Salt accumulation and adenosine triphosphate in carrot xylem tissue.

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 isotope 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), chromatography 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 hydroxide-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 replicate 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