STUDIES IN THE RESPIRATORY AND CARBOHYDRATE METABOLISM OF PLANT TISSUES

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1 New Phytl. (968) 67, STUDIES IN THE RESPIRATORY AND CARBOHYDRATE METABOLISM OF PLANT TISSUES V. CHANGES IN RATE OF CO^ OUTPUT AND IN CONTENT OF VARIOUS PHOSPHATE COMPOUNDS IN POTATOES BY J. BARKER The Btanv Schl, University f Cambridge (Received 6 Nvember 967) SUMMARY The changes f varius phsphate cmpunds were determined during the increase in rate f CO2 utput and f sugar cntent which ccurred in lwsugar ptates at C. The cntents f phsphate cmpunds were als estimated in high and lwsugar ptates at C. The increase in rate f CO2 utput assciated with higher sugar cntent culd nt be attributed whlly t an increase in the amunts f the hexse mn and diphsphates; pssible explanatins are given. The changes bserved in glucsephsphate d nt affrd any explanatin f the mechanism f sweetening and desweetening in ptates. In different metablic data the qutient, glucsephsphate/glucse6phsphate, remained clse t that fr phsphglucmutase in vitr. INTRODUCTION Under certain cnditins the rate f CO, utput f ptat tubers varies directly with the sugar cntent and this relatinship was ascribed t changes in cncentratin f the respirable substrate (Hanes and Barker, 93; Barker, 933, 936). In the present wrk the changes in certain sugar phsphates and glyclytic intermediates have been determined under cnditins in which the rate f CO 2 utput changed markedly. But n cnsistent relatin between rate f CO 2 utput and the cntent f sugar phsphate substrates was bserved. The cnversin f starch int sugar in plant tissues held at lw temperatures is well recgnized but the mechanism f the sweetening prcess is nt knwn (MullerThurgau, 882; ArreguinLzan and Bnner, 949; Samtus, i96; Barker, 965). Hithert the determinatin f the changes in glucsephsphate in plant tissues has prved difficult and it was hped that data fr the alteratins f this ester might help t elucidate the mechanisms invlved. METHODS The methds used fr Experiments i, 2, 4 and 5 were thse f Barker et al. (964b) and Barker and Yunis (965). Fr Experiment 3 the extract was purified by a technique mdified frm that f Isherwd and Barrett (967) befre determining the sugar phsphates, glyclytic intermediates and adensine nucletides: since the latter were estimated by an enzymic methd, their values may include nucletides f ther bases. 495

2 496 J. BARKER EPERIMENTAL RESULTS Experiment i {February 9 6 3). Lwsugar ptates {stck A) transferred frm t i C at days (Fig. i) CO2 utpttt (Fig. I a). Fllwing the preliminary decrease in rate at T' C the CO2 utput changed little fr sme 4 days but then increased t abut three times the initial rate at i' C. This frm f curve fr CO, utput is characteristic f the transfer f lwsugar ptates frm t i" C (Barker, 933). J5 _ a) 6" '.OP p '. p'. ^P ^ x^_ PO (e) p. ' ".. O I _ : _.: 6 ( b) p' 8. 6" p_ p (f) ". 8, V e." _ 5 P ;P (c ), P. " p (g) : O bp., * x fi 5 25 )5 O p.. '.. ".p ". Q O P" x^. \ p ^ 8 O... fl 5 (h) 2 Days 5 Fig. I. (Experiment i.) Changes in ptates at ' C ( x ) and at i' C (O) in (a) rate f CO, utput and (bh) cntents; (b) G6P, (c) F6P, (d)gip, (e) FDP, (f) DHAP, (g) 3PHA and(h) PEP. In this and in Fig. 2 the rate f CO, utput is stated as mg/i g fresh weight/hur; in Figs. 4 the cntents are gi\en as //mles/i g fresh weight and the change frm t C was at days. 2 Sugar cntent. As in the earlier wrk glucse, fructse and sucrse each increased at I C and the relatin between rate f CO 2 utput and sucrse cntent was nearly linear thrugh the rigin with lw sugar cntent (Barker, 933, 936).

