<Emphasis Type="Italic">Amborella trichopoda</Emphasis>, plasmodesmata,and the evolution of phloem loading

Phloem loading is the process by which photoassimilates synthesized in the mesophyll cells of leaves enter the sieve elements and companion cells of minor veins in preparation for long distance transport to sink organs. Three loading strategies have been described: active loading from the apoplast, passive loading via the symplast, and passive symplastic transfer followed by polymer trapping of raffinose and stachyose. We studied phloem loading in Amborella trichopoda, a premontane shrub that may be sister to all other flowering plants. The minor veins of A. trichopoda contain intermediary cells, indicative of the polymer trap mechanism, forming an arc on the abaxial side and subtending a cluster of ordinary companion cells in the interior of the veins. Intermediary cells are linked to bundle sheath cells by highly abundant plasmodesmata whereas ordinary companion cells have few plasmodesmata, characteristic of phloem that loads from the apoplast. Intermediary cells, ordinary companion cells, and sieve elements form symplastically connected complexes. Leaves provided with ¹⁴CO₂ translocate radiolabeled sucrose, raffinose, and stachyose. Therefore, structural and physiological evidence suggests that both apoplastic and polymer trapping mechanisms of phloem loading operate in A. trichopoda. The evolution of phloem loading strategies is complex and may be difficult to resolve.     

Localization of galactinol,raffinose, and stachyose synthesis in Cucurbita pepo leaves

The biochemical pathway of stachyose synthesis was localized by immunocytochemical and ¹⁴C-labeling techniques in mature Cucurbita pepo L. leaves. Galactinol synthase (GaS; EC 2.4.1.123), the first unique enzyme in this pathway, was immunolocalized within the intermediary cells of minor veins in conventionally fixed and cryo-fixed, resin-embedded sections using polyclonal anti-GaS antibodies and protein A-gold. Intermediary cells are specialized companion cells with extensive symplastic connections to the bundle sheath. Gold particles were not seen over the non-specialized companion cells of larger veins or over intermediary cells in young leaves prior to the sink-source transition. In another approach to localization, radiolabel was measured in isolated mesophyll tissue and whole tissue of leaves that were lyophilized following a 90-s exposure to ¹⁴CO₂. Mesophyll, obtained by abrasion of the leaf surface, contained labeled sucrose, galactinol, raffinose and stachyose. However, the latter three labeled compounds constituted a smaller proportion of the neutral fraction than in whole-tissue samples, which also contained minor veins. We conclude that synthesis of galactinol, raffinose, and stachyose occurs in both mesophyll and intermediary cells, predominantly the latter.Abbreviations GaS galactinol synthase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank John Pierce, Phillip Kerr, and Brace Schweiger for the gift of anti-GaS antibody and M.K. Kandasamy for helpful discussions. This research was supported by National Science Foundation grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.     

Are sucrosyl-oligosaccharides synthesized in mesophyll protoplasts of mature leaves of Cucumis melo?

Biosynthesis of sucrosyl-oligosaccharides (raffinose, stachyose) was traced in source leaves of Cucumis melo after ¹⁴C-photoassimilation. The main carbon compound exported was ¹⁴C-labeled stachyose. No oligosaccharide synthesis was detected in young, importing leaves. Mesophyll protoplasts, isolated from mature leaves which had previously photosynthesized ¹⁴CO₂, did not contain ¹⁴C-oligosaccharides but contained [¹⁴C]-sucrose and ¹⁴C-hexoses. Isolated minor-vein-enriched fractions from the same leaves, however, showed nearly 30% of the ¹⁴C of the neutral fraction to be in oligosaccharides. Isolated, viable mesophyll protoplasts incubated with NaH¹⁴CO₃ also failed to incorporate radioactivity into oligosaccharides, although sucrose and galactinol synthesis was unimpaired. Galactinolsynthase activity in leaf extracts and in mesophyll protoplasts was 16.8 mol·h^-1·mg^-1 protein and 13.8 mol·h^-1·mg^-1 protein, respectively. Galactosyltransferase (EC 2.4.1.67), which synthesizes stachyose from raffinose and galactinol, had an activity of 50 nmol·h^-1·mg^-1 protein in leaf extracts and was also present in the minor-vein-enriched fraction, but could not be detected in mesophyll protoplast lysates. The results indicate that mesophyll cells may not be the site of stachyose synthesis although precursor compounds like sucrose and galactinol are synthesized there.Abbreviation HPLC high-performance liquid chromatography     

