Source--sink Relationships and Carbon Metabolism in Tomato Leaves 2. Carbohydrate Pools and Catabolic Enzymes

Levels of the major carbohydrates (hexoses, inositol, sucroseand starch) and activities of the enzymes (invertase, amylaseand starch phosphorylase) were assayed in the leaves and stemsof tomato plants. The transport system of the plants was simplifiedto one leaf and one associated truss either retained or removed.The experiment was carried out at two light intensities, 80and 20 W m^–2. Truss removal immediately increased thelevels of hexoses and starch in leaves under high light, butthose of sucrose and inositol were relatively unaffected withina day. Three days after truss removal the levels of sucrose,hexose and starch had reached a maximum. Under low light, leafsugar levels were lower and did not increase following trussremoval. The stem appeared to act as a substitute sink in thoseplants where the truss had been removed. Invertase and amylase activities were not significantly affectedby the treatments, but loss of starch phosphorylase activityduring the experiment was prevented by truss removal. Tomato, source-sink relationships, carbon, carbohydrate     

Compartmentation of Assimilate Fluxes in Leaves

Abstract: Sugar levels in the apoplast of assimilate exporting leaves were studied in two groups of plant species with contrasting structures of companion cells in minor veins. These species are termed either "symplastic" (with intermediary cells) or "apoplastic" (with transfer or ordinary cells). Sugars were measured in intercellular washing fluid after extracting the apoplast by an infiltration-centrifugation technique. During the course of a day, sugar contents in the apoplast were, in general, lower in species with intermediary cells than in species with transfer or ordinary cells. In "symplastic" species, apoplastic sucrose concentrations were between 0.3 and 1 mM. In "apoplastic" species with transfer cells, they ranged between 2 and 6 mM. Apoplastic hexose contents were between 0.3 and 1 mM irrespective of presumed transport mode. "Symplastic" and "apoplastic" plants differed markedly in their response to a'translocation block. In "symplastic" plants, inhibition of assimilate export left apoplastic concentrations of sucrose and hexoses unchanged, whereas in "apoplastic" plants sugar levels increased, the maximal increase being observed with sucrose. In these plants, concentrations of sucrose were two to six times higher in the apoplast under export inhibition than in control leaves. The data suggest a different role of the leaf apoplast in the compartmentation and export of assimilates in the two plant groups under study.     

Patterns of Assimilate Distribution and Source--sink Relationships in the Young Reproductive Tomato Plant (Lycopersicon esculentum Mill.)

Patterns of distribution of ¹⁴C were determined in 47-day-oldtomato plants (Lycopersicon esculentum Mill.) 24 h after theapplication of [¹⁴C]sucrose to individual source leaves fromleaves 1–10 (leaf 1 being the first leaf produced abovethe cotyledons). The first inflorescence of these plants wasbetween the ‘buds visible’ and the ‘firstanthesis’ stages of development. The predominant sink organs in these plants were the root system,the stem, the developing first inflorescence and the shoot ‘apex’(all tissues above node 10). The contribution made by individualsource leaves to the assimilate reaching these organs dependedupon the vertical position of the leaf on the main-stem axisand upon its position with respect to the phyllotactic arrangementof the leaves about this axis. The root system received assimilateprincipally from leaf 5 and higher leaves, and the stem apexfrom the four lowest leaves. The developing first inflorescencereceived assimilates mainly from leaves in the two orthostichiesadjacent to the radial position of the inflorescence on thevertical axis of the plant; these included leaves which weremajor contributors of ¹⁴C to the root system (leaves 6 and 8)and to the shoot apex (leaves 1 and 3). This pattern of distributionof assimilate may explain why root-restriction treatments andremoval of young leaves at the shoot apex can reduce the extentof flower bud abortion in the first inflorescence under conditionsof reduced photoassimilate availability. Lycopersicon esculentum Mill, tomato, assimilate distribution, source-sink relationships     

