Effect of wounding on ethylene biosynthesis and senescence of detached spinach leaves

Parameters of senescence and ethylene biosynthesis pathway were screened simultaneously in detached spinach leaves and leaf discs. Senescence was enhanced by application of 1-aminocyclopropane-1-carboxylic acid (ACC) and was retarded by amino-ethoxyvinylglycine (AVG). Evidence is presented showing that the bursts of both wound- and climacteric-like ethylene promoted senescence of detached leaves and leaf discs. This ethylene-enhanced leaf senescence was dependent on: (a) ethylene production rates in the tissue; (b) the degree of wounding. Wounding resulted in elevated levels of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), which declined in advanced stages of senescence. The results suggest that wounding might be regarded as one of the primary events in the induction of the senescence syndrome in detached leaves and leaf discs, while ethylene is implicated as a regulator of the rate of the process.     

Characterization and Induction of the Activity of 1-Aminocyclopropane-1-Carboxylate Oxidase in the Wounded Mesocarp Tissue of Cucurbita maxima

1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ethylene-formingenzyme) was isolated from wounded mesocarp tissue of Cucurbitamaxima (winter squash) fruit, and its enzymatic properties wereinvestigated. The enzyme required Fe²⁺ and ascorbate for itsactivity as well as ACC and O₂ as substrates. The in vitro enzymeactivity was enhanced by CO₂. The apparent K_m value for ACCwas 175 µM under atmospheric conditions. The enzyme activitywas inhibited by sulfhydryl inhibitors and divalent cationssuch as Co²⁺, Cu²⁺, and Zn²⁺. ACC oxidase activity was induced at a rapid rate by woundingin parallel with an increase in the rate of ethylene production.The exposure of excised discs of mesocarp to 2,5-norbornadiene(NBD),an inhibitor of ethylene action, strongly suppressed inductionof the enzyme, and the application of ethylene significantlyaccelerated the induction of the activity of ACC oxidase inthe wounded mesocarp tissue. These results suggests that endogenousethylene produced in response to wounding may function in promotingthe induction of ACC oxidase. (Received January 13, 1993; Accepted April 15, 1993)     

Induction of 1-Aminocyclopropane-1-carboxylic Acid Synthase in Wounded Mesocarp Tissue of Winter Squash Fruit and the Effects of Ethylene

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase activityincreased rapidly after wounding of mesocarp tissue of wintersquash fruit (Cucurbita maxima Duch.) and reached a peak at16 h after excision and then declined sharply. The rise in ACCsynthase activity was followed by increases in the endogenousACC content and the rate of ethylene production. The activityof ethylene forming enzyme (EFE) also increased rapidly in theexcised discs of mesocarp of winter squash fruit. ACC synthase activity was strongly inhibited by aminoethoxyvinylglycinewith a Ki value of 2.1 µM. Michaelis-Menten constant ofACC synthase for S-adenosylmethionine was 13.3 µM. Ethylene suppressed the induction of ACC synthase in the woundedmesocarp tissue. The suppression by ethylene increased withthe increasing concentrations of applied ethylene and the maximumeffect was obtained at about 100 µl 1^–1 ethylene,at which point the induction was suppressed by 54%. Ethylenedid not inhibit ACC synthase activity, nor did it suppress theinduction of EFE, but rather it slightly enhanced the latter. (Received August 24, 1984; Accepted October 29, 1984)     

Ethylene formation in sugar beet leaves: evidence for the involvement of 3-hydroxytyramine and phenoloxidase after wounding

Ethylene production by sugar beet (Beta vulgaris L.) leaf discs is inhibited by white (or red, >610 nm) light or by wounding. In contrast, in wounded leaf discs, ethylene production is stimulated by light. The effect of light on wounded leaf discs has been studied by using an in vitro system which mimics the loss of compartmentation in the wounded leaf. Chlorophyll-free extracts from sugar beet leaves stimulate the production of the superoxide free radical ion (as a prerequisite for ethylene formation) by illuminated chloroplast lamellae. The substance from the crude leaf extracts which is active in stimulating the production of the superoxide free radical ion has been identified as 3-hydroxytyramine (dopamine). Exogenous dopamine between 5 mum and 100 mum stimulates ethylene formation by illuminated chloroplast lamellae from methional. It also stimulates the production of the superoxide free radical ion, the formation of which apparently involves both a lamellar phenoloxidase and photosynthetic electron transport as a 1-electron donor, and is cyanide-sensitive.     

