cyt c oxidoreductase activity (j.Ullol. mg protein -1 . min -1) Catalase activity (j.Ullol. mg protein -1 . min -1) Phosphogluconate dehydro,enase activity (j.Ullol. mg protein -1 • min - ) Mitochondrial protein content (mg. g root FW- 1)

80-84 0.Ql 5 ±0.007 0.106±0.009 0.028±0.009

78-81 0.177±0.05 0.091 0.062

0.094±0.007

0.108±0.005

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VESNA HADzl-TMICOVIC SUKAWVIC and MIRJANA VULETIC

Table 2: Specific activities of mitochondrial enzymes in mitochondria isolated from maize plants grown on different nitrogen sources. The results are mean (±SE) of at least three independent experiments.

Enzyme

10.9mMN03-

NAD+-ICDH SDH MDH*-oxaloacetate reduction MDH*-malate oxidation NADH-GDH

GPT

Source of nitrogen 10.9 mM N0 3-

+7.2 mM NH4 +

1

ij1mol. mg protein-I. min-I)

2 ij1mol. mg protein-I. min-I)

0.035±0.OO6 0.014±0.OO5 25.S1 ± 1.31 1.362 ± O.OOS 0.154±0.OO5 0.151±0.005

O.OSl ±0.004 0.070±0.005 46.0S±1.90 1.93±0.22 0.797±0.06 0.S96±0.09

Ratio 2/1 2.3 5.1 1.S

1.4

5.2 5.9

* At optimum pH S.O.

was present (Table 2), suggesting more intensive TCA metabolism in such mitochondria.

Rnpiratory activity ofmitochondria The rates of oxygen consumption and ability to phosphorylate (respiratory control) were different in mitochondria isolated from roots of plants grown in the presence of different nitrogen forms. The respiratory rate, measured with a cocktail of substrates (malate, glutamate and succinate in the presence of TPP), was higher in mixed N-grown mitochondria, in comparison to the mitochondria of plants grown in the presence of nitrate alone (Table 3). When nitrate was the only available nitrogen source, besides the very low respiration rates, mitochondria also exhibited a weak respiratory control and a low ADP/O ratio. In our experiments, when malate was used as a respiratory substrate, a higher value of respiratory control was obtained for mitochondria isolated from mixed-nitrogen grown plants, mainly due to the increased O 2 consumption rate in the «active state» (state 3), state 4 respiration being similar under both conditions (Fig. 1). This result and the ADP/O ratio indicate a more intense phosphorylating activity of mitochondria from plants grown in the presence of ammonia. The involvement of the non-phosphorylating cyanide-insensitive

Table 3: Respiratory activiry of mitochondria isolated from roots of maize plants grown on different nitrogen sources. Oxygen consumption was measured with a cocktail of substrates (10 mM malate, 10 mM glutamate and 10 mM succinate in the presence of 0.1 mM TPP). State 3 respiration was triggered by the addition of 0.1 mM ADP. The results are mean (±SE) of at least three independent experiments.

Parameter

Source of nitrogen 10.9 mM N0 3-

3S.7±4.5 Oxygen uptake (nmol. min -1 . mg protein -1) Respiratory control 1.2S±0.07 ADP/O 1.33±0.1

10.9 mM N0 3- + 7.2 mM NH4+ 55.6±6.3 1.S3±0.OS 1.S2±0.2

pathway, which proceeds via NAD+ -linked malic enzyme (Rustin et al., 1980), was demonstrated by stimulation of malate oxidation with exogenous NAD+ (Fig. 1). Stimulation of oxygen consumption by NAD+ observed in both mitochondrial samples oxidizing malate in state 4 (Fig. lA, B), was much more pronounced in mixed-N mitochondria (258 % in mixed-N vs. 119% in nitrate mitochondria). This stimulation was followed by a decrease in ADP/O ratios. In the presence of cyanide, NAD + was still able to stimulate malate oxidation inhibited by SHAM (Fig. 1 C, D). Such an effect of NAD+ following cyanide treatment was also more pronounced in mitochondria from plants grown on mixed nitrogen (167% in mixed-N vs. 113 % in nitrate mitochondria). This argues in favour of participation of the cyanide-insensitive pathway in malate oxidation in mixed-nitrogen mitochondria. Discussion

The degree of effectiveness of two nitrogen forms on plant growth and nutrient uptake when both sources of nitrogen are present in the solution is dependent on plant species and N0 3- INH4 + ratio (Errebhi and Wilcox, 1990). Since the uptake of nitrate by cotransport with protons increases the pH of the solution, and ammonia uptake coupled to the efflux of protons decreases the pH, acidification of the unbuffered solution containing both nitrogen forms, in our experiments, indicates a preferential uptake of NH4 + by maize plants in this case. Generally, mitochondria isolated from maize roots grown on mixed nitrogen exhibited higher enzyme and respiratory activities compared with mitochondria from nitrate-grown plants. The activities of investigated mitochondrial marker enzymes from plants grown in the presence of both nitrogen forms were similar to reported results for maize roots (CoUt~e et al., 1992), while in nitrate-grown plants their activities were significantly lower. The observed differences are unlikely to be due to different quality of mitochondrial isolates, or to contamination of mitochondria by microbodies or plastids, being equal or even higher in mixed-nitrogen samples. The obtained outer membrane integrity (80 %), equal in both cases, can be considered acceptable, since older maize roots, used in our experiments, are not a very suitable object for

