<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.     

Phloem loading in two Scrophulariaceae species. What can drive symplastic flow via plasmodesmata?

To determine the driving forces for symplastic sugar flux between mesophyll and phloem, gradients of sugar concentrations and osmotic pressure were studied in leaf tissues of two Scrophulariaceae species, Alonsoa meridionalis and Asarina barclaiana. A. meridionalis has a typical symplastic configuration of minor-vein phloem, i.e. intermediary companion cells with highly developed plasmodesmal connections to bundle-sheath cells. In A. barclaiana, two types of companion cells, modified intermediary cells and transfer cells, were found in minor-vein phloem, giving this species the potential to have a complex phloem-loading mode. We identified all phloem-transported carbohydrates in both species and analyzed the levels of carbohydrates in chloroplasts, vacuoles, and cytoplasm of mesophyll cells by nonaqueous fractionation. Osmotic pressure was measured in single epidermal and mesophyll cells and in whole leaves and compared with calculated values for phloem sap. In A. meridionalis, a 2-fold concentration gradient for sucrose between mesophyll and phloem was found. In A. barclaiana, the major transported carbohydrates, sucrose and antirrhinoside, were present in the phloem in 22- and 6-fold higher concentrations, respectively, than in the cytoplasm of mesophyll cells. The data show that diffusion of sugars along their concentration gradients is unlikely to be the major mechanism for symplastic phloem loading if this were to occur in these species. We conclude that in both A. meridionalis and A. barclaiana, apoplastic phloem loading is an indispensable mechanism and that symplastic entrance of solutes into the phloem may occur by mass flow. The conditions favoring symplastic mass flow into the phloem are discussed.     

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.     

Minor vein structure and sugar transport in Arabidopsis thaliana

Leaf and minor vein structure were studied in Arabidopsis thaliana (L.) Heynh. to gain insight into the mechanism(s) of phloem loading. Vein density (length of veins per unit leaf area) is extremely low. Almost all veins are intimately associated with the mesophyll and are probably involved in loading. In transverse sections of veins there are, on average, two companion cells for each sieve element. Phloem parenchyma cells appear to be specialized for delivery of photoassimilate from the bundle sheath to sieve element-companion cell complexes: they make numerous contacts with the bundle sheath and with companion cells and they have transfer cell wall ingrowths where they are in contact with sieve elements. Plasmodesmatal frequencies are high at interfaces involving phloem parenchyma cells. The plasmodesmata between phloem parenchyma cells and companion cells are structurally distinct in that there are several branches on the phloem parenchyma cell side of the wall and only one branch on the companion cell side. Most of the translocated sugar in A. thaliana is sucrose, but raffinose is also transported. Based on structural evidence, the most likely route of sucrose transport is from bundle sheath to phloem parenchyma cells through plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and carrier-mediated uptake into the sieve element-companion cell complex. Received: 5 October 1999 / Accepted: 20 November 1999     

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 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.     

