SUGAR ABSORPTION AND COMPARTMENTATION IN POTATO TUBER SLICES*

Transcription

1 New Phytol. (1968) 67, SUGAR ABSORPTION AND COMPARTMENTATION IN POTATO TUBER SLICES* BY P. J. HARDYt AND G. NORTON Nottingham University School of Agriculture, Sutton Boning ton, Loughborough (Received i July 1966) SUMM.ARY The uptake and utilization of ['"^Cjsucrose, ['*C]glucose and [''*C]fructose by mature and immature potato tuber slices was studied. Both tissues absorbed ['^Cjglucose faster than ['*C]fructose but once absorbed, the hexoses were utilized similarly. In contrast, the greater part of the labelled sugars formed on administration of ['*C]sucrose was sequestered at sites separate from the metabolic centres. Sucrose was taken up from the medium against a concentration gradient and transferred to metabolically inactive storage compartments without being hydrolysed either at the cell surface or at any stage during transit. INTRODUCTION Sucrose absorption has been studied in various plant tissues. Evidence for the splitting of sucrose prior to absorption has been obtained in the following plant tissues: yeast (Cirillo, 1961; Fuente and Sols, 1962), Neurospora spp. (Metzenberg, 1962), Peltigera polydactyla (Harley and Smith, 1956), beech mycorrhizas (Harley and Jennings, 1958), beech tree roots (Lewis and Harley, 1965), vacuum infiltrated leaf discs of Canna indica (Putman and Hassid, 1954), excized tomato roots (Dormer and Street, 1949), Zea mays radicles (Hellebust and Forward, 1962), bean root (Robinson and Brown, 1952) immature and mature sugar-cane storage tissue (Sacher, Hatch and Glasziou, 1963; Hawker and Hatch, 1965). In a few tissues, evidence for sucrose uptake unchanged has been obtained: tobacco leaf discs (Porter and May, 1955), bean pod tissue (Sacher, 1966) and castor bean seedling cotyledons (Kriedemann and Beevers, 1967). The present results show that sucrose can be absorbed from the medium into the storage compartments of cells of potato tuber slices without breakdown either at the cell surface or during passage through metabolic regions. MATERIALS AND METHODS Mature (fully grown and stored at 10 C) and immature (2.5 cm diameter, taken directly from the actively growing parent plant) King Edward tubers were used. ['*C]sucrose, [^"^Cjglucose and ['*C]fructose were obtained from the Radiochemical Centre, Amersham. The specific activity of each was adjusted to 22.7 /(Ci///mole. Slices (9 mm diameter x i mm thick) were cut from tissue cylinders taken from the centre of potato tubers and washed in four changes of distilled water at o" C for a total * This work formed part of a Ph.D. thesis (by P. J. Hardy) in the University of Nottingham, t Present address: C.S.I.R.O. Horticultural Research Section, Private Bag i. Glen Osmond, South Australia.

2 140 p. J. HARDY AND G. NORTON of 20 minutes. 1'ilty mature or immature tuber slices were placed in 10 ml distilled water containing 20 /(Ci (8.9x10'' counts/minute) of [''^Cjsucrose, [''^Cjglucose or ['"^C]- fructose and incubated for 180 minutes at 25 C with constant shaking. The radioactive solutions were then \\ ithdrawn and the slices washed in four changes of ice-water for 20 minutes. They were then killed, extracted and analysed as described by Hardy (1967). Radioactive areas on chromatograms were cut out, eluted, aliquots dried on planchets and counted. CO2 was collected throughout the incubations on strips of filter paper bearing 0.15 ml io"y KOH. The papers were changed at 15-minute intervals and dropped into distilled water. Aliquots of the KOH solutions were dried on glass planchets and counted immediately. All radioactive samples were counted at infinite thinness at 20" efficiency using a gas-flow detector. RESULTS The sugar concentrations of the slices are shown in Table i. The glucose:fructose ratio in both mature and immature slices was appro.ximately unity. The concentrations of all sugars in immature tuber slices were considerably higher than those in mature tuber slices, especially with respect to sucrose. Table 2 shows the total activity in the ethanolextracted water soluble compounds and in respiratory CO,. Table i. Concentrations of sucrose, glucose and fructose in mature and immature potato tuber slices {pmolesififty slices) Sucrose Glucose Fructose Mature Immature Table 2. Total '*C {countsiminute x 10"^) in ethanolextracts and CO. from fifty mature or immature tuber slices after incubation with [^'^ or [^*C]fructose [' *C]sucrose [' 'CJglucose [ l Mature Immature Mature Immature Mature Immature Ethanol extract CO, More ['"^CJglucose than ['"^Cjfructose was absorbed by both mature and immature tuber slices. Mature tuber slices absorbed more [''^Cjhexose than did immature but there was little difference between the quantities of ['"^CJsucrose absorbed by the two tissues. In both tissues the proportion of the absorbed [''^CJglucose converted to CO; was similar to that of [' *C]fructose but was two to four times the proportion of absorbed [' ^Cjsucrose respired. Table 3 shows the percentage distribution of '^C among various fractions of the extracts. In the immature tuber slices supplied with [' *C]glucose or ['"^CJfructose, the labelling of the extracted glucose and fructose, both as free sugars and also combined as sucrose was greater in the hexose corresponding to the ['"^CJhexose supplied. However, when [' *C]sucrose was supplied to these slices the glucose and fructose were equally labelled