Effect of Ethylene on Sucrose Uptake in Root Discs of Sugar Beet

Exogenously-added ethylene stimulated active sucrose uptakein root discs of sugar beet (Beta vulgaris L.) in a log dose-linearresponse manner. The ethylene precursor, 1-aminocyclopropane-1-carboxylicacid (ACC) stimulated both endogenous ethylene production andsucrose uptake. Conversely, an inhibitor of ACC synthesis, aminoethoxyvinylglycine(AVG) inhibited both endogenous ethylene production and sucroseuptake. Exogenously-added ethylene can overcome the AVG effecton sucrose uptake. Root tissue from freshly-harvested sugarbeet plants contain gas-phase ethylene levels slightly belowthat required to stimulate active sucrose uptake. No differenceswere found in gas-phase ethylene levels in the root tissue ofsugar beet cultivars having different concentrations of sucrose.The root tissue has an inherent capacity to synthesize ACC andethylene at high rates. Like ethylene, propylene can stimulate active sucrose uptakein beet root discs, but it is not detected in the gas phaseof the tissue. Acetylene, propane, and ethane had no effecton sucrose uptake. Exogenously-added IAA and ABA each make ethylenesensitivetissue insensitive to ethylene stimulation of sucrose uptake.Other plant hormones have no apparent effect on the ethyleneresponse. The role that ethylene may play on sucrose uptakein root tissue of sugar beet is discussed. (Received February 12, 1986; Accepted April 22, 1986)     

The Effect of Cycloheximide on Absorption of Ions by Bean (Phaseolus vulgaris L) Roots

10^–7 M cycloheximide inhibited bean (Phaseolus vulgaris)root elongation by about 20 per cent but it inhibited absorptionof rubidium, sodium, and phosphate ions to a much greater extent(34–71 per cent). Tips of intact plant roots grown inthe inhibitor showed more inhibition in ion uptake than adjacentproximal portions of the same roots and this is taken to indicatethat 10^–7 M cycloheximide does not exert its effect onion uptake by any uncoupling action. Sodium uptake from 0.5 or 10 mM NaCl solutions by root tipswas inhibited by 10^–7 M cycloheximide to twice the extentthat it was in the elongating region of the root. Assuming thatthe inhibitor affects the plasmalemma more than the tonoplast,Epstein's model of parallel operation of system 1 and system2 at the plasmalemma is supported.     

Preferential Accumulation by Mesophyll Cells at Low and by Veins at High Exogenous Amino Acid and Sugar Concentrations in Commelina benghalensis L. Leaves

The contribution to solute uptake by mesophyll cells and veinsin leaf discs, was assessed through a study of uptake in relationto concentration for ¹⁴C-labelled substrates (sucrose, glucose,arginine, proline, valine and -aminoisobutyric acid) using isolatedmesophyll cells and stripped leaf discs of Commelina benghalensisL. Uptake per unit fresh weight was higher in mesophyll cellsthan in discs at low substrate concentrations (lower than about0·5 mol m^–3). At higher concentrations, uptakeby discs exceeded that by mesophyll cells except for glucoseuptake which was higher in mesophyll cells over the whole concentrationrange. The profiles of uptake versus concentration displayedbiphasic kinetics in mesophyll cells and discs. Comparison ofthe uptake characteristics obtained by iterative fitting confirmedthat the high-affinity systems of uptake prevail in the mesophyllcells, whereas the low-affinity systems are dominant in theveins. The results provide good evidence that, supplementaryto direct vein loading, a pathway via the mesophyll contributesstrongly to the photosynthate loading by veins in stripped discs. Key words: Commelina benghalensis L., amino acid uptake, mesophyll, minor veins, phloem loading, sugar uptake     

Ineffectiveness of cytokinin-induced chlorophyll retention in hypostomatous leaf discs