Long-distance trafficking of macromolecules in the phloem

The fact that macromolecules such as proteins and mRNAs overcome the symplastic barriers between various tissue domains was first evidenced by the movement of plant viruses. We have recently demonstrated that viral infection disengages the symplastic restriction present between the sieve element-companion cell complex and neighboring cells in tobacco plants. As a result, green fluorescent protein, which was produced in mesophyll and bundle sheath cells, could traffic into the sieve tube and travel long distances within the vascular system. In this addendum we discuss the likely existence of a novel plant communication network in which macromolecules also act as long-distance trafficking signals. Plasmodesmata interconnecting sieve elements and companion cells as well as plasmodesmata connecting the sieve tube with neighboring cells may play a central role in establishing this communication network.Key words: companion cells, cucumber mosaic virus, Cucumis melo, plasmodesmata, movement protein, sieve-elementsTranslocation of photoassimilates from the source (site of synthesis) to various sink organs is governed, in part, by short-distance intercellular transfer of assimilates to the loading region of the phloem and long-distance transport within the plant vascular system. Sucrose, which is synthesized in the leaf mesophyll, moves cell-to-cell symplastically through plasmodesmata until it reaches the boundary of the sieve element (SE)-companion cell (CC) complex. In many plant species, the connection between phloem parenchyma (PP)/bundle sheath (BS) cells and CCs is characterized by a sparseness of plasmodesmata (e.g., Solanaceae), and sucrose is exported out of the cells to the apoplast. This type of plants (apoplastic loaders) uses sucrose proton symporters to load the sucrose into the vasculature.¹ Cucurbits are considered one of the model plants for symplastic phloem loading.² This type of plant is characterized by abundant plasmodesmata interconnecting the intermediary cells, which are specialized CCs, with the neighboring BS cells. It is generally accepted that in these plants, phloem loading includes intercellular movement of sucrose through the plasmodesmata, along the entire pathway from the mesophyll cell to the SE-CC complex.Interestingly, the existence of plasmodesmata interconnecting the SE-CC complex and neighboring cells is evident in all plant species that are characterized by an apoplastic phloem-loading mechanism. Moreover, microinjection experiments have indicated that plasmodesmata interconnecting the PP-CC are functional, in that they allow the exchange of small membrane-impermeable fluorescent probes.³ Virus movement through plasmodesmata from the mesophyll into the SEs further supports the notion that the symplastic communication between the CC-SE complex and the neighboring cells is functional.⁴One can assume that in apoplastic-loading plants, it would be an advantage to maintain the SE-CC complex as an isolated domain, with no functional plasmodesmata interconnecting it to the neighboring tissue. Symplastic continuity between the two domains could result in leakage of sucrose out of the vasculature and a significant reduction in the efficacy of sucrose loading. The fact that the two domains are interconnected suggests that any back-leakage of sucrose that might occur is insignificant relative to the likely efficacy of this communication route.What might the advantage be for symplastic communication between the SE-CC complex and the neighboring tissue? Accumulated evidence suggests that at the tissue/organ level, cell-to-cell trafficking of information molecules allows for noncell-autonomous control over a range of processes, whereas at the organismal level, the phloem serves as an information superhighway, delivering a wide range of macromolecules to enable the plant to function as a whole organism.^5–8 We advanced the hypothesis that plasmodesmata interconnecting the CCs and PP/BS cells play a pivotal role in controlling the long-distance trafficking of putative signaling molecules.     

Structural and ultrastructural characteristics of the vascular apparatus of the sensitive plant (Mimosa pudica L.)

Summary 1. In motor organs ofMimosa pudica xylem contains living fibriform elements limited by a thick lignified highly pitted wall, whereas in other parts of the plant (stem, petiole, rachis), xylem and protoxylem vessels are closely associated with parenchyma cells which possess wall ingrowths. These ingrowths, at the apex of which the plasmalemma and the tonoplast touch, are localized like those of transfer cells of C type described byGunning andPate. Nevertheless, xylem parenchyma cells differ from cells of C type in several characteristics. Moreover, in motor organs, phloem contains cells characterized by wall ingrowths, less abundant on the parts adjacent to the sieve tubes; these cells which are localized near collenchyma cells of primary phloem, look like transfer cells of A type defined byGunning andPate; they are absent from internodes, petioles and rachides. 2. In motor organs, three types of vascular cells (companion cells, living xylem fibriform elements and protoxylem parenchyma cells) are characterized by reduced vacuolar volumes and well developed membrane systems, as compared with homologuous cells belonging to other parts of the plant. 3. A symplastic continuity holds from the middle of motor organs to their cortex: it is provided by the presence, in xylem and phloem respectively, of living fibriform elements and collenchyma cells bearing numerous pit fields containing large numbers of plasmodesmata. Several ultrastructural features suggest that the vascular apparatus ofMimosa pudica would be the site of intensive lateral transfer at different levels, specially in motor organs. Possible functions of certain structures observed are discussed in relation to some hypotheses relative to excitatory conduction pathways.     

