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 ac- cumulation is directly coupled, it might be an alternative to oxidative phosphoryla- tion. 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 res- piration, 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 ac- cumulation rates reach maximum values within the first 40 min and continue un- diminished 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 accumula- tion 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 con- trol. 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 con- trols in KCl (Table 1, expt. 3). Thus if salt accumulation under anaerobic con- ditions 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 de- crease 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 phos- phorylation 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.