Regulation of Assimilate Translocation Between Leaves and Fruits in the Tomato

Simultaneous measurement of export from leaves and import tofruits were made on tomato plants reduced to one fully expandedleaf and one fruit. Experimental leaves were exposed to sixlight flux densities (0.5–100 W m^–2) for 24 h whilerapidly growing fruits were kept in the dark at 22 °C. The rates of export of assimilate from these leaves varied from70 to 120 mg C leaf^–1 day^–1 corresponding with ratesof carbon fixation from 3 to 290 mg C leaf^–1 day^–1.Export from leaves with the lowest carbon fixation rates weremaintained by a loss of up to one-sixth of their initial carbon.In contrast, leaves with the highest carbon fixation rates exportedonly half the newly fixed carbon. The rates of import of assimilate to similar-sized fruits (c.16 cm³) were between 80 and 110 mg C fr^–1 day^–1but differed from the export rates of the source leaves. Thespecific growth rates and the specific respiration rates ofthe fruits were related to their initial carbon content at thebeginning of the experiment. Thus, over 24 h, the rate of importwas predetermined by the developmental stage of the fruit unalteredby the rate of current carbon fixation in the source leaf. Translocationof assimilate was regulated by sink demand under both source-and sink-limiting conditions in this short-term situation. The dynamic relationship between assimilate production in leavesand its utilization in fruits is discussed together with therole of sucrose concentration in these organs in regulatingtransport. Lycopersicon esculentumL, tomato assimilate translocation, source-sink relationships     

Effect of Heat Stress on Assimilate Metabolism in Tomato Flower Buds

Assimilate import by flower buds in two cultivars of tomato(Lycopersicon esculentum Mill.) was inhibited by heat stress.With increasing temperature, levels of sucrose in the sourceorgan increased while levels of starch decreased. The transportof radioactive carbon was correlated with the starch contentof the flower buds. In Saladette, a heat-tolerant cultivar,conversion of the imported carbon to starch occurred to a greaterextent than in Roma VF, a heat-sensitive cultivar. Uptake ofsucrose from agar medium by detached flower buds was negativelycorrelated with their internal ratio of sucrose to hexoses.Glucose uptake from agar medium by detached flower buds decreasedwith increasing temperatures. Sucrose hydrolysis was negativelyaffected by high temperatures, and this was more pronouncedin the heat-sensitive than in the heat-tolerant cultivar. Theeffect of heat stress on assimilate translocation from the leavesto the sink organ is discussed. Lycopersicon esculentum Mill., starch, sucrose, heat stress     

14CO2 Fixation, Translocation, and Carbon Metabolism in Rapidly Expanding Leaves ofPopulus deltoides

To examine ¹⁴CO₂ fixation, potential translocation, and carbonflow among leaf chemical fractions of young developing leaves,the shoot tip of 24-leaf cottonwood (Populus deltoides Bartr.ex. Marsh) plants were cut off under water, placed in artificialxylem sap, and treated with ¹⁴CO₂ in continuous and pulse-chaseexperiments. Additional leaves on whole plants were spot treatedon the lamina tip to follow export from the tip only. The analysedleaves ranged in age from leaf plastochron index(LPI) –5to 3, the spot treated leaves from LPI 2 to 5. After 30 minfixation, the specific activity in the lamina tip increasedlinearly with leaf age from LPI –5 to 1 (0.5 to 4.5 kBqmg^–1). Specific activity in the lower lamina increasedslowly with leaf age and did not reach 500 kBq mg^–1 untilLPI –1. Total ¹⁴CO₂ fixed in the lower lamina exceededthat fixed in the tip by LPI –2 because of the large amountof tissue present in the lower lamina. Although the lamina tipfixed high levels of ¹⁴CO₂, pulse-chase studies coupled withautoradiography indicated no vein loading or translocation fromthe tip until about LPI 4 or 5. The ¹⁴C fixed in both tip andlower lamina was incorporated at the site of fixation and wasnot distributed to younger tissue or translocated from the lamina.Although the percentage distribution (¹⁴C in each chemical fractioncompared with the total in all fractions) of ¹⁴C among certainchemical fractions, e.g. sugars, amino acids and proteins, indicatedthat the mesophyll of the tip was more mature than the lowerlamina, physiologically both leaf sectors were immature basedon the expected ¹⁴C distribution in mature tissue. Informationfrom this and other studies indicates that the extreme tip ofa developing cottonwood leaf first begins to export photosynthateabout LPI 4 or 5 on a 24-leaf plant. The first photosynthatetranslocated may be incorporated into the vascular tissues andmesophyll directly below the tip. However, as the tip continuesto mature photosynthate is translocated past the immature lowerlamina into the petiole and out of the leaf. Populus deltoides Bartr. ex. Marsh, eastern cottonwood, translocation, leaf development, ¹⁴C fixation, carbon metabolism     