Kinetin retarded the decrease in chlorophyll content in leafdiscs from 5 species of plants with amphistomatous leaves, wherethe upper surface was exposed to air, but not in Rumex acetosera.When leaf discs were floated so that the lower surface was exposed,the effect of kinetin was less evident. Kinetin also stimulatedtranspiration in leaf discs from Nicotiana tabacum (amphistomatous),but not in leaf discs from Paederia chinensis (hypostomatous).Nor kinetin did retard chlorophyll breakdown in this specieswhen leaf discs were floated so that the stomatal surface wasin contact with the solution. The ineffectiveness of cytokininsin chlorophyll retention in leaf discs from hypostomatous leaveswas not due to reduced uptake of benzylaminopurine-¹⁴C. Chlorophyll retention was severely inhibited by coating theleaf surface with vaseline either in the presence or absenceof kinetin. Leaf discs floated on a solution exposed to CO₂-lessair retained more chlorophyll than those in normal air. Thereis thus a close relationship between stomatal opening (as measuredby stimulation of transpiration) and chlorophyll retention,as influenced by cytokinins. It is suggested that cytokinin-induced chlorophyll retentionand odier effects on leaf tissues could be mediated throughits effects on stomatal opening. (Received January 22, 1976; )     

Effect of Hormones on Sucrose Uptake and on ATPase Activity of Citrus sinensis L. Osbeck Leaves

Carbohydrate accumulation in young, fully expanded leaves ofCitrus sinensis L. Osbeck is affected by the presence of thefruitlet on the shoot. Previous work gave evidence that gibberellinsmay be involved in this 'fruit effect'. In the present workwe have studied the effect of gibberellic acid (GA₃) on ¹⁴C-sucroseuptake by leaf discs and whether its action could be due toa modulation of the plasma membrane ATPase, which maintainsthe H⁺ gradient that drives H⁺/sucrose co-transport. The effect of GA₃ on ¹⁴C-sucrose uptake depended on the osmolarityof the assay medium. At 300 mOsm a reduction in the uptake ratewas observed. The inhibitory effect of the hormone disappearedafter preincubating the leaf discs with para-chloromercuri-phenylsulphonicacid (PCMPS), a sulphydril binding inhibitor. ATPase activityof isolated plasma membrane vesicles was inhibited by IAA treatments,while GA₃ or ABA did not affect this enzyme, even after a 3h preincubation period. However, in the absence of a surfactantin the assay medium, GA₃, together with turgor pressure, modulatedplasma membrane ATPase activity, possibly through modificationsof membrane permeability. The hormone effect on ¹⁴ C-sucroseuptake may involve action on the sucrose carrier.Copyright 1994,1999 Academic Press Abscisic acid, Citrus sinensis, gibberellic acid, indoleacetic acid, orange, osmotic pressure, plasma membrane ATPase, ¹⁴C-sucrose uptake     

Ethylene-Enhanced Transport of Uranine, a Fluorescent Dye, in Rice-Seedling Explants in Relation to Ethylene-Stimulated Coleoptile Growth

Ethylene enhanced the transport of uranine, a fluorescent dye,through the coleoptiles of rice (Oryza sativa L. cv. Sasanishiki)seedlings explants after a 2-h lag period. The uranine transportincreased with increasing concentration of ethylene, reachinga maximum at 1 µ1.liter^–1 ethylene. It decreasedwhen some metabolic inhibitors, 2,4-dinitrophenol, carbonylcyanide 3-chlorophenylhydrazone and N-ethylmaleimide, were appliedto the scutella. Mechanical rupture or heating at the vascular-bundlesides of the coleoptiles also interfered with the ethylene-enhancedtransport of uranine. The wounding and removal of the scutellasimilarly blocked uranine transport. Moreover, strong fluorescenceof uranine was observed on the phloem regions of two vascularbundles of a coleoptile under a fluorescence microscope. Itis suggested that the ethylene-enhanced transport of uraninein the coleoptile of rice-seedling explants occurs through thephloem. On the other hand, the removal of the endosperm or therupture of the vascular bundles inhibited the ethylene-stimulatedelongation of the rice coleoptiles. Moreover, ethylene increaseddry weight of the shoots. These results suggest that the ethylene-stimulatedelongation of rice coleoptiles was supported partly by the ethylene-enhancedtranslocation of materials from the endosperms to the coleoptiles. (Received September 6, 1984; Accepted November 15, 1984)     

General properties and regulation of arginine transporting systems in Saccharomyces cerevisiae

