Effects of Cercospora beticola toxin on ATP level of maize roots and on the phosphorylating activity of isolated pea mitochondria

Plant Science Letters, 18 (1980) 207-214 o Elsevier/North-Holland Scientific Publishers Ltd.

207

EFFECTS OF CERCOSPORA BETKOLA TOXIN ON ATP LEVEL OF MAIZE ROOTS AND ON THE PHOSPHORYLATING ACTIVITY OF ISOLATED PEA MITOCHONDRIA

FRANCESCO MACRi, ANGELO VIANELLO, RAFFAELLA CERANA* and FRANCA RASI-CALDOGNO* Centro di Studio de1 CNR dei Problemi Fitopatologici della Barbabietola da Zucchero, Zstituto di Patoiogia Vegetale, Via Grade&o 6, Z-35100 Padova and *Centro di Studio de1 CNR per la Biologia Cellulare e Molecolare delle Piante, Zstituto di Scienze Botaniche, Via G. Colombo 60, Z-20133 Milan0 (Italy) (Received December 17th, 1979) (Accepted January 31st, 1980)

SUMMARY

The Cercospora beticola toxin (CBT), an inhibitor of membrane transport phenomena, did. not significantly affect ATP and pyruvate levels of maize roots at concentrations up to 40 pg/ml. In contrast, in isolated mitochondrial preparations from pea stem, CBT severely inhibited oxidative phosphorylation at concentrations higher than 10 lg/ml. The oligomycinsensitive ATPase activity of these mitochondrial preparations was completely inhibited by 10 pg/ml CBT. The results suggest that CBT fed to root segments at concentrations from 10 to 40 pg/ml does not influence the energy supply linked to oxidative phosphorylation, but it markedly inhibits mitochondrial functions in cell free systems and, in particular, mitochondrial ATPase and thus phosphorylation. The lack of effect of CBT on ATP and pyruvate levels of root segments is interpreted as being due to the lack of penetration of the toxin in the intact cells. These data are thus consistent with the view that the inhibiting action of CBT on cellular ion transport depends on its direct action on some systems located on the plasmalemma.

INTRODUCTION

Cercospom beticola toxin (CBT) is a phytotoxin Abbreviations:

lacking host-specificity

BSA, bovine mrum albumin; CRT, Cercoepom beticola toxin; DCCD, N,N’-dibyclohexykarb&iiimide; FC, fueicoccin, HEPES, N-2-HydroxyethylpipineN’-2-~thanesulfonic Acid; MES, (2[N-Morpholino] ethane eulfonic Acid); PD, electric potential;

208

produced by Cercospom beticolu Sacc. [l-3] and is endowed with a number of interesting biochemical effects. In fact, CBT is able to inhibit K+ uptake, H’ extrusion and to collapse the transmembrane electric potential (PO) of maize roots, and it also induces a specific inhibition of a plasmalemma K+-Mg*‘dependent ATPase in vitro [4 ] . In addition CBT inhibits the binding of fusicoccin (FC) to a component of a plasmalemma preparation of maize coleoptiles. The fraction that binds fusicoccin shows a high K*-Mg*‘-dependent ATPase activity [ 51. It is generally agreed that the measured PD of plant cells is due both to passive diffusion and energy-dependent electrogenic ion pumps [6] . Since the electrogenic component may be inhibited by various respiratory poisons [ 71, in this paper we examined the effects of CBT on ATP and pyruvate levels of maize roots, on basal and coupled electron transport rate and on phosphorylation of pea internode mitochondria. ATPase activity of pea internode mitochondria was also tested. MATERIALS

