The ATP Content of Carrot Slices Decreased by 20-25

SALT ACCUMULATION AND ADENOSINE TRIPHOSPHATE IN CARROT XYLEM TISSUE BY M. R. ATKINSON,* GAIL ECKERMANN,* MARY GRANT, AND R. N. ROBERTSON

DEPARTMENTS OF AGRICULTURAL BIOCHEMISTRY AND OF BOTANY, THE UNIVERSITY OF ADELAIDE, SOUTH AUSTRALIA Communicated January 3, 1966 Accumulation of potassium or sodium chloride or other metal halides by washed slices of xylem parenchyma from carrot storage roots is associated with increased respiration-the salt respiration. Both salt accumulation and salt respiration are sensitive to inhibitors that block the energy-linked functions of mitochondria, e.g., carbon monoxide in the dark, cyanide, and 2,4-dinitrophenol (for reviews see Robertson' and Briggs, Hope, and Robertson2). It is not known if accumulation of salts by plant tissues is directly coupled to the electron and hydrogen transport of salt respiration or if it is dependent on the hydrolysis of adenosine triphosphate (ATP) formed by the salt respiration. It has been postulated1 that if salt accumulation is directly coupled, it might be an alternative to oxidative phosphorylation. This paper reports some experiments which might distinguish between these two possibilities. A method for analysis of ATP in small samples of carrot discs has now been developed, and the effects of metabolic inhibitors on salt accumulation, salt respiration, and ATP concentration have been studied. Experimental.-Carrot slices were prepared by cutting and washing with frequent changes of deionized water in the first few hours. Subsequently they were kept in aerated deionized water or chloromycetin' (50 ,ug/ml; for details see Table 1) changed daily for 7 days before use. Although the conditions of washing do not favor bacterial growth, it was found by direct counting and by dilution in nutrient agar that slices washed in water were contaminated with 107 to 108 bacteria/gm wet weight of tissue. Chloromycetin reduced the contamination to less than 1% of this number without altering the response to salt or changing the ATP content. Salt accumulation and salt respiration were measured, respectively, by conductivity methods and with Warburg respirometers as described previously.4 The ATP content of extracts was measured in a scintillation counter with luciferin-luciferase.5 Five internal standards and a blank were used for each assay, and the light emission was extrapolated to the time of mixing of sample and enzyme. In these conditions adenosine diphosphate equimolar with the ATP caused less than 0.1% interference. The analyses were confirmed in several cases by a method involving iso- tope dilution with [C'4] ATP and purification of the extracted nucleotide by elution from Dowex-1 with 0.25 N HCl, elution from Nuchar C with ethanol-0.5 N ammonia (2:1, v/v), chromatog- raphy in isobutyric acid-0.5 N ammonia (2: 1, v/v), and electrophoresis in N-tris(hydroxymethyl) aminomethane citrate, pH 4.8. For each assay 20 discs (1 mm X 8 mm) were weighed and threaded on nylon before washing; the set of discs (0.75-1.0 gm) was frozen in liquid nitrogen and ground in 9 ml of cold 0.4 N perchloric acid-0.1 mM Na2EDTA, and the centrifuged extract was brought to pH 7.3 with solid potassium bicarbonate, or, preferably, with 3 M potassium hy- droxide-0.1 M N-tris (hydroxymethyl)methyl-2-aminoethane sulphonic acid. After removal of potassium perchlorate, five 1-ml samples were used for each nucleotide assay. Results.-In the conditions of these experiments, salt respiration and salt accumulation rates reach maximum values within the first 40 min and continue undiminished for at least 5 hr.2 Respiration rates and accumulation rates of rep- licate sets of tissues are shown in Table 1. Despite the increased oxygen uptake due to salt (salt respiration), the ATP content of carrot slices decreased by 20-25 per cent within 15 min of exposure to 40 mM KCl and remained below the control 560 Downloaded by guest on September 29, 2021 VOL. 55, 1966 BIOCHEMISTRY: ATKINSON ET AL. 561