The transport of arginine-¹⁴C by exponentially growing cellsof Saccharomyces cerevisiae (ATCC 9763) was studied in the presenceof various amino acids, ammonium and urea. Arginine transportwas inhibited when the cells were preincubated with these compoundsfor 1 hr. Little or no inhibition of transport occurred whenthe preincubation period was omitted. Kinetics studies revealedthat arginine was transported by two distinct systems havinghigh and low affinities for this amino acid. At given arginineconcentrations the high affinity system was capable of transportingarginine molecules at approximately seventy times the rate ofthe low affinity system. The general requirements for arginine transport revealed energyand temperature dependencies in addition to sensitivity to anumber of metabolic inhibitors. Transfer of cells to N-freemedium was accompanied by increased rates of transport. Thisincrease was shown for the uptake of ten different amino acids.For L-arginine, this increase was prevented by addition of cycloheximide. Analyses of amino acid pools, after various experimental treatments,failed to reveal any consistent correlation between transportrates and the concentrations of individual amino acids or ammonium. It is concluded that arginine transport of S. cerevisiae isregulated by inhibition and repression. In this respect theavailability of ammonium would appear to be of prime importancein the development of transport activity. (Received December 5, 1975; )     

Starch Synthesis from Exogenous Sugars in Tobacco Leaf Discs

Sets of discs were taken from leaves of destarched tobacco plants(Nicotiana tabacum L. cv. xanthii) and floated on solutionsof sucrose or glucose in the dark. Abundant starch was formedin the youngest leaves but there was a marked decline with leafage.By contrast, when replicate sets of discs were floated on waterand illuminated, photosynthetic starch formation was similarin the differently aged leaves. Uptake of sugar, measured bydry weight increases and incorporation of [¹⁴C]sucrose, wasnot dependent on leaf age. The possibility that physiologicalchanges, relating to ageing and import/export status of theleaf, regulate the metabolism of sugar to starch was examined.Increasing retention of sugar in the minor veins is likely tobe a major factor. Invertase activities were measured and foundto be similar in the differently aged leaves. Respiration ratesdeclined with increasing leaf age. Speculations concerning changesin selective permeability of the chloroplast membrane are alsodiscussed.     

Characterization of abscisic Acid-induced ethylene production in citrus leaf and tomato fruit tissues

Abscisic acid (ABA) significantly stimulated ethylene production in citrus (Citrus sinensis [L.] Osbeck, cv Shamouti orange) leaf discs. The extent of stimulation was dependent upon the concentration of ABA (0.1-1 milimolar) and the duration of treatment (15-300 minutes). Aging the discs before applying ABA increased ABA-induced ethylene production due to enhancement of both ethylene-forming enzyme activity and the responsiveness of ABA. Discs excised from mature leaves were much more responsive to ABA than discs excised from young or senescing leaves. ABA stimulated ethylene production shortly after application, suggesting that ABA does not enhance ethylene production via the acceleration of senescence. The stimulating effect of ABA on ethylene production resulted mainly from the enhancement of 1-aminocylopropane-1-carboxylic acid synthesis. Stimulation of ethylene production by ABA in intact citrus leaves and tomato (Lycopersicon esculentum Mill., cv Castlemart) fruit was small but could be increased by various forms of wounding.     

Wound-Induced Ethylene Production and 1-Aminocyclopropane-1-carboxylic Acid Synthase in Mesocarp Tissue of Winter Squash Fruit

Discs (9 mm in diameter and 2 mm in thickness) sliced from mesocarpof winter squash fruit (Cucurbita maxima Duch.) upon incubationat 24°C produced ethylene at an increasing rate after alag period of 3 h. 1-Aminocydopropane-l-carboxylic acid (ACC)synthase activity also increased at a rapid rate after lag periodof less than 3 h, reaching a peak 14 h after incubation andthen declining sharply. The rise in ACC synthase activity precededa rapid increase in ACC formation and ethylene production. Inductionof ACC synthase by wounding in sliced discs was strongly suppressedby the application of cycloheximide, actinomycin D and cordycepin,suggesting that the rise in ACC synthase activity may resultfrom de novo synthesis of protein. ACC synthase extracted from wounded tissue of winter squashmesocarp required pyridoxal phosphate for its maximum activity.The optimum pH of the reaction was 8.5. Km value for S-adenosylmethioninewas 120 µM. The reaction was markedly inhibited by aminoethoxyvinylglycinewith Ki value being 2.7 µM. (Received March 23, 1983; Accepted May 23, 1983)     

