The effect of temperature on the energetic activity of maize mitochondria

s

AN p ELSEVIER

CiENCE

Plant Science 114 (1996) 29-33

The effect of temperature on the energetic activity of maize mitochondria Tamara

Pobezhimova*,

Victor Voinikov,

Nina Varakina

Siberian Insiitute of Plant Physiology and Biochemistry, Siberian Branch of the Russian Academy of Sciences, P. 0. Box 1243, Irkutsk 664033, Russia

Received I August 1995; revision received 19 October

1995; accepted

21 November

1995

Abstract The energetic activity of isolated maize mitochondria oxidizing cr-ketoglutarate has been studied in relation to temperature and duration of in vitro incubation. An increase of temperature from 0°C to 37°C in the incubation solution

has been shown to result in organelle inactivation. At 27°C the mitochondria cannot oxidize cr-ketoglutarate as early as after 30 min of incubation. The reduction in the activity of mitochondria during the incubation under enhanced temperature is suggested to be caused by inactivation of the ol-ketoglutarate dehydrogenase complex. The activity of mitochondria can be reconstituted by the addition of thiamine pyrophosphate to the incubation solution in the presence of ATF’. Keyworris:

Zea mays;

Mitochondria;

Temperature;

Thiamine

1. Introduction The effect of high temperature on various organisms including plants results in significant changes in gene expression, protein synthesis and enzymatic activity [l-4]. Cell organelles, including mitochondria, are involved in cellular response to enhanced temperature [5-71. A number of heat shock proteins have been found in mitochondria and the activity of these organelles was shown to change significantly under high temperature [8]. These changes in the mitochondrial activity are most often recorded by isolating the mitochondria Abbreviations: CCCP, carbonyl cyanide mchlorophenylhydrazone; NAD+, nicotinamide adenine dinucleotide; TPP, thiamine pyrophosphate. * Corresponding author. 0168~9452/96/$15.00

0

pyrophosphate

from heat-treated plant cells [6-71. However, the functioning of isolated mitochondria under enhanced temperature in vitro remains unexamined. The aim of the present work was to examine the changes in the energetic activity of mitochondria incubated in vitro under enhanced temperature. In this case, the transfer of isolated mitochondria kept on ice to high temperature can be a convenient experimental model system. 2. Materials and metbods Three-day-old

etiolated maize seedlings (Zeu grown on wet filter paper in a thermostat at 27”C, were used in this work. Mitochondria were isolated by a method described elsewhere [lo]. The mitochondrial pellet

muys L., VIR-36 variety),

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T. Pobezhimova et al. /Plant Science 114 (19%)

was resuspended at 0°C in the following solutions: 1, (medium A): sucrose (250 mM), MgC12 (1 mM), KH,PO, (18 mM), cr-ketoglutarate (5 mM), malonate (3 mM), pH 7.4; 2, medium A plus NAD+ (0.2 mM); 3, medium A plus TPP (0.2 mM); 4, medium A plus TPP (0.2 mM) and ATP (4 mM); 5, medium A plus ATP (4 mM); 6, medium A plus NAD+ (0.2 mM), TPP (0.2 mM) and ATP (4 mM). Each of the suspensions obtained was incubated for either 30 min or 1 h at 27°C and then the energetic activity of mitochondria was analyzed. The activity of mitochondria resuspended in medium A was analyzed immediately following isolation and incubation for 30 min at 0°C 17°C 27°C and 37°C. In another series of experiments the mitochondria were incubated for 30 min at 27°C in solution 3 (medium A plus TPP) and divided into two fractions; the first was supplemented with ATP (4 mM) and the other one with NAD+ (0.2 mM) and, after 30 min additional incubation, the mitochondrial activity was analyzed. The activity of mitochondria was recorded polarographically at 27°C with a Clark-type electrode in a 1.4 ml cell. The reaction solution contained KC1 (125 mM), KH2P0, (18 mM), MgQ(1 mM), and EDTA (5 mM), pM 7.4. CYKetoglutarate (5 mM) was used as a substrate for oxidation. The protein content was determined by the Lowry method [l 1). Polarograms were used to calculate the rate of phosphorylating respiration (state 3), the rate of non-phosphorylating respiration (state 4), the respiratory control and the ADP:O ratio [ 121. All experiments were performed in five to six biological replicates. The data obtained were statistically analyzed and standard deviations of the means (f m) were determined. 3. Results The freshly isolated mitochondria demonstrated a high energetic activity with a high degree of coupling (Fig. 1A). The incubation of mitochondria for 30 min at 0°C in medium A failed to cause any significant change in oxidative phosphorylation of organelles (Fig. 1B). Increasing the temperature in the incubation solution to 17°C resulted in