value for at least 4 hr (Table 1, expts. 1, 2, and 3). The decreased level of ATP might have resulted either from increased hydrolysis during salt accumulation or from decreased phosphorylation of adenosine diphosphate through interaction of KCl with systems that generate ATP in carrot slices, or from both. While meas- urement of the turnover of the terminal phosphate in ATP might permit a choice between these alternatives, preliminary experiments with [P32]orthophosphate indicate that the turnover is very rapid. Uncertainty about the specific activity of the cytoplasmic pool of orthophosphate during the first minutes of exposure to [P32]orthophosphate in the presence and absence of salt has prevented this com- parison of turnovers. It is relevant that Khan and Barker6 have observed decreases in phosphoenolpyruvate from 7.3 to 4.7 mgmoles/gm wet weight and in 3-phospho- glycerate from 18.4 to 12.5 mjumoles/gm wet weight on exposure to KCl of carrot slices that had been washed for 7 days. When slices in 40 mM KCl were transferred to anaerobic conditions, salt accumulation stopped within 3 min. The concentration of ATP fell, but even after 20 min was still 32 per cent of the aerobic control in water in one experiment (Table 1, expt. 1) and 54 per cent of the control in another (Table 1, expt. 6). Figure 1 shows the changes in rate of salt uptake and in ATP content when carrot slices in 40 mM KCl were transferred from air to nitrogen. Salt uptake had ceased within a few minutes, but after 30 min the ATP content was still about a third of that in the aerobic control. On addition of 4 1A\ mesoxalonitrile 3-chlorophenylhydrazone ("m-chloro car- bonylcyanide phenylhydrazone," CCP) to tissue in 40 mM KCl, salt accumulation stopped almost completely within 1 min, but even after 30 min exposure to this effective uncoupler of energy-linked reactions of mitochondria7 the ATP content of the tissue was 55 per cent of that of controls in water and 69 per cent of that of controls in KCl (Table 1, expt. 3). Thus if salt accumulation under anaerobic conditions or in the presence of CCP had stopped through depletion of the supply of ATP to a metabolic "pump," much of the ATP (one third to two thirds) must have been unavailable to the pump. Table 1 also shows the effect of arsenite, and iodoacetamide, on the ATP content of carrot slices; both inhibit salt accumulation and respiration, but again the decrease in ATP content was only about 50 per cent. Thus there is no evidence that uptake had ceased through complete depletion of ATP in the tissue. Alternatively these inhibitors of salt accumulation could have interfered with reactions dependent on transfer of electrons from substrates to oxygen. The results reported so far would be consistent with a requirement for ATP in salt accumulation if only that nucleotide generated by mitochondrial oxidative phosphorylation could be used for this process. Oligomycin inhibits rephosphorylation of the adenosine diphosphate that enters mitochondria. At a concentration of 6 gg/ml (100 ,ug/gm fresh weight of slices) oligomycin caused a 21 per cent decrease in ATP content of slices within 30 min (Table 1, expt. 7). In these conditions salt accumulation continued undiminished for at least 30 min and sometimes for an hour; longer exposures caused a gradual decrease in rate to about a half after 2 hr. Absence of inhibition by oligomycin of salt accumulation in conditions where this antimetabolite decreases the ATP content of the slices is evidence against an involvement of mitochondrial ATP. Downloaded by guest on September 29, 2021 562 BIOCHEMISTRY: ATKINSON ET AL. PROC. N. A. S.