3 CO2 utptit and changes in phsphates 497 Sugar phsphates and dihydrxyacetnephsphate {DHAP) (Fig. i, bf). The transfer t i" C prduced a large initial increase fllwed by a decrease in the cntents f glucse 6phsphate (G6P) and fructse6phsphate (F6P) and smaller changes f glucseiphsphate (GiP). Neither fructsei, 6diphsphate (FDP) nr DHAP increased initially at i^ C. I (a) r (e) _ (f) c _. 8 : (d) 5 5 (h) CD 5 2 Dys 2 Fig. 2. (Experiment 2.) Changes in ptates at i ' C ( x ) and at i' C { ) in (a) rate utput and (bh) cntents; (b) G6P, (c) F6P, (d) GiP, (e) FDP, (f) DH.\P, (g) and (h) PEP. f CO. After 7 days at T C G6P had decreased nearly t the i C level but then increased thrughut the experiment. FDP and DHAP als increased after sme 7 days at i" C. Other glyclytic intermediates (Fig. i, g and h). Fllwing the transitin frm t

4 498 J. BARKER C the cntents f 3pbsphglycerate (3PGA), 2phspbglycerate (2PGA) and phsphenlpyruvate (PEP) each decreased t a lw value but subsequently changed little. Cmment. Large changes in the cntents f G6P and F6P thus ccurred initially at i' C withut change in the rate f CO 2 utput. The bexse mnphsphates always changed in this manner at i C in ur wrk and the reactin, which is cnsidered elsewhere (Barker, 968), appears nt t have been reprted previusly. (a) (d) 3 ;.8 2 _ ~.8.'. 5 ' p ' _ (e). ". ' (b) (f) " 25 y _ '?,.' t y 5 5 (c (g) L_ Days 8 ' 4 Fig. 3. (Experiment 3.) Changes in ptates at ' C ( x ) and at " C (O) in cntents (a), (cg); and in percentage change in GP/G6P (b); (a) G6P, (c) GiP, (d) FDP, (e) DHAP, (f) 3PGA and (g) PEP. 5

5 CO2 utput and changes in phsphates 499 During the large rise f CO2 utput frm 5 days nwards at i C G6P, FDP and DHAP each appeared t increase in cntent. Experiment 2 {May 963). Lwsugar ptates {stck A) trayisferred frm t i C at days (Fig. 2) CO2 utput (Fig. 2a). The CO, utput changed as in Experiment i. Sugar phsphates and DHAP (Fig. 2, bf). Again G6P and F6P first increased and then decreased at C but in this experiment FDP and DHAP als changed similarly. There was little alteratin in GiP. I (a) (c) 5.8" (b) (d) '.D 5 8 ' 4 4 Days I I I Fig. 4. (Experiment 3.) Changes in ptates at ^ C ( x ) and at i" C () (a) ATP, (h) ADP, (c) AMP and (d) ttal adensine nucletide. in cntents; After 7 days at C G6P increased until abut days, while FDP appeared t rise nly between 4 and 22 days. Other glyclytic intermediates (Fig. 2, g and h). Bth 3PGA and PEP again decreased markedly initially; subsequently PEP fell slwly. Experiment 3 {January 966). Lwsugar ptates {stck B) transferred frm t C at days (Figs. 3 and 4) The transfer t C prduced the usual initial increase fllwed by a decrease in

