NOTE ON THE INCORPORATION OF ACETATE AND THE TCA CYCLE IN MYCORRHIZAL ROOTS OF BEECH

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1 New PhytoL (1968) 67, NOTE ON THE INCORPORATION OF ACETATE AND THE TCA CYCLE IN MYCORRHIZAL ROOTS OF BEECH BY B. B. CARRODUS AND J. L. HARLEY Universities of Melbourne and Sheffield {Received 26 January 1968) During the course of work upon the absorption of ammonium ions by mycorrhizal roots of beech, the progress of incorporation of [2-'*C]acetate was observed. The methods used were essentially similar to those of Harley and Beevers (1963). Samples of 1-4 g fresh weight of mycorrhizas were used. The extraction in 80 ethanol was PCA Succ. Mai. Cit. Phosphate Compound 1 r Water Fresh Amount Tube number Fig. I. Titratable acids in mycorrhizas. Tube numbers refer to samples obtained by gradient elution from Dowex AG i x io resin. 557

2 558 B. B. CARRODUS AND J. L. HARLEY Fig. 2. Incorporation of [2-''*C]acetate into total extract of mycorrbizas over a period of IO minutes.,.^mino acids; #, organic acids; x, neutral fraction. 60r Time (mmutes) Fig. 3. Incorporation of [2-'''C]acetate into non-volatile organic acids over a period of 3 hours. X, Citrate; :, succinate;, malate.

3 Acetate., TCA cycle and mycorrhiza 559 carried out at C to obviate the formation of 2,5-pyrrolidone carboxylic acid. The extract was brought to small volume under reduced pressure at 40 C and passed through Dowex AG 50W-X8 resin (hydrogen form) to remove cationic compounds and then through Dowex AG i x 10 (formate form) to remove anions. After washing, the cationic resin was eluted with N NH4OH and the eluate separated on paper in two dimensions, using «-butanol:acetic acid:water in the proportions 120:30: t;o (by volume) and saturated aqueous phenol:0.88 SG ammonia 200:1 (by volume) with a trace of KCN. The resin containing anionic compounds was subjected to gradient elution using zero-8 N formic acid. After evaporating the fractions to dryness, the acids were located by titration against ^ NaOH in a current of CO^-free air. The amounts of non-volatile organic acids were estimated in samples of 3-4 g of fresh mycorrhizas. It was confirmed that there was no significant change in their quantities over periods of up to 20 hours, when the mycorrhizas were kept in aerated water I 100 o u g 80 CL Time (minutes) Fig. 4. Changes in specific activities of acids over a period of 3 hours, x, Citrate;, malate., succinate; at 20' C (Fig. i). I'^CHjCOOH was fed to samples of i g of fresh mycorrhizas, in small Erlenmeyer flasks, at a concentration of 5 ^moles with 5 //Ci/20 ml. The temperature was 20^^ C. Fig. 2 gives the results of a study of the progress of incorporation of ' *C from acetate over a period of 10 minutes. It will be observed that the percentage labelling of the acid fraction can reasonably be assumed to extrapolate to ioo o at zero time. The radioactive labelling in the neutral fraction is low throughout but that in the basic (amino) fraction rises so as to constitute nearly 70% of the total label at 10 minutes. In samples allowed to absorb [2-' * ]acetate for longer periods up to 3 hours, about 70^0 of the total radioactivity was found in the amino fraction. Fig. 3 shows that after 30 minutes, citrate is the acid most heavily labelled and that succinate is slightly more heavily labelled than

4 560 B. B. CARRODUS AND J. L. HARLEY rnalate. This is irrespective of the fact that the sizes of the pools of these acids are in the diminishing order malate, citrate and succinate. The rise in radioactivity is then in the order expected for the operation of the TCA cycle irrespective of the pool sizes. Fig. 4 gives the calculated specific activities of these three acids from the experiment shown in Fig. 3. The values for citrate and succinate rise fairly steadily but there is a lag in the rise of specific activity in malate. These results give evidence of the probable operation of the TCA cycle in mycorrhizal roots of beech. Future work is aimed at determining the relative parts played by sheath and core in this process. REFFRENCE HARLEV, J. L. & BEEVERS, H. (1963). Acetate utilization by maize roots. PL Physiol., Lancaster, 38, 117.

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