TABLE 1 EFFECTS OF INHIBITORS AND ANAEROBIC CONDITIONS ON SALT ACCUMULATION, RESPIRATION, AND ADENOSINE TRIPHOSPHATE CONTENT OF CARROT XYLEM SLICES Respiration KCI rate* ATP Content Accumulation Rate* (pmoles mpuMoles/ Percent pMoles/ Percent Expt. Treatment 02/gm/hr) gmt of control gm/hr of control 1 Water 3.2 25 4 1 100 40 mM KCl (20 min) 4.3 20 (23, 17) 80 3.4 100 N2 atmosphere (20 min) 0 19 ± 1 76 40 mM KCl-N2 atmosphere (20 0 8 41 1 32 0 0 min) 2 Water 1.3 20 i 2 100 40 mM KCl (15 min) 2.4 15 i 3 75 3.4 100 1 mM Sodium arsenite-40 mM 0.7 10 (11, 9) 50 0 0 KCl (15 min) 1 mM Sodium arsenite 1.0 3T Water 3.2 40 ± 3 100 40mMKCl(4hr) 4.9 32±2 80 4.4 100 44 ,M CCP (30 min) 7.0 26 ± 2 65 40mMKCl(4hr)-4uMMCCP 8.6 22±2 55 0.2 5 (30 min) 4 Water 2.4 40 mM KCl 3.5 3.3 100 0.1% Ethanol (1.5 hr) 24 ± 1 100 6 ;&g Oligomycin/ml of 0.1% 17 ± 1 71 ethanol (1.5 hr) 5 mM Iodoacetamide-0.1% 0.9 12 ± 1 50 ethanol (1.5 hr) 6 ,.g Oligomycin/ml of 0.1% 0.7 9 (9, 9) 37 ethanol-5 mM iodoacetamide (1.5 hr) 40 mM KCl-6 ,ug oligomycin/ 0.5 0.2 6 ml of 0.1% ethanol-5 mM iodoacetamide 40 mM KCl-5 mM iodoaceta- 0.6 0.5 15 mide-0.1% ethanol 5 Water 2.6 25 (24, 25.5) 100 40 mM KCl 4.5 3.1 100 0.1% Ethanol (1 hr) 3.4 28 42 112 6 gg Oligomycin/ml of 0.1% 3.4 18 ± 1 72 ethanol (1 hr) 40 mM KCl-0.1 % ethanol 4.7 2.9 94 40 mM KCl-6 ,ug oligomycin/ 3.7 1.7 55 ml of 0.1% ethanol 6t Water 2.6 41±1 100 40 mM KCl(1 hr) 4.5 3.3 100 0.1%Ethanol (1 hr) 3.4 47 ± 2 115 6 ,ug Oligomycin/ml of 0.1 % 4.2 38 ± 1 93 ethanol (1 hr) 40 mM KCl-N2 atmosphere 22 ± 2 54 0 0 (20 min) 40 mM KCl-6 jg oligomycin/ 5.4 ml of 0.1% ethanol 40 mM KCl-0.1% ethanol 4.9 7t 0.1% Ethanol (30 min) 19 ± 1 100 40 mM KCl (1 hr)-0.1% etha- 16 ± 1 84 nol (30 min) 6 ,ug Oligomycin/ml of 0.1% 15 ± 1 79 ethanol (30 min) 40 mM KCl (1 hr)-6 ug oligo- 12 ± 1 63 mycin/ml of 0.1% ethanol (30 min) * The steady rates maintained after the initial uptake period,2 except where iodoacetamide was added and the values were taken 90 min after the addition. e Mean 4i S.E.M. Slices washed in chloromycetin (50 pg/ml). Downloaded by guest on September 29, 2021 VOL. 55, 1966 BIOCHEMISTRY: ATKINSON ET AL. 563

Discussion.-The results reported in this * l paper suggest some deductions about the re- lations between electron transport, oxidative N2 phosphorylation, and ion transport or accumu- 12 lation in this tissue. In these experiments uncouplers or respira- 25 1 10 tory-chain inhibitors which prevented salt accumulation also decreased the ATP content of @ 0 8 20 the tissue, but at least a third remained. This 0 0 0 8 observation indicates that the salt accumula- E * 0 tion had not stopped due to lack of ATP unless . 6 E we postulate that this residual nucleotide was : unavailable to, or unconnected with, the ion X 4o transport mechanism. The alternative is a E act specially close connection between the ion trans- 0 port mechanism and either the electron trans-_ port chain itself or its coupled oxidative ATP formation. This alternative was suggested by 0 60 9 120 earlier work on inhibitors (particularly the light- Time in KCI (min.) reversible inhibition by carbon monoxide) and FIG. 1.-ATP content (I--- or 1 -o-) and salt uptake (-A- or uncouplers (particularly 2,4-dinitrophenol), -A-) at various times after exposure but it had not been possible to distinguish be- of carrot slices to 40 mM KC1. At the on time indicated, one set of slices(- , tween dependence the electron transport *-) was stirred with oxygen-free system and dependence on the coupled for- nitrogen. mation of ATP. The decrease in ATP in the presence of KCl could be due either to an increased rate of utilization if the salt accumulation were dependent on ATP, or to an uncoupling effect of salt so that ATP formation decreased despite the increase in oxygen uptake. The second of these two possibilities would be consistent with the earlier postulate that ion transport and phosphorylation both depend on the electron transport chain, are both inhibited by uncouplers, but are alternative consequences of electron transport. Oligomycin is known to inhibit production of ATP in isolated plant mitochondria without inhibiting the alternative ion transport mechanisms and provides a means of distinguishing between dependence on the electron transport chain and dependence on ATP formed by oxidative phosphorylation. In these experiments, oligomycin decreased the ATP content of the tissue within 30 min, while salt accumulation continued undiminished for nearly an hour. Since the amount of salt accumulated was 3-4 ttmoles/gm of tissue/hr and the decrease of ATP content was only 10 mgmoles/gm of tissue, it is unlikely that the continued accumulation was due to transport coupled with hydrolysis of preformed ATP. The simplest explana- tion is that, as in isolated mitochondria, ion transport is dependent on electron transport but not on oxidative phosphorylation. The original hypothesis that the salt transport mechanism depended on separation of hydrogen ions and electrons in the electron transport system was based on the stoichiometry of the two processes9; the number of hydrogen ions and electrons, calculated from the salt respiration, was approximately equal to the number of ions accumulated. In the present experiments the ratio (jsmoles of KCl accumu- Downloaded by guest on September 29, 2021 564 BIOCHEMISTRY: KANEKO AND DOI PROC. N. A. S.