6 5OO J. BARKER G6P tgetber with falls in 3P(iA and PEP. FDP increased greatly during the first 5 days prir t a decrease fllwed by anther increase. Adensine triphsphate (ATP) had already increased after 2 days at i^ C and the rise cntinued thrughut the experiment. Adensine diphsphate (ADP) may have decreased initially but rse frm 5 days nward. There was a large increase in the ttal adensine nucletide at i" C. Experiments 4 {January 962) and 5 {Marcii 963). Highsugar pcjtates {stcks B and C) transferred frm i t C and lzvsugar ptates held at C (Table i) Ptates f high sugar cntent were transferred frm i t C, at which temperature the initial rate f CO, utput was high in relatin t that fr cmparable lw sugar ptates at C (Table i). There was n difference in cntent f G6P in high and lwsugar ptates at C (Table i) but there appeared t be sme difference in FDP. Table i. Rates f CO 2 utput and cntents f G6P, FDP and sucrse in high and lwsugar ptates at C as /.iinlesji g fresh weight Experiment 4 5 SuKur Higb Lw High Lw CO2 utput hur G6P FDP SucrO! 6 3 DISCI'SSION Changes in rate f CO2 utput and in cntent f sugar phsphates In lwsugar ptates transferred frm t i C bth the rate f CO, utput and the sugar cntent increase in time curves f characteristic frm. The cnclusin frm the earlier wrk was that the increase in sugar cntent increased the respiratin because f a rise in cncentratin f respirable substrate (Barker, 9^3, 936). The present data are, I believe, the first accurate results t be published fr the changes in phsphate cmpunds during the characteristic rises in rate f CO2 utput and in cntent f sugar at i' C. Initially at i C there were large increases fllwed by decreases in the cntents f G6P and F6P; in Experiment 2, but nt in Experiment i, these changes w ere accmpanied by rises and falls f FDP and DHAP. In Experiment 3 FDP increased initially fr 5 days but the change in DHAP was small. Nevertheless the rate f CO, utput remained virtually unchanged during these large alteratins f sugar phsphates and f DHAP. Pssible explanatins f these data are cnsidered elsewhere (Barker, 968). After 4 r 5 days at r C the rate f CO, utput increased by abut threefld in sme 5 days and in Experiment i this rise was accmpanied frm 7 days by prgressive increases in the hexse mn and diphsphates; the rises in phsphate cmpunds were, hwever, relatively smaller than the increase in CO, utput. Mrever, in Experiment 2, thugh bth G6P and FDP were higher after 22 than after 7 days, the sugar phsphates did nt increase cntinuusly thrughut this perid as did the CO, utput; the rises f the phsphate cmpunds w ere again relatively smaller than that f the CO, utput. Althugh there was a large increase f FDP at i C in Experiment 2, the cntent may have decreased between 5 and 9 days in cntrast with the rise in CO2 utput during this perid.

7 CO2 utput and changes in phsphates In Experiments 4 and 5 at " C the CO 2 utput f sweet ptates was sme fur times that f the lwsugar ptates. Yet the cntents f G6P f lw and highsugar ptates were similar, althugh the amunt f FDP appeared t be smewhat higher in the sweet ptates (Table i). Thus neither at i' C nr at " C culd the large changes in rate f respiratin be adequately explained in terms f changes f the ttal cellular cntents f the respirable substrates, G6P and FDP. The data fr the changes after 5 days at i C are thus similar t thse fr the initial perid at i' C (Barker, 968) in shwing a lack f crrelatin between changes in respiratin and alteratins in the cntents f the phsphate esters. One pssible explanatin was that the cncentratin f sugar phsphates near the respirable enzymes was small cmpared with the ttal cellular cntent. I thus visualize that the cntents f G6P and FDP near the respirable enzymes did in fact increase markedly between 5 and 2 days at C, as did the CO 2 utput, but that these changes in the phsphate esters were nly partly reflected in changes f the ttal cntent f these cmpunds. This interpretatin thus invlves the cncept f 'lcalizatin' f substrate r enzyme currently invked in certain theries f the mechanism f the Pasteur effect (Lynen et al., 959; Lynen, 963; Barker, Khan and Slms, 964a). An alternative explanatin was that the cntents f G6P and FDP near the respirable enzymes were already f saturating cncentratin in lwsugar ptates and that the increases in rate f CO 2 utput were pssibly due t the increase in sugar affecting the rate f respiratin thrugh changes in the cncentratin f adensine di and triphsphates. But in Experiment 3 the cntent f ATP had already increased after 2 days at i" C and this rise cntinued thrughut the experiment. The cntent f ADP either decreased r remained unchanged initially at i" C but increased after 5 days. These determinatins thus prvide n evidence that the increase in CO> utput was related t a decrease f ATP as ccurred in Chlrella (Syrett, 958). If the adensine nucletides were, in fact, cntrlling the rate f respiratin, ne pssibility is that the cntrl was due t a decrease in the amunt f ATP near the respiratry centres and this alteratin must have ccurred withut a crrespnding change in the ttal cellular cntent f ATP. Anther pssibility is that adensine triphsphate culd increase the rate f respiratin thrugh its influence as a substrate in the hexkinase and phsphfructkinase reactins. Thus frm abut 5 days nwards at i C the bserved increase f ATP culd hasten the reactins catalysed by these enzymes and s accelerate respiratin (Fig. 3). But the cntent f ATP appeared t have increased substantially during the flrst 2 days at i' C withut a rise f respiratin. The accumulatin f lactate in anxia and f citrate in xygenpisning indicate that glyclysis ccurs in ptat tubers (Barker and Saifl, 952; Barker and Mapsn, 955). If, then, the rate f glyclysis increases during the rise f CO, utput in ptates at I C, pyruvate shuld accumulate. The chrmatgraphic determinatin f pyruvate shwed, hwever, n increase f pyruvate under these cnditins (Barker and Mapsn, '955) The mechanisms f sweetening and desweetening Sugar begins t accumulate in ptates after 24 days at i C but it is nt knwn whether the prcess is hydrlytic, invlving amylase r phsphrylytic, invlving starch phsphrylase (Barker, 965). Starch phsphrylase appears nt t increase in cntent in ptates held at lw temperatures (ArreguinLzan and Bnner, 949) but the phsphrylase reactin shuld be accelerated if either inrganic phsphate increased t;i