Degradation of stachyose,raffinose, melibiose and sucrose by different tempe-producing Rhizopus fungi

Forty-six strains of tempe-forming Rhizopus species were screened for their ability to grow on raffinose as the sole carbon source. Six of the strains showed good growth and sporulation. These isolates were one Rhizopus oligosporus, one Rhizopus microsporus var. chinensis, three Rhizopus oryzae and one Rhizopus stolonifer. These six moulds and R. oligosporus strain NRRL 2710 were investigated for their metabolism of the raffinose family of -galactoside carbohydrates. Degradation experiments were performed in submerged culture in a medium containing soybean -protein, sodium phytate and either stachyose, raffinose or melibiose. R. oryzae and R. stolonifer completely consumed the tested carbohydrates as carbon source. R. microsporus var. chinensis failed to hydrolyse the -galactosidic bonds of raffinose, stachyose or melibiose, whereas it was able to use sucrose and the fructose moiety of raffinose or stachyose for growth. R. oligosporus NRRL 2710 was unable to hydrolyse any of the tested carbohydrates. The results of the oligosaccharide degradation experiments could be verified during tempe production from soybeans with the selected fungal species.     

Significance of minor-vein anatomy to carbohydrate transport

Plant species which translocate distinct combinations of carbohydrates in the phloem were investigated to assess whether differences in minor-vein anatomy were associated with differences in carbohydrate composition of the phloem sap. In Vicia faba L., a species in which the minor-vein companion cells are modified into transfer cells, sucrose alone was found to be the translocated form of carbohydrate. In Vicia, phloem transport of sucrose was inhibited by pretreatment of leaves with p-chloromercuribenzenesulfonic acid (PCMBS), a known inhibitor of the sucrose carrier. In contrast, in Ocimum basilicum L., a species in which the minor-vein companion cells are of the symplasmically linked intermediary cell type, both sucrose- and raffinose-family oligosaccharides were exported in the phloem. In this species, no PCMBS sensitivity was observed for phloem transport of either sucrose- or raffinose-family oligosaccharides, although a PCMBS-sensitive sucrose carrier was detected in leaf tissues. This carrier did not appear to be involved in phloem loading, rather, it appeared that phloem loading occurred via the symplasm in this species. In the polyoltranslocating species Petroselinum crispum L., the same insensitivity to PCMBS was seen, suggesting that symplasmic phloem loading also occurred. The companion cells were symplasmically connected to the surrounding bundle-sheath cells by numerous H-shaped plasmodesmata but were not intermediary cells, and no raffinose oligosaccharides were exported by Petroselinum. Taken together, the data indicate that apoplasmic transport may be responsible for phloem loading in species in which sucrose alone is exported. However, in those plant species in which a combination of sucrose and any other carbohydrate, including the polyols, is translocated, symplasmic phloem loading may predominate.Abbreviation PCMBS p-chloromercuribenzenesulfonic acid This work was supported by National Science Foundation Grant DCB 8901785 to M.A.M. and by a National Science Foundation Graduate Minority Fellowship to L.L.F. The authors gratefully acknowledge the help of Dr. William W. Thomson in preparing the micrograph.     

Ultrastructure of minor veins inCucurbita pepo leaves

Summary The minor veins ofCucurbita pepo leaves were examined as part of a continuing study of leaf development and phloem transport in this species. The minor veins are bicollateral along their entire length. Mature sieve elements are enucleate and lack ribosomes. There is no tonoplast. The sieve elements, which are joined to each other by sieve plates, contain mitochondria, plastids and endoplasmic reticulum as well as fibrillar and tubular (190–195 diameter) P-protein. Fibrillar P-protein is dispersed in mature abaxial sieve elements but remains aggregated as discrete bodies in mature adaxial sieve elements. In both abaxial and adaxial mature sieve elements tubular P-protein remains undispersed. Sieve pores in abaxial sieve elements are narrow, lined with callose and are filled with P-protein. In adaxial sieve elements they are wide, contain little callose and are unobstructed. The intermediary cells (companion cells) of the abaxial phloem are large and dwarf the diminutive sieve elements. Intermediary cells are densely filled with ribosomes and contain numerous small vacuoles and many mitochondria which lie close to the plasmalemma. An unusually large number of plasmodesmata traverse the common wall between intermediary cells and bundle sheath cells suggesting that the pathway for the transport of photosynthate from the mesophyll to the sieve elements is at least partially symplastic. Adaxial companion cells are of approximately the same diameter as the adaxial sieve elements. They are densely packed with ribosomes and have a large central vacuole. They are not conspicuously connected by plasmodesmata to the bundle sheath.     