Phloem loading in cucumber: combined symplastic and apoplastic strategies

Phloem loading, as the first step of transporting photoassimilates from mesophyll cells to sieve element‐companion cell complex, creates a driving force for long‐distance nutrient transport. Three loading strategies have been proposed: passive symplastic loading, apoplastic loading and symplastic transfer followed by polymer‐trapping of stachyose and raffinose. Although individual species are generally referred to as using a single phloem loading mechanism, it has been suggested that some plants may use more than one, i.e. ‘mixed loading’. Here, by using a combination of electron microscopy, reverse genetics and ¹⁴C labeling, loading strategies were studied in cucumber, a polymer‐trapping loading species. The results indicate that intermediary cells (ICs), which mediate polymer‐trapping, and ordinary companion cells, which mediate apoplastic loading, were mainly found in the fifth and third order veins, respectively. Accordingly, a cucumber galactinol synthase gene (CsGolS1) and a sucrose transporter gene (CsSUT2) were expressed mainly in the fifth/third and the third order veins, respectively. Immunolocalization analysis indicated that CsGolS1 was localized in companion cells (CCs) while CsSUT2 was in CCs and sieve elements (SEs). Suppressing CsGolS1 significantly decreased the stachyose level and increased sucrose content, while suppressing CsSUT2 decreased the sucrose level and increased the stachyose content in leaves. After ¹⁴CO₂ labeling, [¹⁴C]sucrose export increased and [¹⁴C]stachyose export reduced from petioles in CsGolS1i plants, but [¹⁴C]sucrose export decreased and [¹⁴C]stachyose export increased into petioles in CsSUT2i plants. Similar results were also observed after pre‐treating the CsGolS1i leaves with PCMBS (transporter inhibitor). These results demonstrate that cucumber phloem loading depends on both polymer‐trapping and apoplastic loading strategies.     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Phloem Loading in Coleus blumei in the Absence of Carrier-Mediated Uptake of Export Sugar from the Apoplast

Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier-mediated transport of export sugar from the apoplast into the sieve element-companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p-chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of ¹⁴C-photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to ¹⁴CO₂. The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides.     

Apoplastic Transport of 14C-Photosynthates Measured under Drought and Nitrogen Supply

Using water infiltration of the plant and individual shoots with the subsequent intercellular liquid extraction by the pressure chamber, dynamics of the movement ¹⁴C-photosynthates from cell to apoplast, and ¹⁴C distribution among photosynthetic products in mesophyll cells and apoplast were studied. The relative quantity of ¹⁴C-photosynthetes in leaf apoplast depended on growing conditions; drought increased, and nitrate supply decreased it. When the middle leaves absorbed ¹⁴CO₂, photosynthates moving down in stem phloem appeared in intercellular space, where they were transported up by transpiration stream. ¹⁴C-photosynthates entering to the apex and young leaves were utilized a accumulated, and photosynthates transported to the mature leaves were reloaded into the phloem and reexported. Thus, photosynthates circulated through the plant and were redistributed to the plant organs according to their transpiration. In leaf apoplast photosynthetic sucrose was partly hydrolyzed to glucose and fructose. This increased under high nitrogen supply. The result indicate that apoplast sucrose hydrolysis is the basic cause of the reduction of photosynthate flux from leaves when the nitrate concentration in soil increases.     

Energized Uptake of Sugars from the Apoplast of Leaves: A Study of Some Plants Possessing Different Minor Vein Anatomy

