THE METABOLISM OF [i,3-"c] ACETONE BY HIGHER PLANT TISSUES

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1 THE METABOLISM OF [i,3-"c] ACETONE BY HIGHER PLANT TISSUES BY E. A. COSSINS* Depart77ient of Biological Sciences, Purdue University, Lafayette, Indiana, U.S.A. [Received 23 April 1963) SUMMARY The metabolism of [i,3-i-'c] acetone by plant tissues has been examined. Under the experimental conditions used, '^C of meth}'! labelled acetone was rapidly incorporated into CO2, organic acids, amino acids, lipids and into the insoluble residue. In pea cotyledons and beet leaves, acetate and formate together contained the bulk of the radioactivity of the organic acid fraction. The possibility is discussed that acetone, accumulating during the early stages of germination in peas may be subsequently metabolized as seedling development takes place. INTRODUCTION Acetone has been detected among post-climacteric volatiles evolved by several species of ripening fruits (Biale, 1950; Ulrich, 1958). This ketone has also been found in relatively large amounts during the germination of pea seeds (Cossins and Turner, 19630). Although little is known of the pathway for acetone formation in plant tissues, Cossins (1962) reported labelling of acetone, following short periods of [2-"C] ethanol metabolism in pea cotyledon slices. Cossins and Turner (19636) suggested that as acetone and acetaldehyde appeared to be freely interconvertible in pea cotyledons, synthesis of acetone might occur via condensation of acetyl CoA involving acetoacetate as an intermediate. Millerd and Bonner (1954) have reported the formation of acetoacetate by homogenates of spinach leaves supplied with acetate and coenzyme A. Reports in the literature, indicate that in animal tissues, acetone is actively metabolized. Using intact rats, Price and Rittenburg (1950) have demonstrated a cleavage of acetone to form a 2-carbon fragment, which enters the 'acetyl' pool. Sakami (1950) has shown that the methyl groups of acetone may be used by rat tissues for the synthesis of the 3-carhon of serine and the methyl groups of methionine and choline. Using rat tissues, Rudney (1954) showed that propanediol phosphate was an intermediate in acetone metabolism. In these experiments all three carbon atoms of acetone were involved in carbohydrate synthesis. The possibility of acetone metabolism by higher plant tissues was discussed by Cossins and Turner (19630). In a later paper, Cossins and Turner (1963^) reported stimulations m carbon-dioxide output by pea cotyledon slices fed with dilute acetone solutions. The increased carbon-dioxide output was accompanied by increases in the acetaldehyde contents of the tissues. The possibility of acetone metabolism occurring in germinating pea cotyledons and m beet tissues has now been examined using dilute carrier-free [i,3-i4c] acetone solutions. * Present address: Department of Botany, University of Alberta, Edmonton, Canada. 28