Metabolism of the Raffinose Family Oligosaccharides in Leaves of Ajuga reptans L. (Inter- and Intracellular Compartmentation)

We recently suggested that leaves of the frost-hardy species Ajuga reptans L. (Lamiaceace) contain two pools of raffinose family oligosaccharides (RFO): a large long-term storage pool in the mesophyll, possibly also involved in frost resistance, and a transport pool in the phloem (M. Bachmann, P. Matile, F. Keller [1994] Plant Physiol 105: 1335-1345). In the present study, the inter- and intracellular compartmentation of anabolic RFO metabolism was investigated by comparing whole-leaf tissue with mesophyll protoplasts and vacuoles. The studies showed the mesophyll to be the primary site of RFO synthesis in A. reptans. Mesophyll protoplasts were capable of RFO formation upon in vitro 14CO2 photosynthesis. Sucrose-phosphate synthase, galactinol synthase, and the galactinol-independent galactosyltransferase, which is responsible for RFO chain elongation, were located predominantly in the mesophyll protoplasts. The percentage of stachyose synthase in the mesophyll changed greatly during the cold-acclimation period (from 26% at the beginning to 88% after 20 d). The remainder was most probably in the intermediary cells of the phloem. Compartmentation studies in which mesophyll protoplasts were compared with vacuoles isolated from them showed that, of the components of the RFO storage pool, galactinol synthase, stachyose synthase, myo-inositol, galactinol, and sucrose were extravacuolar (most probably cytosolic), whereas galactinol-independent galactosyltransferase and higher RFO oligomers (with degree of polymerization 4) were vacuolar. Raffinose was found in both locations and might serve as a cryoprotectant.     

Paraveinal Mesophyll of Soybean Leaves in Relation to Assimilate Transfer and Compartmentation : III. Immunohistochemical Localization of Specific Glycopeptides in the Vacuole after Depodding

Immunohistochemical staining was used to determine the cellular distribution of two glycosylated polypeptides (molecular weights of 27 and 29 kilodaltons) which are normally present at low levels in soybean (Glycine max var `Wye') leaves but which markedly accumulate after depodding. These polypeptides, which comprise a substantial portion of the total leaf soluble protein of depodded plants, were exclusively located in the vacuoles of paraveinal mesophyll and associated bundle sheath cells. These results support the unique role of the soybean leaf paraveinal mesophyll in the transport and spatial compartmentation of nitrogen reserves in relation to seed filling.     

Demonstration of the Intercellular Compartmentation of l-Menthone Metabolism in Peppermint (Mentha piperita) Leaves

The metabolism of l-menthone, which is synthesized in the epidermal oil glands of peppermint (Mentha piperita L. cv. Black Mitcham) leaves, is compartmented; on leaf maturity, this ketone is converted to l-menthol and l-menthyl acetate in one compartment, and to d-neomenthol and d-neomenthyl glucoside in a separate compartment. All of the enzymes involved in these reactions are soluble when prepared from whole-leaf homogenates. Mechanical separation of epidermal fragments from the mesophyll, followed by preparation of the soluble enzyme fraction from each tissue, revealed that the neomenthol dehydrogenase and the glucosyl transferase resided specifically in the mesophyll layer, whereas the menthol dehydrogenase and substantial amounts of the acetyl transferase were located in the epidermis, presumably within the epidermal oil glands. These results suggest that the compartmentation of menthone metabolism in peppermint leaves is intercellular, not intracellular.     

Movement of 14C- and 11C-labelled Assimilate in Wheat Leaves: the Effect of IAA

The rate of translocation of naturally-loaded, radiolabelledassimilate has been studied in leaves of wheat treated withIAA. The velocity was measured directly by following the movementof ¹¹C-labelled material along the leaf. The kinetics of translocationwere also estimated by using a two-compartment model to calculatethe rate constant of disappearance of ¹⁴C from the fed area.IAA was applied at three different sites on the leaf and atvarious times up to 24 h before ¹⁴CO₂ feeding. No effects ofIAA were observed on: (1) direction and velocity of transport;(2) loading of assimilate into the phloem; or (3) the site andkinetics of unloading. These results are discussed with referenceto the movement of IAA along the transport path and the useof different tissues as experimental systems.     