Solutions of sucrose, glucose, raffinose, and stachyose were fed via the petiole to detached leaves of plant species known to transfer sugars during photosynthesis into the phloem using either the apoplastic or the symplastic pathway of phloem loading. Symplastic phloem loaders, which translocate raffinose-type oligosaccharides and sucrose in the phloem, and apoplastic plants, translocating exclusively sucrose, were selected for this study. As the sugars arrived with the transpiration stream in the leaf blade within little more than a minute, dark respiration increased. Almost simultaneously, fluorescence of a potential-indicating dye, which had been infiltrated into the leaves, indicated membrane depolarization. Another fluorescent dye used to record the apoplastic pH revealed apoplastic alkalinization that occurred with a slight lag phase after respiration and membrane depolarization responses. Occasionally, alkalinization was preceded by transient apoplastic acidification. Whereas membrane depolarization and apoplastic acidification are interpreted as initial responses of the proton motive force across the plasma membrane to the advent of sugars in the leaf apoplast, the following apoplastic alkalinization showed that sugars were taken up from the apoplast into the symplast in cotransport with protons. This was true not only for glucose and sucrose, but also for raffinose and stachyose. Similar observations were made for sugar uptake not only in leaves of plants known to export sugars by symplastic phloem loading but also of plants using the apoplastic pathway. Increased respiration during sugar uptake revealed tight coupling between respiratory ATP production and ATP consumption by proton-translocating ATPase of the plasma membrane, which exports protons into the apoplast, thereby compensating for the proton loss in the apoplast when protons are transported together with sugars into the symplast. The extent of stimulation of respiration by sugars indicated that sugar uptake was not limited to phloem tissue. Ratios of the extra CO₂ released during sugar uptake to the amounts of sugars taken up were variable, but lowest values were lower than 0.2. When a ratio of 0.2 is taken as a basis to calculate rates of sugar uptake from observed maxima of sugar-dependent increases in respiration, rates of sugar uptake approached 350 nmol/(m² leaf surface s). Sugar uptake rates were half-saturated at sugar concentrations in the feeding solutions of about 10–25 mM indicating a low in vivo affinity of sugar uptake systems for sugars.     

Efflux of sucrose from minor veins of tobacco leaves

Mature leaves import limited amounts of nutrient when darkened for prolonged periods. We tested the hypothesis that import is restricted by the apoplast-phloem loading mechanism, ie., as sucrose exits the phloem of minor veins it is retrieved by the same tissue, thus depriving the mesophyll of nutrient. When single, attached, mature leaves of tobacco (Nicotiana tabacum L.) plants were darkened, starch disappeared from the mesophyll cells, indicating that the supply of solute to the mesophyll was limited. Starch was synthesized in mesophyll cells of darkened tissue when sucrose was applied to the apoplast at 0.1–0.3 mM concentration. Efflux from minor veins was studied by incubating leaf discs on [¹⁴C]sucrose to load the minor veins and then measuring subsequent ¹⁴C release. Efflux was rapid for the first hour and continued at a gradually decreasing rate for over 13 h. Net efflux increased when loading was inhibited by p-chloromercuribenzene-sulfonic acid, anoxia, isotope-trapping, or reduction of the pH gradient. Neither light nor potassium had a significant effect on the rate of labeled sucrose release. The site of labeled sucrose release was investigated by measuring efflux from discs in which sucrose had previously been loaded preferentially by either the minor veins or mesophyll cells. Efflux occurred primarily from minor veins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - Mops 3(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid - SE-CC sieve element-companion cell complex     

Carbohydrate Metabolism in Photosynthetic and Nonphotosynthetic Tissues of Variegated Leaves of Coleus blumei Benth

Mature, variegated leaves of Coleus blumei Benth. contained stachyose and other raffinose series sugars in both green, photosynthetic and white, nonphotosynthetic tissues. However, unlike the green tissues, white tissues had no detectable level of galactinol synthase activity and a low level of sucrose phosphate synthase indicating that stachyose and possibly sucrose present in white tissues may have originated in green tissues. Uptake of exogenously supplied [¹⁴C]stachyose or [¹⁴C]sucrose into either tissue type showed conventional kinetic profiles indicating combined operation of linear first-order and saturable systems. Autoradiographs of white discs showed no detectable minor vein labelling with [¹⁴C]stachyose, but some degree of vein labeling with [¹⁴C]sucrose. Autoradiographs of green discs showed substantial vein loading with either sugar. In both tissues, p-chloromercuribenzenesulfonic acid had no effect on the linear component of sucrose or stachyose uptake but inhibited the saturable component. Both tissues contained high levels of invertase, sucrose synthase and α-galactosidase and extensively metabolized exogenously supplied ¹⁴C-sugars. In green tissues, label from exogenous sugars was recovered as raffinose-series sugars. In white tissues, exogenous sugars were hydrolysed and converted to amino acids and organic acids. The results indicate that variegated Coleus leaves may be useful for studies on both phloem loading and phloem unloading processes in stachyose-transporting species.     

Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in <Emphasis Type="Italic">Plantago major,Plantago maritima</Emphasis>, <Emphasis Type="Italic">Prunus persica</Emphasis>, and <Emphasis Type="Italic">Apium graveolens</Emphasis>

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem.     