Nitrate Accumulation and its Relation to Leaf Elongation in Spinach Leaves

The leaf elongation rate (LER) of spinach leaves during theday was twice that during the night when grown at a photon fluxdensity of 145 µmol m^–2 s^–1. All leaves showedthe same LER-pattern over 24 h. Due to low turgor, LER was lowin the afternoon and in the first hours of the night until wateruptake restored full turgor. Osmotic potential remained constantdue to increased nitrate uptake and starch degradation in thisperiod. LER increased to high rates in the second part of thenight and in the morning. The lower rate in the dark comparedto the light was not caused by the lower night temperatures,as increased photon flux density during growth resulted in equalrates in the light and the dark. Increased relative humiditydecreased LER and afternoon rates were most sensitive to waterstress. A ‘low light’ night period did not changeLER-pattern during the night or on the following day. We concludethat nitrate is not an obligatory osmoticum during the nightand can be exchanged for organic osmotica without decreasingLER. During the night the turgor is first restored by increasingwater uptake, nitrate uptake and starch degradation. This resultedin increased leaf fresh weight in this period. Thereafter, elongationincreased by simultaneous uptake of nitrate and water. Nitrateconcentration was, therefore, constant in the older leaves.In the younger leaves nitrate concentration increased to replacesoluble carbohydrates. The vacuoles of the old leaves were filledwith nitrate before those of the young leaves. Key words: Spinacia oleracea L., nitrate accumulation, osmotic potential, organic acids     

Aldolase--An Important Enzyme in Controlling the Ribulose 1,5-bisphosphate Regeneration Rate in Photosynthesis

This study was done to explore an enzymatic mechanism for thephotosynthetic carbon reduction cycle whereby the rate of synthesisof ribulose 1,5-bisphosphate (RuBP) could be changed while thelevels of intermediates other than 3-phosphoglycerate and RuBPwere kept constant. Chloroplast aldolase was purified to homogeneityfrom spinach leaves. When the enzyme was assayed in the directionof fructose 1,6-bisphosphate synthesis in the presence of theconcentrations of the substrates reported in vivo, the activitywas severely inhibited by physiological concentrations of RuBP.The aldolase reaction proceeded with a sequential mechanism.The K_m for dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphatewere 0.45 mM and 40 µM, respectively. The activity wascompetitively inhibited by RuBP with respect to dihydroxyacetonephosphate. The K^I was 0.78 mM. The maximum activity of aldolasein spinach leaves was calculated as 1,360µmol (mg Chl)^–1h^–1 An equation to express the reaction for the synthesisof fructose 1,6-bisphosphate by aldolase was constructed topredict the metabolic rate of this reaction in vivo. The calculationclearly showed that aldolase is an important enzyme in controllingthe rate of RuBP regeneration. (Received March 25, 1991; Accepted August 12, 1991)     

Adenylate Uptake by Proplastids from Cultured Cells of Tobacco {Nicotiana tabacum L. cv. BY2) Indicates That an Adenylate Translocator is Present in All Types of Plastid

The presence of an adenylate translocator in the envelope membranesof proplastids isolated from the cultured cells of tobacco (Nicotianatabacum L. cv. BY2) was examined by means of transport experimentsusing the silicone oil filtering centrifugation technique. Itwas observed that proplastids can import [³H]ATP, [³H]ADP, [³H]AMPand less specifically ADP-[¹⁴C]Glc which can eventually be usedfor starch biosynthesis. The effects of specific inhibitorsof the mitochondrial adenylate translocator, i.e. atractyloside,bongkrekic acid and carboxyatractyloside were tested. Similarto the case of amyloplasts isolated from the cultured cellsof sycamore and chloroplasts isolated from spinach leaves, onlyATP and ADP-Glc uptake were shown to be partially inhibitedby carboxyatractyloside. On the other hand, neither atractylosidenor bongkrekic acid exerted a significant inhibitory effecton adenylate uptake. (Received August 8, 1992; Accepted November 26, 1992)     