Nitrogen form Influence on Root Mitochondria

A

71

B malate

malate

~

10 J.1M 02

1min

RC 1.79 ADP/O 1.76

RC 1.17 ADP/O 1.50

RC 1.92 ADP/O 1.88 /

C /

/

ADP

RC 1.27 ADP/O 1.66

ADP

D

malate

RC 1.71 ADP/O 1.58

RC 1.82 ADP/O 1.75

malate

RC 1.37 2 ADP/O 1.60 RC 1.40 ADP/O 1.60

/

KCN

NAD+ ADP

SHAM

Fig. 1: Oxidation of malate and the effect of NAD + on malate oxidation by root mitochondria isolated from maize plants grown on different nitrogen sources: NH4 +IN03 - (A, C) and N03 - (B, D). NAD+ was added during state 4 rate (A, B), and afrer cyanide treatment (C, D). The numbers on the traces refer to nmol O 2 consumed.min-I.mg protein-I. The concentrations were: 30mM malate, 200l1M NAD+, O.lmMADP, ImMKCN, ImMSHAM,pH7.5.

mitochondrial isolation. A similar value of 84 % of outer membrane integrity was reported for mitochondria isolated from 3-day-old maize root tips (Couee et al., 1992). In the presence of high NH4 + concentrations, glutamine generation in the GSIGOGAT system was shown to be insufficient to assimilate NH4 + in roots, and mitochondrial enzyme GDH involvement in NH4 + assimilation and detoxification in maize roots was proposed (Oaks et al., 1980). Our study demonstrates an increased activity of GDH and GPT in the presence of ammonium. In addition to increased specific activity of GDH, increased GDH affinity for NH4 + when it was present in the nutrient solution, indicates an increased amination activity of this enzyme in such conditions. Such an aminating role of GDH was reported for a number of plant tissues grown in increased external ammonium ion concentration (Zheng-Qiang et al., 1992; Osuji and Madu, 1995), as well as for plants under stress, leading to increased ammonia concentration in the tissues (Nauen and Hart-

mann, 1980). Also, recent results of Osuji and Madu (1995) have shown that ammonium ion-dependent isomerization of GDH is a critical reaction step in the synthesis of glutamate, by regulating the assembly of subunits to form the hexameric structure of enzyme. Consequendy, we can conclude that in the presence of high ammonium concentration GDH is involved in ammonia assimilation, catalyzing predominandy glutamate generation in the aminating reaction. Increased activity of the other enzyme involved in nitrogen metabolism, GPT, in the presence of ammonium ion, also argues in favour of such an aminating role of GDH. Glutamate synthesized in the mitochondria by GDH can function as an amino donor for amino acid synthesis (especially aspartate and alanine), as well as for the formation of glutamine in a reaction with glutamine synthetase outside mitochondria. Increased activity of GPT in our experiments, as well as the results obtained on corn and pea shoots in the presence of NH4 + (Yamaya and Matsumoto, 1985), demonstrate that the

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VESNA HAob-T~n)VJc SUKAl.OVlC and MIRJANA VULETIC