Stachyose Synthesis in Source Leaf Tissues of the CAM Plant Xerosicyos danguyi H. Humb

Leaf tissues from Xerosicyos danguyi H. Humb., a succulent member of the Cucurbitaceae, were found to possess both galactinol synthase activity and the capacity for photosynthetic production of stachyose, the phloem transport oligosaccharide common to other nonsucculent cucurbits. The amounts of stachyose isolated from leaf tissues, and the extractable activity of galactinol synthase, were somewhat higher in leaf tissues obtained from plants operating in the Crassulacean acid metabolism (CAM) mode (well watered plants) compared to leaf tissues from plants operating in the CAM-idling mode (water-stressed plants). In contrast, in leaf discs, the photosynthetic incorporation of label into stachyose following pulse labeling with ¹⁴CO₂ was similar for stressed and for nonstressed tissues. Stachyose could be extracted from, and was synthesized photosynthetically by, leaf discs which contained no vascular tissues, indicating that synthesis of stachyose can occur in photosynthetic mesophyll cells of Xerosicyos.     

Distribution and immunolocalization of stachyose synthase in Cucumis melo L.

Indirect evidence for the site of stachyose biosynthesis has been provided by determining the occurrence and distribution of stachyose, raffinose and galactinol, the donor of the galactosyl moiety for stachyose synthesis, in Cucumis melo L. cv. Ranjadew. Studies of enzyme activities for the synthesis of these sugars and their distribution in different plant organs and isolates has led to the conclusion that stachyose is synthesized mainly in mature leaves and seeds. Nevertheless, stachyose-synthase activity varied with leaf age, the developmental stage of a plant, the growing season and the plant cultivar used. No stachyose or stachyose-synthase activity could be detected in isolated mesophyll protoplasts and chloroplasts, whereas both were found in a minor-vein-enriched fraction isolated from mature leaves. The conclusion that stachyose biosynthesis is associated with minor veins was confirmed by immunolocalization of the enzyme. Positive specific immunoreactivity of stachyose synthase with polyclonal anti-stachyose-synthase antibodies, labeled with protein A-gold, was detected in intermediary cells of leaf minor veins. The implication of this local synthesis of the main transport sugar for phloem loading in mature leaves of Cucumis melo is discussed.Abbreviation RUBPCase ribulose-1,5-bisphosphate carboxylase This work was supported by Deutsche Forschungsgemeinschaft. The excellent assistance of Ms. B. Müller in preparing the samples for electron microscopy is gratefully acknowledged. The authors thank Professor H.J. Schneider-Poetsch for anti-RuBPCase antibodies.     

Raffinose oligosaccharide concentrations measured in individual cell and tissue types in Cucumis melo L. leaves: implications for phloem loading

Raffinose, stachyose, and galactinol are synthesized in intermediary cells (specialized companion cells) of the minor-vein phloem of cucurbits. To better understand the role of these carbohydrates and the regulation of their synthesis and transport, we measured the concentrations of each of the components of the raffinose oligosaccharide synthetic pathway in mesophyll and sieve element-intermediary cell complexes (SE-ICCs) in the leaves of melon (Cucumis melo L. cv. Hale's Best Jumbo). These concentrations are consistent with a polymer-trapping mechanism for phloem loading, with sucrose diffusing from mesophyll into intermediary cells and being made into raffinose and stachyose, which are too large to diffuse back to the mesophyll. To determine carbohydrate concentrations, we developed a method involving microdissected tissues. Blind endings of areoles, and mesophyll surrounding these veins, were separately removed from lyophilized leaf tissue. Carbohydrates were quantitated by high-performance liquid chromatography with pulsed amperometric detection. A small amount of mesophyll remained attached to the blind endings; the carbohydrate contribution of these cells to the vein sample was eliminated by subtraction, based on the amount of chlorophyll. Volumes of cells and subcellular compartments were calculated by morphometric analysis and were used to calculate carbohydrate concentrations. Assuming no subcellular compartmentation, the additive concentration of sugars in the SE-ICCs of minor veins is about 600 mM. Stachyose and raffinose concentrations are about 330 mM and 70 mM, respectively, in SE-ICCs; concentrations of these sugars are much lower in mesophyll (0.2 and 0.1 mM). This is consistent with the view that stachyose and raffinose are unable to pass through the plasmodesmata between intermediary cells and bundle-sheath cells. Sucrose levels appear to be higher in the SE-ICC (about 130mM) than in the mesophyll (about 10 mM), but if compartmentation is taken into account the gradient for sucrose is probably downhill from mesophyll to intermediary cells. Flux through plasmodesmata between the bundle sheath and intermediary cells was calculated and was found to be within the range of values of flux through plasmodesmata reported in the literature.Abbreviations BS-IC bundle sheath-intermediary cell - PC plasmodesmatal channel - SE-ICC sieve element-intermediary cell complex - SEL size exclusion limit We would like to thank Gayle Volk, Philip Laible, Canan Inan, Esther Gowan, Richard Medville, Nathan Wilson, Jessica Plant, and Steven Boese for their help, Thomas Owens, M.V. Parthasarathy, and Ian Merwin for use of equipment, and Nancy Haritatos for suggestions. This research was supported by U.S. Department of Agriculture Competitive Grant 94-37306-0351 (R.T.), the Swiss National Foundation (F.K.), and a NSF/DOE/USDA Cornell Plant Science Center fellowship (E.H.).     