2 Metabolism of acetone 29 MATERIALS AND METHODS Preparation of plant material Pea seeds {Pisum sativum) variety Alaska, were soaked for 18 hours in tap water at 20 C. The seeds were surface sterilized by rinsing in 0.1% mercuric chloride solution, followed by three washes in sterile distilled water and then placed between layers of moist filter paper in sterile dishes and germinated at 25" C in darkness. After 2 days and 5 days germination the testas were carefully removed and the cotyledons sliced as described earlier (Cossins and Turner, 1963^). Plumules, removed from 5-day-old pea seedlings, were cut into sections approximately i mm in thickness using a chilled razor blade. The leaves and roots of beet plants {Beta vidgaris) were prepared as follows. The lamina of fully expanded beet leaves was cut into sections 2 mm wide using a chilled razor blade. The leaf sections were kept in darkness before treating with [i,3-"c] acetone solutions. Cylinders of beet root tissue were removed using a 6 mm cork borer. Disks I mm thick were then cut from the cylinders, washed in distilled water and kept at 2 C before use. Eeeding experiments [i.s-'^^c] acetone supplied by New England Nuclear Corporation, Boston, Massachusetts was diluted with distilled water to give 70 pim of acetone per millilitre of solution. The methods employed in feeding [i,3-i4c] acetone to the tissues and extraction of the products of acetone metabolism were essentially those described by Cossins and Beevers (1963a). Slices, 0.5 g (fresh weight), of the plant materials were incubated with 7 JM of [i,3-^''c] acetone with a count rate of approximately 700,000 counts/min. The incubations were carried out in large Warburg flasks (125 ml) at 30 C in the dark. The only liquid in the flasks was o.i ml of acetone solution delivered from a micro syringe directly onto the plant material which just covered the bottoms of the flasks. Analytical methods Carbonate-free 20% (w/v) NaOH solution was added to the centre wells to absorb ^'*C02. The absorbed carbonate was converted to BaC03 and assayed for radioactivity on sintered porcelain planchettes with the use of a Mylar window continuous gas flow Geiger-Mliller tube. The counts were corrected for background and self-absorption. At the end of the experimental treatments, the tissues were killed by addition of 20 ml of an ice-cold 80% ethanol: concentrated ammonia mixture (50:1 v/v) and ground finely in a hand blender. After centrifugation the residue was washed successively with 10 ml of 50% ethanol and 10 ml of distilled water. The combined supernatants, referred to as 'ethanol solubles' in Tables i and 2, were then taken to dryness under vacuum at 40 C and the lipid material removed by washing the dried extract, twice, with 15 ml of anhydrous diethyl ether. Material not soluble in the ether washes, was dissolved in distilled water and separated into three fractions using ion exchange resins (Canvin and Beevers, 1961). The amino acids were separated from the organic acids and sugars by passing the water-soluble extract through a 6 x i cm column of Dowex AG 50W-X8 (hydrogen form). The organic acids and sugars passing through the column were collected in strong ammonia to prevent loss of acetate and formate by volatility.

3 3O E. A. CossiNS The amino acid fraction was eluted from the Dowex AG 50W-X8 column, using 50 ml of 2 N NH4OH solution. It was further fractionated into acidic amino acids, mainly aspartic and glutamic acids, and into the neutral and basic amino acids using Dowex i- Xio (acetate form) as described earlier (Cossins and Beevers, 19630). The organic acid and sugar fraction was separated by descending paper chromatography using basic solvent systems (Isherwood and Hanes, 1953; Kennedy and Barber, 1951). Acetate and formate were identified after steam distillation by co-chromatography with authentic acetate and formate. Autoradiography of the organic acid and sugar fraction was achieved using Kodak No-Screen X-ray film after stabilizing the volatile acids by spraying the dried chromatograms with 0.5 M K2HPO4 solution (Kennedy and Barber, 1951). In no case was radioactivity found in the sugars separated. The radioactive areas on the chromatograms were eluted using distilled water and aliquots of the eluates placed on metal planchettes to determine the amount of radioactivity present. Similarly, aliquots of the fractions recovered from the ion exchange resins were placed on metal planchettes, dried and counted After thorough drying at 100" C samples of the insoluble residues were combusted by the method of Stutz and Burris (1951), using the wet combustion reagents of Van Slyke and Folch (1940). The BaC03 so formed was assayed for radioactivity as described above. Table i. Metabolism of [1,2,-^^C] acetone by pea seedling tissues Plant tissues incubated at 30' C in air for 4 hours, radioactivity expressed as counts/min. 2-day-old cotyledon slices 5-day-old cotyledon slices 5-day-old plumule sections Fraction ",, of "C «;, of "C % of "C Counts/mm, incorporated Counts/min. incorporated Counts/min. inco"rporated Ethanol solubles Organic acids Acidic ammo acids j Neutral and basic ' amino acids iiooo 'f Not active ]^ l"^-,, ' Carbon dioxide li Total "C incorporated RESULTS Pea seedling tissues It is evident from Table i that the pea seedlings incorporated the "C of [i 3-14C] acetone mto a variety of compounds. In the tissues examined, approximately 16-21% of the total 14C incorporated was evolved as i^co.. The greatest incorporation of ^C occurred m the 2-day-old cotyledon shces. In these tissues, there was a striking incorporation mto the organic acids and into the insoluble residual material. The radioactivity of the hpid, acidic ammo acid, and neutral and basic amino acid fractions was considerably lower. In the extracts prepared from 5-day-old pea cotyledons incubated with [1,3-1^] acetone (1 able i), the neutral and basic amino acids contained 32% of the total "C incorporated by the tissues. The organic acids contained 22% of the incorporated ic with lower percentages being present in the other fractions separated In 5-day-old pea plumules, the [i,3-i4c] acetone supplied was converted mainly into the insolube residue and into UQO,. Although the organic acids and amino acid fractions contained ^^C, the lipid material was not radioactive.