Ethylene production by wounded tissue of citrus fruit

Segments cut from young immature fruits and albedo discs excisedfrom both immature and mature fruits of Satsuma mandarin ormature fruits of Natsudaidai produced much ethylene during incubationat 26?C in the dark. Ethylene formation was markedly acceleratedby the application of abscisic acid but markedly delayed by3,5-dibromo-4-hydroxybenzoic acid. Both the stimulation andretardation decreased greatly during the course of incubation.Both compounds seem to be associated with the early stages ofethylene formation by wounded citrus fruit tissues. Albedo discs were fed ¹⁴C methionine labeled at one of threedifferent positions. Of the three radioanalogs (carbon-2, carbon-3and methyl carbon), the label at the 3 position was preferentiallyincorporated into ethylene. This agrees with the former observationthat ethylene is derived from carbon-3 and -4 of methionine.Incorporation of label into ethylene from L-[3-¹⁴C] methioninewas strongly inhibited by L-canaline, L-ethionine, 2,4-dinitrophenoland cycloheximide. Ethylene evolution was also strongly inhibitedby 2,4-dinitrophenol, KCN, NaN₃ and cycloheximide, but lesscompletely by L-canaline and L-ethionine. These results supportthe view that ATP and pyridoxal phosphate are utilized in activationof methionine to form ethylene. (Received October 25, 1977; )     

Characterization of Hexose Transporter for Facilitated Diffusion of the Tonoplast Vesicles from Pear Fruit

Tonoplast vesicles were prepared from the flesh tissue of maturepear fruit. Sugar uptakes into the vesicles determined by twodifferent methods, the membrane and the gel filtration methods,were quite similar. The uptake was highest for glucose and subsequently,in order, for fructose, sucrose and sorbitol. It was not stimulatedby addition of ATP, although the vesicles could create a protongradient. However, the uptakes were significantly inhibitedby p-chloromercuribenzene sulphonate (PCMBS, SH-reagent andinhibitor of sugar transporter). Further, the PCMBS-sensitiveuptakes of glucose and fructose saturated with their increasedconcentrations. Thus, these PCMBS-sensitive uptakes are mediatedby the transporter of facilitated diffusion. The uptakes ofglucose or fructose each had two K_m values. K_m values for glucosewere 0.35 and 18 mM, and those for fructose were 1.6 and 25raM. The uptake of 0.2 mM glucose was inhibited by 2 mM fructoseand that of 2 mM fructose was inhibited by 2 mM glucose, butneither was inhibited by sucrose or sorbitol. O-methyl-glucose(OMG) also inhibited both the glucose and fructose uptakes.Therefore, the same transporter may mediate both glucose andfructose uptakes at lower concentrations; this hexose transportsystem differed from the sucrose and sorbitol transport systems. ¹Research Fellow of the Japan Society for the Promotion of Science. ²Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan.     

Characterization of the Permeability of Excised Apple Tissue 2

Sorbitol uptake from a bathing solution into the compartmentedspace and into the diffusible or apparent free space of excisedparenchyma tissue from apple fruit (Pyrus malus L. cv. GoldenDelicious) was investigated. Uptake into the two cell compartmentswas measured after washing of ^l4C-loaded tissue for 1 h withan osmoticum-free bathing solution. Compartmental analysis showedthat this treatment released sorbitol taken up into the cytoplasmof the cell, which was considered to be part of the apparentfree space. Uptake of sorbitol into the apparent free space was dependenton the osmotic concentration of the incubation medium. Usingmannitol up to 200 mM, uptake decreased by 60%, and increasedagain above 600 mM mannitol, the external concentration whereturgor was eliminated. Uptake in the compartmented space wasabout 3 times lower and was hardly affected by the externalosmotic concentration. PCMBS inhibited sorbitol transport intothe apparent free space by 25% at 100 mM mannitol, but at 600mM the inhibitor had no effect. The results indicate that sorbitoltransport across the plasma membrane is possibly facilitatedby a turgor-sensitive carrier. Uptake of ^l4C-sorbitol into thefreely diffusible space of tissue discs also increased by 200%after storage of unripe fruit for 70 d. This increase in agedtissue did not occur when uptake was measured at 4C or in thepresence of 200 mM PEG. Enhanced uptake was concomitant withan increased release of endogenous sugars from aged tissue. It would appear that the effect of a hypotonic bathing solutionon the permeability of excised apple tissue is related to structuralchanges, such as stretching of the plasma membrane. This effect,which becomes more marked as unripe fruit ages, is probablybrought about by turgor-driven relaxation of the tissue. Itmay increase non-specific leakage of sugars but could also bea factor affecting carrier-mediated transport of sorbitol atthe plasma membrane. Key words: Apple, sugar transport, sorbitol, plasma membrane, apoplast