Phloem Loading of Sucrose: pH Dependence and Selectivity

Autoradiographic, plasmolysis, and ¹⁴C-metabolite distribution studies indicate that the majority of exogenously supplied ¹⁴C-sucrose enters the phloem directly from the apoplast in source leaf discs of Beta vulgaris. Phloem loading of sucrose is pH-dependent, being markedly inhibited at an apoplast pH of 8 compared to pH 5. Kinetic analyses indicate that the apparent K_m of the loading process increases at the alkaline pH while the maximum velocity, V_max, is pH-independent. The pH dependence of sucrose loading into source leaf discs translates to phloem loading in and translocation of sucrose from intact source leaves. Studies using asymmetrically labeled sucrose ¹⁴C-fructosyl-sucrose, show that sucrose is accumulated intact from the apoplast and not hydrolyzed to its hexose moieties by invertase prior to uptake. The results are discussed in terms of sucrose loading being coupled to the co-transport of protons (and membrane potential) in a manner consistent with the chemiosmotic hypothesis of nonelectrolyte transport.     

Low night temperatures inhibit galactinol synthase gene expression and phloem loading in melon leaves during fruit development

Low night temperatures seriously affect plant growth and fruit quality. To investigate the effect of low night temperatures on the expression of galactinol synthase genes (GOLS) and phloem loading of raffinose family oligosaccharides, particular stachyose and raffinose (RFO represents stachyose and raffinose in this paper) and to gain a better understanding of the relationship between the phloem loading of RFO and fruit development, melon (Cucumis melo L.) plants at the fruit development stage were treated with temperatures of 28/12°C or 28/9°C (day/night) with 28/15°C as the control. Both the CmGOLS1 and CmGOLS2 gene expression and the activity of galactinol synthase were clearly repressed after treatments with 9 and 12°C at night, and the effect of 9°C was more obvious. Furthermore, low night temperatures inhibited photosynthesis and caused the lower amounts of sucrose to supply the RFO synthesis. However, the total soluble sugar, RFO, and sucrose contents were increased in leaves subjected to low night temperatures. It is supposed that low night temperature blocked symplastic phloem loading, which led to the accumulation of RFO in the leaf cells. With increasing content of RFO in the leaves, the expression of GOLS genes was inhibited according to the principle of feedback, and therefore the decreased expression of GOLS limited RFO synthesis and was indirectly harmful to phloem loading, thereby affecting fruit development.     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.     

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.     

摘除雌花对甜瓜成熟叶片中糖及相关酶活性的影响

甜瓜有果株的成熟叶片中蔗糖、葡萄糖、果糖含量与无果株的无显著差异,水苏糖与棉子糖含量略低于无果株,肌醇半乳糖苷(合成水苏糖的前体)含量显著低于无果株,蔗糖磷酸合成酶(SPS)和肌醇半乳糖苷合成酶活性与无果株的无显著差异,水苏糖合成酶活性显著高于无果株。     

Stachyose synthesis in leaves of Cucurbita pepo

An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC2.4.1.67), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (K_m 7.7 mM) and the acceptor raffinose (K_m 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co²⁺, Hg²⁺, Mn²⁺ and Ni²⁺ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (K_i 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves ofC. pepo proceeds via this galactosyltransferase and not via α-galactosidase.