52 J. BARKER r GiP decreased. Initially at i' C GiP increased either markedly (Figs, i and 3) r slightly (Fig. 2) but the change appeared t fllw that f G6P. Thus the qutient GP/G6P was 7.

8 52 J. BARKER r GiP decreased. Initially at i' C GiP increased either markedly (Figs, i and 3) r slightly (Fig. 2) but the change appeared t fllw that f G6P. Thus the qutient GP/G6P was 7.5% at C and 3.5% initially at i" C in Experiment i and 4.% at C and i.5" 3t i" C in Experiment 2. Subsequently at i" C GiP remained abve the C value (Figs, i and 3) r decreased belw the ' C amunt (Fig. 2). Thus while the phsphrylytic breakdwn f starch might be accelerated by the decrease in GiP at I C in Experiment 2, the reverse was the case fr Experiments i and 3. Excluding xygen prevents sweetening in ptates at T" C (Barker, 965) and this might be explained if sucrse was frmed directly frm starch via GiP and uridine diphsphglucse; the cnversin f starch t sugar might then be inhibited in anxia thrugh a lack f adensine and uridine triphsphates resulting in failure t frm uridine diphsphglucse frm GiP. If, then, the phsphrylytic breakdwn f starch t GiP cntinued in anxia, GiP shuld accumulate. In three experiments at i C cnducted t test these pssibilities, GiP accumulated in anxia in ne experiment but did nt change in the ther tw experiments. Sugar decreases in sweet ptates transferred frm i t " C and this change is inhibited in anxia, pssibly because f a decrease in GiP caused by a shrtage f ATP (Barker, 965). But the cntent f GiP was unchanged in anxia in ne experiment and decreased nly a little in a secnd. The cnversin f sugar t starch by starch phsphrylase r uridine transglycsylase in highsugar ptates at " C shuld be facilitated by high cntent f GiP. But in several experiments the cntents f GiP in ptates desweetening rapidly at C was f the same rder as that in lwsugar ptates at ^^ C. A feature f the data under cnsideratin is that the qutient GP/G6P changed little in ptates in different metablic states. A pssible explanatin is that the cntent f phsphglucmutase is sufficiently high near the chief pl f G6P in the cell t maintain the GP/G6P qutient near the equilibrium value. Thus the increases and decreases f GiP, which might be expected t ccur in ptates in the varius metablic states cnsidered abve, wuld be cnfined t small regins f the cell and s wuld nt be recrded in determinatins f the ttal cellular cntents f the hexse mnphsphates. ACKNOWLEDGMENTS This wrk was cnducted partly at the Btany Schl with the aid f a grant fr a special research awarded by the Department f Scientific and Industrial Research and partly at the Lw Temperature Research Statin, with facilities prvided by the Directr, Dr E. C. BateSmith. These facilities are gratefully acknwledged, as is als the interest and encuragement shwn by the Agricultural Research Cuncil. I wish t thank Dr T. Slms fr advice and Mr M. Askham, Mr P. Curtis, Mr R. Jakes and Mr B. Rystn fr their technical assistance. REFERENCES ARREGUiNLz.'iNO, B. & BNNER, J. (949). Experiments n sucrse frmatin by ptat tubers as influenced by temperature. PI. PhysiL, Lancaster, 24, 72. KER, J. (933). Analytic studies in plant respiratin. IV and V. The relatin f the respiratin f ptates t the cncentratin f sugars and t the accumulatin f a depressant at lw temperatures. Part I. The effect f temperaturehistry n the respiratin/sugar relatin. Part 2. The frm f the nrmal respiratin/sugar relatin and the mechanism f depressin. Prc. R. Sc, B, 2, 36.