Translocation of 14Carbon Within and Between Leaves

Plants of tobacco and a variegated variety of Pelargonium wereused to investigate some aspects of translocation of ¹⁴Carbonwithin and between leaves, following assimilation of ¹⁴CO₂ byone of the leaves. Bi-directional transport in leaves is consideredto result from import into immature regions and exports frommature regions. In variegated leaves the chlorotic areas behavelike immature areas in the sense that they continue to importtranslocate from outside the leaf to a greater extent than adjacentgreen areas. However, some transport occurs from green to chloroticareas via the veins in the same leaf. Using masking techniqueson tobacco leaves it was shown that labelled carbon failed tomove across darkened mesophyll. This was taken to indicate thatthe mechanism resulting in translocation from leaves was locatedin the veins. Labelled carbon was shown to leave the veins ofimporting leaves along their entire length. A simplified technique for freeze-drying plant material priorto wax-embedding is described and some of the limitations ofheat-drying as a preliminary to gross autoradiography of leavesare discussed.     

Carbon Translocation in the Tomato: Pathways of Carbon Metabolism in the Fruit-

The rate of carbon import by tomato fruits has been relatedto their carbon metabolism by examining the effects of fruittemperature on the metabolism of imported assimilates. ¹⁴C–sucrose,–glucose, –fructose, –malic acid and –citricacid were injected individually into young growing tomato fruitswhich were subsequently maintained at 25 or 5 °C for 48h. Fruit temperature greatly affected the proportions of ¹⁴Clost from the fruits by export and respiration. Only 40 percent of the injected ¹⁴C from ¹⁴C–sugars and 20 per centfrom ¹⁴C–acids was recovered from fruits at 25 °C.Less than 10 per cent of the injected ¹⁴C was exported, thebalance being respired. In contrast, more than 50 per cent ofthe injected ¹⁴C was recovered from cooled fruits, in whichthe import rate of carbon was presumably reduced, and 20–36per cent of injected ¹⁴C was exported. Cooling enhanced thesynthesis of ¹⁴C–sucrose from injected ¹⁴C–hexosesand inhibited the incorporation of ¹⁴C into starch and insolubleresidue. When ¹⁴C–sugars were injected, radioactivityexported from the cooled fruits was detected as sucrose in thephloem of the peduncles; radioactivity was also detected instems and roots when fruits were cooled. In almost fully–grownfruits injected ¹⁴C–compounds were metabolized less readilythan in smaller fruits. Conversion of ¹⁴C–hexoses to ¹⁴C–sucrosewas again enhanced by cooling (5 °C, but was less in fruitsmaintained at 35 °C than in controls. Lycopersicon esculentum, tomato, fruit, translocation, carbon metabolism     

The Regulation of Carbon Transport and the Carbon Balance of Mature Tomato Leaves

Rates of carbon transport from a single mature tomato leaf inthe light period (day transport) and the dark period (nighttransport) were estimated from the rate of carbon fixation inthe light period, the rate of respiration in the dark periodand the changes in carbon contents over these two periods. Plantswere grown initially at 40 W m^–2 light intensity witheither 350 vpm (nonenriched plants) or 1000 vpm CO₂ (enrichedplants). Various light flux densities or CO₂ concentrationswere then applied to the experimental leaves in the light periodduring the experiment When leaves were temporarily exposed to contrasting light fluxdensities both day transport and night transport were linearlyrelated to the rate of carbon fixation. If leaves were shadedbelow the light compensation point for up to five days, or transferredto contrasting CO₂ concentrations for up to ten days, the linearrelationship between carbon fixation and carbon transport nolonger held. During acclimatization, therate of wbon fixationwas simply related to thecurrent light flux density and CO₂concentration, but the rate of carbon transport changed withtime. Day and night transports responded differently to changesin environment: night transport was more related to the contentof reserve, particularly starch, than to the rate of concurrentwbon fixation. It is concluded that the rate of carbon transport of a maturetomato leaf in a single photoperiod is regulated not merelyby the rate of concurrent carbon fixation but by the contentof reserve in the leaf. The latter results from previous cumulativewbon fixation and carbon transport. As a result of changingthe rate of carbon transport, a balance of carbon input andoutput was achieved within 10 days of acclimatization in a maturetomato leaf.     