major route for glutamate oxidation by mitochondria pro- stricted due to the increased level of NADH (Neuburger et ceeds through transamination reactions, and not via the dea- al., 1984), and malate oxidation is catalyzed by NAD+ -malic enzyme, being associated with the cyanide-resistant, nonmination reaction catalyzed by GDH. The nitrogen assimilating reaction of GDH may be en- phosphorylative pathway. The increased non-phosphorylative hanced by increased rates of synthesis of 2-oxoglucarate or in- pathway in mitochondria from plants grown in the presence creased rates of utilization of glutamate (Yamaya et al., 1986). of ammonium, obtained by NAD+ activation in the presence On the basis of the obtained increased specific activities of of KCN, could be explained by their enhanced capacity to the investigated TCA-cycle enzymes ICDH, SDH and transpon electrons via the alternative pathway due to rapid MDH, in the presence of NH4 +, i.e. more intensive TCA oxidation of substrates in the TCA cycle (Lambers and Atkin, metabolism, we propose that in the presence of excess NH4 +, 1995). Pyruvate produced in malate oxidation via NAD+amino acid biosynthesis utilizes the citric acid cycle interme- malic enzyme is recycled (Neuburger et al., 1984), thus allowdiate 2-oxoglutarate to synthesize glutamate in a reductive ing the TCA cycle to continue. To conclude, NH4 + in the presence of N0 3- favours the amination reaction catalyzed by GDH. NADH oxidation by this reaction could be determined by increased rates of panicipation of GDH in the amination reaction in maize NAD+ -ICDH activity in the presence of ammonium, this root mitochondria, making them the site of more intense enzyme being sensitively controlled in plants by the NAD+ / amino acid synthesis. Increased requirements of carbon skeleNADH ratio, as already suggested by Nauen and Hartmann ton and energy in biosynthetic reactions intensify the catabo(1980). The amination reaction with GDH allows the regen- lic and respiratory functions of such mitochondria. eration of NAD+, bypassing the respiratory chain (ZhengQiang et al., 1992). As a consequence, the regulatory enzyme Acknowledgements of the TCA cycle, NAD+ -ICDH, is stimulated, increasing This work was supported by research grants in biotechnology the TCA metabolism. Measurements of oxygen consumption by our mitochon- (no. 1201) and biophysics (no. 1901) from the Ministry for Science dria with a mixture of substrates demonstrated increased and Technology of Republic of Serbia. The authors are grateful to Dr. Zc:ljko Vucinic for his interest and helpful discussions. respiratory rates and phosphorylative activity of these organelles when obtained from plants grown in the presence of ammonia. Lower values obtained when nitrate was the only References nitrogen source are probably due to increased demands for electrons in N0 3- assimilation and, consequendy, dimi- AIlBI, H.: Catalase. In: BERGMEYER, H. U. (00.): Methods in Enzynished mitochondrial electron transpon as proposed by matic Analysis, Vol. 2, pp. 764-767. Verlag Chemie, Weinheim, Bloom et al. (1992). The increased respiration rate in the ISBN 3-527-25596-6 (1974). presence of ammonium is in accordance with more intensive BERGMEYER, H. U. and E. BERNT: Colorimetric assay ofTonhazy. In: BERGMEYER, H. U. (00.): Methods in Enzymatic Analysis, TCA metabolism, presumably providing an excess of respiraVol. 2, pp. 764-767. Verlag Chemie, Weinheim, ISBN 3-527tory substrates to such mitochondria. On the other hand, the 25596-6 (1974). increased phosphorylative activity of mitochondria from plants grown in the presence of ammonia, providing ATP for BLOOM, A. j., S. S. SUKRAPANNA, and R L. WARNER: Root respiration associated with ammonium and nitrate absorption and asmore intensive amino acid and protein synthesis in root tissimilation by barley. Plant Physiol. 99, 1294-1301 (1992). sue, is consistent with the proposed role of mitochondria in BULEN, W. A.: The isolation and characterization of glutamate deammonia assimilation. However, respiratory activity is less hydrogenase from corn leaves. Arch. Biochem. Biophys. 62, affected by the presence of ammonium compared with TCA 402-414 (1956). cycle enzyme activities. This discrepancy could be explained Coun, I., M. JAN, J.-P. CARnE, R BROUQUISSE, P. RAYMOND, and by the fact that a pan of the reducing power in the form of A. PRADET: Effects of glucose starvation on mitochondrial subpopulation in the meristematic and submeristematic regions of NADH delivered through an increasing TCA cycle activity is maize root. Plant Physiol. 100, 1891-1900 (1992). used in the GDH reaction and therefore is not available for DoucE, R, E. L. CHRISTENSEN, and W. D. BONNER: Preparation of the respiratory chain. intact plant mitochondria. Biochim. Biophys. Acta 275, 148-160 Malate is known to participate in the maintenance of cytoom). plasmic pH, being increased in response to alkalinization of ERREBHI, M. and G. E. WILCOX: Plant species response to ammothe cytoplasm and cation uptake (Osmond and Popp, 1983). nium-nitrate concentration ratios. J. Plant Nutr. 13, 1017-1029 Also, malate is an energy rich compound, whose decomposi(1990). tion provides reducing equivalents, and it is an intermediate EsTABROOK, R w.: Mitochondrial respiratory control and the polaof both the glycolytic and oxidative-pentose pathways occurrographic measurement of ADP/O ratios. Methods Enzymol. 10, 41-47 (1967). ring in the cytoplasm. Oxygen consumption by mitochondria oxidizing malate can be carried out by NADH oxidation pro- HAob-T~KOVlC SUKAl.OVlC, Y.: Nitogen assimilating enzyme activities in maize in response to the supply of different nitrogen sourduced by both MDH and NAD+ -linked malic enzyme. In ces. J. Sci. Agric. Res. 57. 43-54 (1994). the presence of ADP (state 3), the oxygen consumption rate, HAYES, K. M., H. M. LuETHY, and E. J. ELTHON: Mitochondrial significandy higher in N~ +/N03 - mitochondria compared malate dehydrogenase from corn. Plant Physiol. 97. 1381-1387 with N03- mitochondria, is in agreement with the obtained (1991). increased activity of MDH producing NADH; this in turn is JOURNET, E. P. and R DOUCE: Enzymatic capacities of purified cauoxidized in the phosphorylative pathway. Under conditions liflower bud plastids for lipid synthesis and carbohydrate metaboof exhausted ADP (state 4), malate oxidation by MDH is relism. Plant Physiol. 79,458-463 (1985).

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