3 Sugar absorption in potato slices 141 Table 3. Percentages of total ''^C in various fractions of ethanot e.\ tracts from mature or immature tuber slices incubated with [' '^Cjsucrose, [' '^( ']gliicose Basic fraction.\cidic + neutral traction Phosphorylated compounds Sucrose Glucose moiety Fructose moiety Glucose Fructose Citric acid Malic acid or [-C]: Mature ^ sucrose Immature Mature Immature [' CltVuctose.Mature Immature in both moieties. Since these slices absorbed ['"^CJglucose faster than ['"'Cjfructose this shows that the [''^Cjsucrose could not have been hydrolysed prior to absorption. When ['"''Cjsucrose was fed to either mature or immature tuber slices the proportion of the total extracted radioactivity found in free glucose and fructose was as great or greater than when ['"^Cjglucose or [''*C]fructose were supplied, yet the percentages of total '*C in compounds which become labelled on supplying [' *C]he\oses (basic compounds, phosphorylated compounds, acidic compounds and CO,) were much smaller. This shows that the greater proportion of the labelled glucose and fructose formed from [''*C]sucrose was located in compartments physically separate from the sites where synthesis of amino acids, organic acids, phosphate esters and respiration occurred. These differences in the labelling of metabolic intermediates following uptake of [' '^Cjhexoses as compared with [' *C]sucrose also confirm that the [''*C]sucrose was not hydrolysed at the cell surface Table 4. Specific activities (counts I minute I ptnole) of CO j, glucose, frttctose and of glucose and fructose obtained by hydrolysis cjf sucrose from mature or immature tuber slices incubated with [^*C]sucrose, [^'^C]glttcose or [^*C]fructose C0,x6 Glucose Fructose Glucose moiety Fructose moiety [' 'CJsucrose [' 'CJglucose [' 'C]fructose Mature Immature Mature Immature Mature Immature Table 4 shows that when ['^CJglucose or ['^CJfructose were fed to either mature or immature tuber slices the specific activity of CO, (collected in the final 11; minutes of the incubations) was much higher than the specific activities of the extracted sugars (on a carbon basis). This shows that both [''*C]hexoses were metabolized without equilibrating with the bulk of the endogenous sugars. When ['"^CJsucrose was supplied, the specific activity of COT was again higher than that of the extracted hexoses but the disparity was not nearly so great. The sucrose synthesized in the mature tuber slices presented with ['"^C]hexoses had a higher specific activity than that of the e.xtracted free hexoses (Table 4) showing that the hulk of the cell glucose and fructose did not participate in sucrose synthesis