AND METHODS

Chemicals. CBT and oligomycin were dissolved in absolute ethanol to give stock solutions, and these were added to the incubation mixtures to give final toxin and oligomycin concentrations as specified in each experiment. The concentration of ethanol in the assays did not exceed 1%. Controls also contained the same amount of ethanol. Oligomycin, ADP and malate were purchased from Sigma Co., St. Louis, MO, U.S.A. Na2-ATP, NADP, glucose 6-phosphate dehydrogenase, hexokinase and lactic dehydrogenase were purchased from Boehringer GmbH, Mannheim, F.R.G. “Pi was purchased from Radiochemical Center, Amersham, England. Plant material. Pea (Pisum satiuum L. var. Alaska) and maize (Zeu mays L. cv. Dekalb XL 342) seeds were germinated and grown in the dark at 25°C for 5-6 days over aerated 0.5 mM CaC12. Oxygen uptake measurements. Etiolated pea internodes were ground in a mortar containing an ice-cold grinding medium composed of 20 mM HEPES, 0.4 M sucrose, 5 mM Na-EDTA, 0.1% BSA (pH 7.6) (1 : 3, w/v). The brei were strained through 8 layers of gauze and the filtrate was centrifuged at 28 000 g for 5 min. The pellet was re-suspended in one half of initial volume of grinding medium and centrifuged at 2 500 g for 3 min. The supematant was then centrifuged at 28 000 g for 5 min and the pelleted mitochondria were re-suspended in 2 ml of a solution of 0.4 M sucrose in 20 mM HEPES (pH 7.5). Oxygen uptake was monitored at 20°C with a platinum electrode assembly of the Clark type [ 81. Protein content was estimated by the biuret method

[91*

209

Asscryof ATPase activity and of phosphorylating activity of pea mitochondria. Pea internode mitochondria were prepared according to AbouKhalil and Hanson [lo], For ATPase activity assay the reaction medium contained 50 mM KCl, 1 mM MgCll, 1 mM Na2-ATP, 20 mM MES-Tris (pH 8.7) and 100 ~1 of diluted mitochondrial suspension (approx. 3 tig protein) in a final volume of 1 ml, CBT or oligomycin were added to the reaction mixture. The samples were pre-incubated with the inhibitors at 0°C for 10 min. The reaction was started by addition of ATP and carried out at 20°C for 30 and 60 min. Unincorporated 32Pi was removed from “Piprocedure [ 111, modified according to Cross et al, [ 12]- ATP formation by mitochondria was measured as 32Pi incorporation into glucose 6-phosphate in the presence of a hexokinase trap. The,reaction medium contained in a final volume of 1 ml: 0.4 M sucrose, 3 mM Na2-ADP, 3 mM MgCl2,5 mM KCl, 30 mM glucose, 0.035 mg hexokinase/ml, 10 mM KP04 buffer (pH 7.4) and “Pi (approx. 16 000 cpm/pmol Pi). The reaction was started by addition of the mitochondrial suspension (approx. 40 pg protein) and was carried out at 20°C for.30 and 60 min. Unincorporated ‘lpi was removed from 32Pi labelled glucose 6-phosphate by a two-phase separation (isobutanol-benzene and HC104) [ 131. The amount of ?‘Pi incorporated into 32Pi-labelled glucose 6-phosphate was determined by liquid scintillation spectroscopy. Measurement of ATP and pyruvate levels.Maize root segments, 5 mm long, were cut between 5 and 15 mm from the tip and washed in 0.5 mM CaS04 for 30 min. The segments (200 mg) were incubated in 5 ml of 0.5 mM CaS04 in the presence or absence of CBT for 1 h at 28°C under shaking (70 rev./ min). The tissue was homogenised in a mortar with 0.8 N perchloric acid at 0°C. The homogenate was centrifuged for 5 min at 28 000 g and the pellet washed with 0.8 N perchioric acid. The supernatant was neutral&d with KOH buffered with 120 mM triethanolamine-HCl/NaOH buffer at pH 7.6 and re-centrifuged. The enzymic assay of ATP and pyruvate was performed on the supematant according to Lamprecht and Trautschold [ 141 and to Biicher et al. [ 151, respectively. Measurement of ion leakage from pea root segments. Samples of 0.5 g of pea root segments were incubated in 10 ml of distilled water in the presence or absence of CBT (10 pg/ml) at 30°C with shaking. Ion leakage from the roots was measured as previously described [ 41. RESULTS AND DISCUSSION

The major effect of CBT on transport phenomena is the inhibition of electrogenic, energy-linked, H’ extrusion associated with K’ uptake [4]. CBT also inhibits a K’-Mg’+-activated, DCCD-sensitive, plasmalemma ATPase [4]. Since the activity of a K’/H’ electrogenic exchange system is inhibited if the availability of cellular energy is affected [ 161, we deter-

210 TABLE I EFFECT

OF CBT ON ATP AND PYRUVATE

Each value is a mean of three experiments. Values in nmol/g fresh wt.