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Fig. I. Oxidative phosphorylative activity of maize seedling mitochondria incubated in vitro under different temperature conditions. A, freshly isolated mitochondria; B, 0°C 30 min; C, 17°C 30 min; D, 27°C. 30 min; E, 37°C 30 mm. The isolated mitochondria were incubated in medium A (see Materials and methods). The figures on the right of the curves indicate the rate of oxygen uptake (nmol O,/min per mg protein). S, substrate (a-ketoglutarate, 5 mM); M, mitochondria (0.5 mg). The arrows indicate the addition of ADP (200 pmol) and CCCP (I PM), respectively.

a more drastic drop of the phosphorylative respiration rate, respiratory control and ADP:O ratio (Fig. 1C). Incubation of mitochondria for 30 min at 27°C resulted in their complete inactivation (Fig. 1D). Increasing the incubation temperature to 37°C also resulted in mitochondrial inactivation (Fig. 1E). In either case, adding the uncoupler mchlorophenylhydrazone; (carbonyl cyanide CCCP) to the inactivated mitochondria failed to accelerate respiration (Fig. 1). Consequently, a rise in incubation temperature of isolated mitochondria results in a decrease in the activity of mitochondria oxidizing a-ketoglutarate and this decline is most pronounced at 27°C and above.

T. Pobezhimova et al. /Plant

The addition of NAD+, TPP and ATP to medium A prevented some of the mitochondrial inactivation during incubation at 27°C (Table 1). Following 30 min of incubation, there was a decrease in state 3 respiration, respiratory control and ADP:O ratio, though both oxidative activity and coupling of oxidative phosphorylation, when maintained at a relatively high level, remained constant during further incubation (up to 60 min) at 27°C (Table 1). Based on these results, the involvement of each component: NAD+, TPP and ATP was analyzed in its ability to maintain the energetic activity of mitochondria during incubation at 27°C. NAD+, ATP and TPP when added separately did not prevent inactivation of the organelle (Fig. 2). The effects of incubating in ATP and TPP were the same as having these two metabdlites plus NAD+ present. To elucidate the role of TPP, additional experiments were performed and these results appear in Fig. 3. Freshly isolated mitochondria resuspended in solution 3 (medium A plus TPP) possess a high activity (Fig. 3A); however, after 30 min incubation at 27°C they lose their energetic activity (Fig. 3B). When inactivated mitochondria incubated at 27°C for 30 min in the presence of TPP were supplemented with ATP, their activity was observed to recover following an additional 30 mm incubation (Fig. 3C). The addition of NAD+ instead of ATP failed to restore the mitochondrial activity (Fig. 3D).

Table I Energetic activity of mitochondria incubated in vitro at 27°C for O-60 min Duration of Oxygen uptake (nmol O+in per mg protein) incubation (mm) State 3 state 4 0 30 60

86.0 f 1.7 44.2 f 4.7 38.9 + 3.6

20.0 zt 0.5 18.5 zt 1.3 15.0 f 1.3

Respiratory control

ADPO ratio

4.30 f 0.04 3.02 f 0.07 2.40 f 0.14 1.93 f 0.10 2.60 f 0.11 1.98 l 0.04

The mitochondria were incubated in medium A (see Materials and methods) plus TPP, ATP, NAD+; cr-ketoglutarate (5 mM) is the substrate; values are given as standard deviations of the mean ( f m), n=6.

Science 114 (19%)

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Fig. 2. The effect of incubation conditions on mitochondria phosphorylating activity (state 3). A, freshly isolated mitochondria, medium A; B, medium A; C, medium A plus ATP; D, medium A plus ATP, NAD+ and TPP; E, medium A plus ATP, NAD+; F, medium A plus NAD+; G, medium A plus ATP, TPP H, medium A plus TPP. B-H, all incubations were at 27T for 30 min.

4. Disclrssioo The results obtained in this study indicate that temperature conditions of in vitro incubation of isolated maize mitochondria affect the energetic activity of these organelles oxidizing o-ketoglutarate. The mitochondrial activity decreased as the incubation temperature increased. This effect was most pronounced at 27°C and above. The addition of CCCP to the inactivated mitocondria failed to accelerate respiration. This suggests that the reduction in respiration as a result of mitochondrial incubation between 17 and 37°C is caused by a decrease in the rate of electron flow in the inactivated organelles. The reduction in the activity of isolated mitochondria during prolonged storage on ice is well known [9] and is accounted for by a progressive loss of NAD+ content [13]. Therefore, it would be reasonable that mitochondrial inactivation during incubation at 27°C is also related to NAD+ depletion. However, the results obtained in our experi-

T. Pobezhimova et al. /Plant Science 114 (19%)

Fig. 3. The effect of incubation conditions on mitochondrial activity. A, freshly isolated mitochondria, medium A plus TPP; B, 27’C, 30 mitt, medium A plus TPP; C, 27”C, 30 min, medium A plus TPP, then ATP was added and the activity was measured following an additional 30 mm incubation; D, 27”C, 30 mm, medium A plus TPP, then NAD+ was added and the activity was measured following 30 min incubation. S, substrate (a-ketoglutarate, 5 mM); M, mitochondtia (A-C, 0.6 mg each; D, 0.7 mg). The values on the right of the curves indicate the rate of oxygen uptake (nmol O#nin per mg protein). The arrows indicate the addition of ADP (200 pmol) and CCCP (1 PM), respectively.