Metabolism of phytol-U-14C and phytanic acid-U-14C in the rat

The metabolism of uniformly-labeled (14)C-phytol, (14)C-phytenic acid, and (14)C-phytanic acid was studied in the rat. Conversion of both phytol and phytenic acid to phytanic acid was demonstrated. Tracer doses of phytol-U-(14)C given orally were well absorbed (30-66%), and approximately 30% of the absorbed dose was converted to (14)CO(2) in 18 hr. After intravenous injection, 20% appeared in (14)CO(2) in 4 hr. Phytanic acid-U-(14)C given intravenously was oxidized at a comparable rate (22-37% in 4 hr) and was as rapidly oxidized as palmitic acid-1-(14)C (21% in 4 hr). Metabolism of these substrates was also studied in rats previously maintained on a diet containing 5% phytol by weight, which causes accumulation of phytanic acid, phytenic acid, and, to a lesser extent, phytol in blood and tissues. Despite the large body pools of preformed, unlabeled substrate in these animals, the fraction of an administered dose of phytol-U-(14)C or phytanic acid-U-(14)C converted to (14)CO(2) was not significantly diminished. These studies indicate that the rat has an appreciable capacity to degrade the highly branched carbon skeleton of phytol and its derivatives. Twenty-four hours after administration of phytol-U-(14)C, the lipid radioactivity remaining in the body was widely distributed among the tissues, highest concentrations being found in liver and adipose tissue. Four hours after intravenous administration of phytanic acid-U-(14)C, all of the major lipid classes in the liver contained radioactivity, most in triglycerides and phospholipids and least in cholesterol esters and lower glycerides. There was no demonstrable incorporation of mevalonate-2-(14)C or acetate-1-(14)C into liver phytanic acid when they were given intravenously to a rat previously fed phytol. Endogenous biosynthesis, if it occurs at all, must be extremely limited.     

Compartmentation in Vicia faba Leaves: I. Kinetics of C in the Tissues following Pulse Labeling

Leaflets of Vicia faba were pulse-labeled with ¹⁴CO₂ to follow the subsequent movement of photosynthate between leaf tissues. Samples were taken during a ¹²CO₂ chase, quick frozen, freeze-substituted, and embedded in methacrylate. Paradermal sections provided tissue samples consisting only of upper epidermis, palisade parenchyma, spongy parenchyma and veins, spongy parenchyma, or lower epidermis. Most CO₂ fixation occurred in the palisade parenchyma, but its ¹⁴C content declined rapidly after labeling. Concomitant with the decline of activity in the palisade parenchyma, there was an increase in activity in the spongy parenchyma and upper epidermis and a slow increase in the lower epidermis. Activity in the palisade parenchyma and spongy parenchyma eventually reached similar levels and remained constant. Tissue samples containing veins were consistently the most radioactive, and activity in those samples showed a decline. Very little change occurred in the insoluble fraction from any tissue. The results support previous assumptions regarding the pathway of assimilate transport to the veins, and demonstrate the rapidity of such transport. Sucrose is apparently the principal mobile compound.     

Light versus Dark Carbon Metabolism in Cherry Tomato Fruits: II. Relationship Between Malate Metabolism and Photosynthetic Activity

The possible relationship between malate metabolism and photosynthetic activity in green tomato fruit tissues (Lycopersicum esculentum var. cerasiforme Dun A. Gray) was investigated. Initial experiments consisted of vacuum-infiltrating ¹⁴C-3 or ¹⁴C-4-malate into isolated tissues in darkness and then incubating the tissues under photosynthetic conditions. Other experiments involved a short pulse with ¹⁴C-bicarbonate in darkness to label the malate pool(s), followed by a chase in the light in the presence of nonradioactive bicarbonate. Both series of experiments were followed by the separation and identification of labeled metabolic intermediates.