Control CBT (10 rglml) CBT (40 pg/ml)

LEVELS

OF MAIZE ROOT SEGMENTS

Variation is expressed as standard deviation.

ATP

Pyruvate

122 ? 10.3 117 I 15.6 120 + 9.1

77.1 f 15.5 78.1 f. 14.9 72.2 + 13.2

mined the ATP and pyruvate contents in maize root segments treated with CBT. Table I shows that CBT had no significant effect on ATP and pyruvate levels of maize roots at the concentration from 10 to 40 pg/ml after 1 h of incubation. After the same incubation period and at 10 pg/ml of CBT concentration, K’ uptake in vivo and ATPase activity of plasmalemma-enriched preparations were inhibited to the extent of 40% and 25% respectively [4] . CBT also inhibits the K’-Mg”-dependent, DCCD-sensitive, ATPase activity of plasmalemma of several other higher plant sources, including pea [ 171. On the other hand, CBT, as previously reported for maize roots [ 4 ] , induced

25

-

I

1

I 30

60

Time

90

I

I 120

(min)

Fig. 1. Effect of CBT on ion leakage of pea roots. Samples of 0.5 g of pea root segments were incubated in 10 ml of CBT solution (10 rg/ml) at 30°C under shaking. Ion leakage was measured as increase of conductivity of the bathing solution. Control, (rl--rl ); CBT-treated, (-¤).

Fig. 2. Effect of CBT on electron transport and coupled electron transport of pea internode mitochondria. The figures next to each trace are expressed as natoms 0, . min-’ . mg protein-‘. The reaction medium contained 20 mM HEPES, 0.4 M sucrose, 5 mM Na-Kphosphate, 5 mM MgCl, and 1.5 mg of mitochondrial protein in a final volume of 2 ml. The substrate was 20 pmol of malate. State 3 rates were obtained by adding 200 nmol ADP. CBT was added during oxidative phosphorylation (trace A and B), or with the substrate (trace C), or 30 min before adding the substrate (trace E). Trace D represents the mitochondria pre-incubated for 30 min without CBT.

212

a rapid leakage of ions from pea roots (Fig. 1). Therefore, as a second means of investigating whether CBT may influence ATP synthesis, we have tested its effect on volume changes, oxygen uptake and phosphorylating activity of isolated pea internode mitochondria. CBT at a concentration up to 20 pg/ml did not affect the volume of mitochondria within a 15 min interval (data not shown). No early (l-2 min) detectable effect was found on coupled electron flow in the presence of CBT at 10 pg/ml concentration, with malate as a respiratory substrate (Fig 2, trace A). CBT at 40 pg/ml did not influence basal electron transfer (trace C), but inhibited phosphorylation-dependent oxygen uptake (trace B). Inhibition of oxidative phosphorylation was also observed at 10 pg/ml concentration of CBT when the mitochondrial preparation was pre-treated for 30 min with the toxin (trace D and E). The same pattern was obtained using succinate or NADH as substrates. These results, suggesting that CBT affects the mitochondrial phosphorylating system, are supported by the data of Table II showing that CBT at 10 and 40 pg/ml completely inhibited mitochondrial phosphorylation coupled to malate oxidation. The CBT ability of interacting with mitochondrial structures is confirmed by the effect of the toxin on the ATPase activity of mitochondria. In fact the data of Table III show that CBT markedly inhibited the oligomycinsensitive ATPase activity of mitochondrial preparations. Therefore, the inhibition of CBT on mitochondrial phosphorylation is likely dependent on its capacity to inhibit ATPase activity of mitochondrial preparations. Taken as a whole, the present results show that the effects of CBT on cell structures do not depend on its interference with the energy supply linked to oxidative phosphorylation, albeit the toxin interacts with isolated mitochondria. The lack of any detectable effect on ATP level and pyruvate level (the latter taken as an indicator of the glycolysis) of maize roots can be interpreted as due to the lack of penetration of the toxin into the cell [4] . These results are in agreement with the view that CBT primarily interacts with the plasmalemma, possibly by influencing plasmalemma ATPase and thus the TABLE II INHIBITION OF OXIDATIVE PHOSPHORYLATION IN ISOLATED MITOCHONDRIA INDUCED BY CBT AND OLIGOMYCIN