4 142 p. J. HARDY AND G. NORTON DISCUSSION If hydrolysis of sucrose at the cell surface was necessary for absorption of sugar from sucrose solutions, e.xogenously supplied ['"^CJsucrose and ['"^Cjhexoses would he expected to behave similarly. From the differences in labelling of various intermediates on administration of ['"^CJsucrose as compared with ['"^CJhexoses it is concluded that the [' *C]sucrose was absorbed unchanged. The results of ['^Cjhexose administration showed that those exogenously supplied sugars were utilized in the synthesis of amino acids, phosphate esters, organic acids and sucrose and in respiration in metabolic compartments separate from the bulk of the stored cell glucose and fructose. In contrast, the greater part of the glucose and fructose which became labelled on supplying [''^Cjsucrose was sequestered in storage compartments separate from the metabolic centres. This compartmentation of sugars in metabolically active and inactive or storage pools, in cells of potato tuber slices was demonstrated by Laties (1964) who suggested that these compartments were probably the cytoplasm and vacuole respectively. Hatch (1964) adduced evidence to show that sucrose phosphate formation in metabolic compartments of cells of sugar-cane storage tissue was necessary for the transfer of sucrose to the storage compartments. The only known route for the synthesis of sucrose phosphate is by means of the reaction catalysed by uridine di-phosphate (UDP)-glucosefructose-6-phosphate glucosyl transferase. For the conversion of the absorbed ['"Cjsucrose to sucrose phosphate it would first have to be broken down and then converted to UDP-glucose and fructose-6-phosphate in the metabolic compartment. Both enzymes known to be capable of breaking down sucrose in plants (invertase, UDP-glucose-fructose glucosyl transferase) would produce free labelled hexoses and these, being in the metabolic compartment, would be utilized in metabolic transformations as were the exogenously supplied [''^CJhexoses. It is therefore concluded that the [''*C]sucrosefroni which were formed the glucose and fructose sequestered in storage compartments was not broken down and resynthesized, nor converted to sucrose phosphate at any stage before reaching the storage compartments, where, as implied by the equal labelling of glucose and fructose, it was in all probability inverted. Whilst the present work does not prove that sucrose cannot be transferred from the metabolic to storage compartments as sucrose phosphate, it shows that the synthesis of sucrose phosphate is not a pre-requisite for the penetration of sucrose into storage compartments in this tissue. ACKNOWLEDGMENT One of us (P. J. Hardy) acknowledges receipt of a Ministry of Agriculture postgraduate studentship. REFERENCES CiRlLLO, V. P. (1961). Sugar transport in microorganisms. A. Rev. MicrobioL, 15, 197. DORMER, K. J. & STREET, H. E. (1949). The carbohydrate nutrition of tomato roots. Ann. Bot., N.S., I3i 199- FuENTE, G. DE LA & SoLS, A. (1962). Transport of sugars in yeasts. II. Mechanisms of utilisation of disaccharides and related glycosides. Biochim. biophvs. Acta, 56, 49. HARDY, P. J. (1967). Sucrose breakdown and synthesis in the ripening grape berry. Aust.J. biol. Sci.,iS>t 465- HARLEY, J. L. & JENNINGS, D. H. (1958). The effect of sugar on the respiratory response of beech mycorrhizas to salts. Proc. R. Soc, B, 148, 403.

5 Sugar absorption in potato slices 143 H.\RLEY, I. L. & SMITH, D. C. (1956). Sugar absorption and surface carbohydrase acti\it\- of Peltigera polydactyla. Ann. Bot., N.S., 20, 513. HATCH, M. D. (1964). Sugar accumulation by sugar-cane storage tissue: the role of sucrose phosphate. B/oc/jem. J., 93, 521. H.\WKER, J. S. & HATCH, M. D. (1965). Mechanism of sugar storage by mature stem tissue of sugarcane. Physiologia PL, 18, 444. HELLEBUST, J. A. & FORWARD, D. F. (1962). The invertase of the corn radicle and its acti\ it\- in successi\e stage of growth. Can. J. Bot., 40, 113. KRIEDEMANN, P. & BEEVERS, H. (1967). Sugar uptake and translocation in the castor bean seedling. II. Sugar transformations during uptake. PI. PhysioL, Lancaster, 42, 174. LATIES, G. G. (1964). The relation of glucose absorption to respiration in potato slices. PI. PhysioL, Lancaster, 39, 391. LEWIS, D. H. & H.ARLEY, J. L. (1965). Carbohydrate physiology of mycorrhizal roots of beech. II. Utilization of exogenous sugars by uninfected and mycorrhizal roots. Nezi- Phytol., 64, 238. METZENBERG, R. L. (1962). \ gene affecting the repression of in\ertase and trehalase in A'enrospora. Archs Biochem. Biophys., 96, 468. PORTER, H. K. & M.\Y. L. H. (1955). Metabolism of radioacti\ e sugars by tobacco leaf discs. J. exp. Bot., 6, 43-. PUTMAN, F. \V. & H.ASSID, W. Z. (1954). Sugar transformations in leaves of Canna indica. I. Synthesis and inversion of sucrose. _/. biol. Chem., 207, 885. ROBINSON, E. & BROWN, R. (1952). The development of enzyme compliment in growing root cells. J. exp. Bot., 3, 356. S.^CHER, J..A. (1966). The regulation of sugar uptake and accumulation in bean pod tissue. PL PhysioL, Lancaster, 41, 181. SACHER, J. \., HATCH, M. D. & GL.\SZIOU, K. T. {1963). Sugar accumulation cycle in sugarcane. III. Physical and metabolic aspects of the cycle in immature storage tissues. PI. PhvsioL, Lancaster, 38, 348. K N.P.

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