9 CO2 utput and changes in phsphates 53 BARKER, J. (936). Analytic studies in plant respiratin. VI. The relatin f the respiratin f ptates t the cncentratin f sugars and t the accumulatin f a depressant at lw temperatures. Part 3. The relatin f the respiratin t the cncentratin f sucrse. Prc. R. Sc, B, 9, 453. B.WKER, J. (965). Studies in the respiratry and carbhydrate metablism f plant tissues. V'III. The effect f xygen n starch frmatin and disslutin in ptates. Nezv Phytl., 64, 2. B.WKER, J. (968). Studies in the respiratry and carbhydrate metablism f plant tissues. IV. The influence f a decrease in temperature n the cntents f certain phsphate esters in phint tissues. Nei Phytl., 67, 487. B.^RKER, J., ISHERWOOD, F. A., JAKES, R., SOLOMOS, T. & YOUNIS, M. E. (964b). Studies in the respiratry and carbhydrate metablism f plant tissues. IV. Determinatin f certain phsphate cmpunds in plant extracts. J. exp. Bt., 5, 284. BARKER, J., KHAN, M. A. A. & SOLOMOS, T. (964a). Mechanism f the Pasteur effect. Nature, Lnd., 2., 26. BARKER, J. & MAPSON, L. W. (955). Studies f the respiratry and carbhydrate metablism f plant tissues. VII. E.xperimental studies with ptat tubers f an inhibitin f the respiratin and f a 'blck' in the tricarbxylic acid cycle induced by xygen pisning. Prc R. Sc, B, 43, 523. BARKER, J. & SAIFI, A. F. EL (952). Studies f the respiratry and carbhydrate metablism f plant tissues. I. Experimental studies f the frmatin f carbn dixide, lactic acid and ther prducts in ptat tubers under anaerbic cnditins. Prc. R. Sc, B, 4, 362. BARKER, J. & YOUNIS, M. E. (965). Studies in the respiratry and carbhydrate metablism f plant tissues. VI. Further studies t' the influence f idacetate as a stimulant f respiratin in strawberry leaves.^, exp. Bt., 6, 59. HANES, C. S. & BARKER, J. (93). The physilgical actin f cyanide, i. The effects f cyanide n the respiratin and sugar cntent f the ptat at 5 C. Prc R. Sc, B, 8, 95. ISHERWOOD, F. A. & B.\RRETT, F. C. (967). Analysis f phsphate esters in plant material. Bicheni Jf., 4, 922. LvxEN, F., H.\RTM.AN, G., ETTER, K. F. & ScHUEGRAF,. \. (959). In Ciba Fundatin Sympsium n Regulatin f Cell Metablism (Ed. by G. E. W. Wlstenhlme & C. N. O'Cnnr), p Churchill, Lndn. LYNEN, F. (963). Cntrl ^Iechanisrns in Respiratin and Fermentatin (Ed. by B. Wright), p Rnald Press, Waltham, Massachussetts. ML'LLERTHURGAU, H. (882). Ober die Natur des in sussen Kartffeln sich Vrfidenen Zucher. Laiidtc. jfbr, II, 75. SAMOTUS, B. (i96). The rle f phsphrus in bichemical transfrmatin f starch in ptat tubers during the vegetative perid and different temperatures f strage. Rep. Rckefeller Fdn (unpublished). SYRETT, P. J. (958). Respiratin rate and internal adensine triphsphate cncentratin in Chlrella. Archs Bichem. Biphys., 75, 7.

STUDIES IN THE RESPIRATORY AND CARBOHYDRATE METABOLISM OF PLANT TISSUES

Nezv Phytl. (968) 67, 2522. STUDIES IN THE RESPIRATORY AND CARBOHYDRATE METABOLISM OF PLANT TISSUES II. THE PASTEUR EFFECT IN POTATOES AND APPLES BY J. BARKER AND M. A. A. KHAN* The Btany Schl, Cambridge