4 Metabolism of acetone 31 Beet tissues After incubation with [i,3-^4(;;;] acetone for 6 hours at 30" C, ^'C was distributed throughout the fractions separated from beet tissues (Table 2). The greatest incorporation of i*c was shown by the leaf slices with large amounts of activity present in the organic acids. From both leaf and root tissues, ^''CO^ was evolved during the experimental period. In the root disks, the insoluble residue contained over 50% of the total incorporated. Formation of [^^C] formate and [^^C] acetate from [i,3-^''c] acetone by pea and beet tissues The organic acid fractions prepared from 2-day-old pea cotyledons, 5-day-old pea cotyledons and beet leaf slices were separated by paper chromatography as described above. In all cases P^C] acetate and [^''C] formate were present in the extracts (Table 3). In addition the pea cotyledon extracts contained labelled citrate and malate. P*C] citrate was, however, not detected in the beet leaf extracts but here there was a striking incorporation of ^''C into malate. Table 2. Metabolism of [i,3-'^*c] acetone by beet tissues Plant tissues incubated at 30'^' C in air for 6 hours, radioactivity expressed as counts/min. Fraction Ethanol Organic acids Acidic amino acids Neutral and basic amino acids Lipids Residue Carbon dioxide Total ^''C incorporated Beet leaf slices Beet root discs % of "C % of "C Counts/min. Counts/i incorporated Counts/min. incorporated I II OO Table 3. Distribution of ^"^C from [1,3-^^C] acetone in the organic acid fraction of pea and beet tissues Fraction Organic acids Citrate Malate Formate Acetate z S3 10 Radioactivity expressed as counts/min. Pea cotyledons* Pea cotyledons* Beet leaf slicesf 2-day-old 5-day-old (counts/min.) (counts/min.) (counts/min.) i Not active gooo * After incubation with [i,3-"c] acetone for 4 hours at 30 C in air. t After incubation with [i,3-^*c] acetone for 6 hours at 30 ' C m air. DISCUSSION The results clearly show that, under the experimental conditions [1,3-^^C] acetone was metabolized by the tissues investigated. The metabolism of methyl-labelled acetone was in all cases accompanied by evolution of "COa. This observation is m agreement with earlier experiments (Cossins and Turner, 1963&) where acetone feedmg to pea cotyledon slices was accompanied by stimulations in the rate of carbon-dioxide output. N.P. C

32 E. A. COSSINS Cossins and Turner (1963a) have reported changes in the concentrations of ethanol, acetaldehyde and acetone which accompany germination of pea seedlings.