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ments, the depletion of TPP in isolated maize mitochondria incubated at 27°C in vitro contributes to the drastic decline in oxidation of CYketoglutarate because of inactivation of the (Yketoglutarate dehydrogenase complex. During 27°C incubation, the ability of mitochondria to oxidize ar-ketoglutarate can be maintained by activating the dehydrogenase complex by the addition of TPP plus ATP to the incubation solution. It should be noted that TPP can activate the (Yketoglutarate dehydrogenase complex only in the presence of ATP, i.e. this process is energy dependent. The active transport of TPP into the mitochondria seems to demand energy expenses. This transport is either directly associated with ATP hydrolysis or provided with the energy of the transmembrane potential, which is generated by mitochondria at the expense of using ATP and the substrate of oxidation present in the incubation medium. Furthermore, it should be noted that the oxidation of cr-ketoglutarate in the presence of TPP by mitochondria incubated at 27°C for 30-60 min failed to attain the activity observed in freshly isolated organelles. This fact could suggest that the in vitro incubation of mitochondria at 27°C causes failure not only of the ol-ketoglutarate dehydrogenase complex but also of other structures involved in the oxidation of ar-ketoglutarate and in the transport of electrons entering the respiratory chain as a result of the oxidation of this substrate. Acknowledgements

ments failed to provide support for this suggestion, because the presence of NAD+ in the incubation solution failed to increase mitochondrial activity during incubation at 27°C. On the other hand, it is known that plant mitochondria readily oxidize ar-ketoglutarate when the reaction solution contains TPP [9] and, in contrast, the absence of TPP leads to the very slow oxidation of ar-ketoglutarate [9,14]. This is attributed to the fact that TPP is one of the cofactors of the ar-ketoglutarate dehydrogenase complex 1151.Highly purified preparations of this complex obtained from plant mitochondria require the addition of TPP for functioning [9]. In our experi-

This research was supported by the Russian Foundation of Basic Research (project 93X1421695). References VI P.W. Hochachka and G.N. Somero, Biochemical Adaptation. Princeton University Press, New Jersey, 1984.

121R.T. Nagao, J.A. Kimpel, E. Vierling and J.L. Key, The heat shock response: a comparative analysis, in: B.J. Miflin (Ed.), Oxford Surveys of Plant Molecular and Cell Biology, Oxford University Press, New York, 1986, pp. 384-483. I31 J.A. Kimpel, R.T. Nagao, V. Goekjian and J.L. Key, Regulation of the heat shock response in soybean seedlings. Plant Physiol., 94 (1990) 988-995.

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CRC Press, Boca Raton. FL, USA, 1991. T.P. Pobezhimova, N.N. Varakina and V.K. Vojnikov, Energetic activity of isolated mitochondria and respiration of intact cells of maize suspension culture under hyperthermia. Physiol. Biochem. Crop Plants (USSR), 22, 6 (1990) 537-542. T-Y. Lin and A.H. Markhart III, Temperature effects on mitochondrial respiration in Phaseolur aeutifolius A. gray and Phaseolus vulgaris L. Plant Physiol., 94 (1990) 54-58. N.N. Varakina, T.P. Pobezhimova and V.K. Voinikov, Effect of hyperthermia on energy-related activity of mitochondria and growth of maize seedlings. Soviet Plant Physiol., 38 (2) (1991) 304-311. M. Chou, Y-M. Chen and C-Y. Lin, Thermotolerance of isolated mitochondria associated with heat shock proteins. Plant Physiol., 89 (1989) 617-621. R. Deuce, Mitochondria in Higher Plants: Structure, Function, and Biogenesis, Academic Press, Orlando, San Diego, New York, London, Toronto, Monreal, Sydney, Tokyo, 1985.

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[IO] V.K. Voinikov, N.N. Varakina and T.P. Pobezhimova, Reconstruction of energy-producing activity in in vitro inactivated mitochondria from maize seedlings. Soviet Plant Physiol., 38 (3) (1991) 530-537. [II] O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randoll, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [12] R.W. Estabrook, Mitochondrial respiratory control and the polarographic measurement of ADPO ratio, in: R.W. Estabrook and M. Pullman (Eds.), Methods in Enzymology, Vol. IO, Academic Press, New York, London, 1967, pp. 41-47. [13] M. Neuburger and R. Douce, Slow passive diffusion of NAD between intact isolated mitochondria and suspending medium. Biochem. J., 216 (1983) 443-450. [14] E.P. Journet, W.D. Bonner and R. Deuce. Glutamate metabolism triggered by oxaloacetate in intact plant mitochondria. Arch. B&hem. Biophys., 214 (1982) 366375. 1151 M. Dixon and E.C. Webb, Enzymes. Longman Group Ltd., London, 1979.