PEA

Data are expressed as Hmol of ‘*Pi incorporated into ATP per mg mitochondrial for 30 and 60 min. Means of two experiments are reported. Treatment

30 min

60 min

None Malate (25 mM) + Oligomycin (0.25 rg/ml) + CBT (10 fig/ml) +CBT (40 MS/ml)

0.04 6.17 0.06 0.04 0.05

0.04 9.34 0.05 0.04 0.05

protein

212 TABLE III EFFECT OF CBT ON OLIGOMYCIN-SENSITIVE MITGCHONDRIA

ATPase OF PEA INTERNODE

Data are the means of three experiments, Variability is expressed as standard deviation. One gram of fresh tissue yielded 70 rg of mitochondriai protein. ATPase activity (rmol Pi g fresh wt-’ l

Control CBT: 2 pg/ml 10 rg/mI 20 rglml 40 rglmi 80 rglml Oligomycin (0.1 pglml)

7.94 3.93 2.39 2.26 2.16 1.58 2.22

f 0.40 ?Z0.25 t 0.30 f 0.14 * 0.22 * 0.27 t 0.13

96 inhibition l

30 min“ )

50 70 72 73 80 72

electrogenic proton transport and related processes [ 4,5]. Circumstantial evidence that supports this hypothesis is provided by the finding that CBT inhibits the binding of FC, a phytotoxin which seems to interact with a K’-Mg”-activated, DCCD-sensitive, plasmalemma ATPase [ 51. ACKNOWLEDGMENTS

We thank Prof. E. Man-e for discussions and comments on the manuscript. We also thank Prof. L. Merlini and G. Nasini, Centro di Studio delle Sostanze Organiche Naturali, CNR, Milan, for a generous gift of CBT and D. Ferrara for technical assistance. REFERENCES 1 G. Assante, R. Locci, L. Camarda, L. Merlini and G. Nasini, Phytochem., 16 (1977) 243. 2 C. Baiis and M.G. Payne, Pbytopatb., 61(1971) 1477. 3 E. ScblSsser, Phytopath. Medit., 10 (1971) 154. 4 F. Macrf and A. Vianello, Physiol. Plant Path. 15 (1979) 101. 5 L. Tognoli, N. Beffagna, P. Pesci and E. Ma&, Plant. Sci. Lett., 16 (1979) 1. 6 N. Higinbotham, Annu. Rev. Plant. Physiol., 24 (1973) 25. 7 N. Higinbotbam, 5.8. Graves and R.F. Davis, J. Membrane Biol., 3 (1970) 210. 8 R.W. Estabrook, Methods Enzymol., 10 (1967) 41. 9 A.G. Gornall, C.J. Bardawill and M.M. David, J. Biol. Cbem., 177 (1949) 751. 10 S. Abou- Kbalil and J.B. Hanson, Arch. Biocbem. Biopbys., 183 (1977) 581. 11 C.H. Fiske and Y. Subbarow, J. Biol. Cbem., 66 (1925) 375. 12 J.W. Cross, W.R. Briggs, U.C. Dohrmann and P.M. Rayle, Plant Physiol., 61 (1978) 581. 13 E.C. Slater, Methods Enzymol. 10 (1967) 25. Bestimmung with Hexo14 W. Lamprecht and I. Trautscbold, Adenosin-5-tripbospbat kinase und Glucose+phoepbat debydrogenase, in H.U. Bergmeyer (Ed.), Methoden der Enzymatiscben Analyse, Verlag Cbemie, Weinbeim, 1970, p. 2024.

214 15 T. Biicher, R. Czock, W. Lamprecht and E. Latzko, Pyruvate, in U.H. Bergmeyer (Ed.), Methods in Enzymatic Analysis, Verlag Chemie, Weinheim - Academic Press, New York, San Francisco and London, 1963, p. 253. 16 E. Marre, Integration of solute transport in cereals, in D.L. Leidman and R.G. Wyn Jones (Eds.), Recent Advances in the Biochemistry of Cereals. Academic Press, London and New York, 1979, p. 3. 17 L: Tognoli and E. Marre, G. Bot. It. (1980), in press.