5 32 E. A. COSSINS Cossins and Turner (1963a) have reported changes in the concentrations of ethanol, acetaldehyde and acetone which accompany germination of pea seedlings. After rising to a maximum after 24 hours germination, the acetone content fell to a low level after 48 hours. If this rapid decrease in acetone concentration was due to metabolism of acetone, then at this stage of germination, cotyledons supphed with [i,3-^''c] acetone should possess the ability to metabolize this compound even when supplied in only micromolar amounts. Direct evidence for the metabolism of acetone at this stage of germination in peas is given in Table i. Furthermore the ability to metabolize acetone was still apparent in the later stages of germination and seedling development in peas. BregoflF and Delwiche (1955) have reported on the importance of formate in betaine and choline synthesis by beet leaf disks. [^"^C] formate was incorporated into the methyl groups of betaine and choline in experiments carried out in the light or dark. It is therefore of interest to determine whether the methyl groups of acetone might similarly be involved in choline and betaine synthesis by these tissues. Although labelling in choline and betaine was not detected during the present experiments, the beet tissues showed a high rate of acetone metabolism, especially by the leaf slices. The large amounts of radioactivity in malate and acetate with no appreciable radioactivity in citrate remain unexplained but may reflect the relative pool sizes of these acids in the leaves investigated. The presence of [i^c] acetate and [i^'c] formate in the organic acid extracts might indicate a splitting of acetone into Ci and C2 fragments as occurs in animal tissues (Price and Rittenburg, 1950; Sakami, 1950). The labelling of the organic and amino acids in pea tissues is consistent with metabolism of the [i^c] acetate by established pathways. The possibility that the methyl groups of acetone might be involved in serine and methionine biosynthesis, which occurs when [i^c] methanol is fed to these tissues (Cossins and Beevers, 1962, 1963^) is being investigated in current experiments. ACKNOWLEDGMENTS This work was supported by National Science Foundation Grant G It is a pleasure to acknowledge the advice and interest of Professor Harry Beevers during the course of this work. REFERENCES M ('95o).Post-harvest physiology and biochemistry.of fruits. Amm. Rev. Plant PhysioL, i, 183. rnetssandpafhwiri.so/cs lastss COSSINS, E. A (1962). Utilization of ethanol-2-c" by pea slices. Nature, Lond., 104, ' ' ' ' ' ' ^ ^ ^ : T ^ ^ ^ t ' ' ' ^ ^ ' - ' ^ C " ' d h^^a^» by higher plant ' ' ^''''''^'''' /'''^\''^^*'" metabol.stn in plant tissues. Plant Phvswl., 38, 375. Ns' I \ & TURN'R E^'R? ^ f ^n?i"" ^ n^ethanol-ci^ n^ethalci^ in higher h h plant l tisiues. i In I ^r'ep'aration) ^'' 7p: Bot 142^0 ^"^^"^' n^etabohsm of ethanol in germinating pea seedlings. J. p^ St^ j^^^?i^s,^r A^&HANES, C. S. (1953). Separation and estimation of organicacids on paper chromatogratns. E^D'A & BoilrTv"- ^ ^1^''^- ^"P" <=hromatography of volatile acids. Analyt. Chem, 23, ^' ^ ^R'-'^-^'^H^*^"*'''''^^*"'^*' "'"plants. ^rc/t.bwc^e;n., 49, 343. "*, D. T. & RITTENBURG, D. (1950). The metabolism of acetone.^, btol. ChJii., isg, 449.

6 Metabolism of acetone 33 RuDNEY, H. (1954). Propanediol phosphate as a possible intermediate in the metabolism of acetone. J. biol. Cheni., 210, 361. SAKAMI, W. (1950). The formation of formate and labile methyl groups from acetone. J. biol. Chem., 187, 369- STUTZ, R. E. & BuRRls, R. H. (1951). Photosynthesis and metabolism of organic acids in higher plants. Plant Physiol., 26, 226. ULRICH.R. (1958). Postharvestphysiology of fruits. Annn.Rev. Plant PhvsioL,<), 385. VAN SLYKE, D. D. & FOLCH, J. (1940). Manometnc carbon determination. J. biol them., 136, 509.

Analytical Method for 2, 4, 5-T (Targeted to Agricultural, Animal and Fishery Products)

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