lated)/(,Mmoles oxygen uptake in salt respiration) ranges from 1.7 to 3.1 and is within the requirements of the hypothesis that the maximum possible would be 4. In addition, the two observations (1) that salt reduces the ATP level, probably by preventing its oxidative formation, and (2) that oligomycin prevents ATP formation without inhibiting ion accumulation are also consistent with the hypothesis that salt accumulation and oxidative phosphorylation are alternative consequences of the charge separation of electron transport. Summary.-The effects of anaerobic conditions and of inhibitors (sodium arsenite, iodoacetamide, mesoxalonitrile 3-chlorophenylhydrazone, and oligomycin) on ATP content, respiration rate, and salt accumulation rate of carrot tissue weie investi- gated. The best hypothesis to explain the observations is that the ion transport mechanism in this tissue is directly coupled to the electron transport system, does not require the intervention of ATP, and may be an alternative to ATP formation.

Help with microbiological tests by Dr. A. Rovira and information from 1)r. N. Good on the advantages of N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid as a buffer are grate- fully acknowledged. * Present address: School of Biological Sciences, Flinders University of South Australia, Bed- ford Park, South Australia. I Robertson, R. N., Biol. Rev., 35, 231 (1960). 2 Briggs, G. E., A. B. Hope, and R. N. Robertson, in Electrolytes and Plant Cells (Oxford: Blackwell Scientific Publications, 1961), p. 152. 3 Leaver, C. J., and J. Edelman, Nature, 207, 1000 (1965). 4Robertson, R. N., and J. S. Turner, Australian J. Exptl. Biol. Med. Sci., 23, 63 (1945). 5 Strehler, B. L., and J. R. Totter, in Methods of Biochemical Analysis, ed. D. Glick (New York: Interscience Publishers, 1954), vol. 1, p. 341. 6 Khan, M. A. A., and J. Barker, unpublished results. 7Heytler, P. G., Biochemistry, 2, 357 (1963). 8 Millard, D. L., J. T. Wiskich, and R. N. Robertson, these PROCEEDINGS, 52, 996 (1964). 9 Robertson, R. N., and M. J. Wilkins, Australian J. Sci. Res. B., 1, 17 (1948).

ALTERATION OF VALYL-SRNA DURING SPORULATION OF BACILLUS SUBTILIS* BY ICHIRO KANEKO AND RoY H. Doi DEPARTMENT OF BIOCHEMISTRY AND BIOPHYSICS, UNIVERSITY OF CALIFORNIA (DAVIS) Communicated by G. Ledyard Stebbins, January 5, 1966 During sporulation of Bacillus subtilis an active, vegetative cell is converted to a dormant spore by a complex series of biochemical events.1 This is a case of unicellu- lar morphogenesis in which differential expression of the genome is evident.2 3 The control factors involved in these events are still unknown. In an analysis of the RNA of vegetative cells and spores, it was observed that the sRNA from these two forms had different elution patterns from a M\fAK coluMnl.4 The role of sRNA in protein synthesis is well established;5 more recently sRNA has been proposed as having a regulatory function in the translation of messenger RNA.6 7 Differential synthesis of proteins, as during morphogenesis, may depend on the functional con- Downloaded by guest on September 29, 2021