PLASMODESMATAL FREQUENCY AND OTHER STRUCTURAL ASPECTS OF ASSIMILATE COLLECTION AND PHLOEM LOADING IN LEAVES OF SONCHUS OLERACEUS (ASTERACEAE), A SPECIES WITH MINOR VEIN TRANSFER CELLS

Leaves of Sonchus oleraceus (Asteraceae) were examined with the electron microscope to determine plasmodesmatal frequencies and other structural features relating to the collection of photoassimilate and its subsequent loading into minor veins. Few plasmodesmata occur between mesophyll cells, which contain chloroplasts that are sometimes connected to both the plasmalemma and the tonoplast by membranous tubules. The minor veins consist of tracheary elements, sieve-tube members, vascular parenchyma cells, and companion cells. The latter two cell types are transfer cells, with some of the fingerlike wall ingrowths in companion cells being traversed lengthwise by plasmodesmata. The frequencies of plasmodesmata at the mesophyllbundle sheath boundary and within are higher at some interfaces than at corresponding interfaces in nine other species, including some that previously had been characterized as loading assimilate via the symplast. It is thus premature to designate all species containing transfer cells in their minor veins as loading assimilate only via the apoplast.     

Pathway of assimilate transfer between mesophyll cells and minor veins in leaves of Cucumis melo L.

Photoassimilating mature leaves of Cucumis melo exported carbon at a rate of 1.7 mg C·dm^-2·h^-1. Radiolabeling with ¹⁴C showed that stachyose and raffinose are the main carbohydrates translocated. Autoradiograms indicated that sieve elements of the abaxial phloem of minor veins are the sole conduits for carbon export from mature leaves and carbon import into immature leaflets. Sieve elements of the abaxial phloem are associated with intermediary cells which are intimately connected with the surrounding mesophyll cells by numerous plasmodesmata. Photoassimilate, labeled with ¹⁴C, was released into the leaf apoplast and could be trapped in a buffer solution circulating over the abraded adaxial epidermis. Carbon efflux was 1% of the carbon-export rate. A comparable distribution of ¹⁴C among the sugars, amino acids and organic acids, recovered from the free space and from leaf extracts, was recorded. The composition of released ¹⁴C-labeled carbohydrates in the free space resembled the pattern of photoassimilate, but differed clearly from the translocate. Release of organic compounds into the leaf apoplast was stimulated by chelating agents like Na-ATP, ethylenediaminetetraacetic acid and ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid; a correlation between carbon efflux into the apoplast and carbon export from the leaf was not detected. It is suggested that the release of organic compounds into the leaf apoplast of Cucumis melo is the consequence of a general leakage from mesophyll and vascular parenchyma cells. A selective release of transport oligosaccharides was not observed. The experimental results presented here do not preclude a symplastic transfer of assimilates in mature leaves.Abbreviations EDTA ethylenediaminetetraacetate - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetate - PCMBS p-chloromercuribenzenesulfonic acid     

Sugar Transport into Storage Tubers of Stachys sieboldii Miq.: Evidence for Symplastic Unloading and Stachyose Uptake into Storage Vacuoles by an H+-Antiport Mechanism*

Following assimilation of ¹⁴CO₂ by leaves of Stachys sieboldii, ¹⁴C-stachyose is translocated into the tubers. Stachyose is accumulated and stored in the vacuoles of the pith parenchyma. Protoplasts and vacuoles were isolated and the uptake of sugars was examined. Uptake of sucrose and sucrosyl oligosaccharides of the raffinose family by protoplasts was very low compared to glucose. Transport parameters for glucose indicated a carrier mediated transport in the lower concentration range which was superimposed by diffusion at higher concentrations (> 10 mM). The very low sugar uptake by protoplasts and the sparse enzyme activities of stachyose synthase in the storage parenchyma as well as acid invertase and α-galactosidase in the cell walls indicated symplastic unloading of stachyose in the tubers. Experiments on ¹⁴C-stachyose uptake by isolated vacuoles confirmed previous observations by Keller (1992). Isolated vacuoles exhibited ATP and PP hydrolysis and were capable of generating a proton gradient across the tonoplast by a V-type H⁺-ATPase and H⁺-PPase. This was demonstrated by fluorescence quenching of quinacrine. Fluorescence could be restored by the addition of gramicidin and partly recovered by the addition of stachyose; mannitol, sorbitol and glucose had no effect. Fluorescence recovery depended on the concentration of stachyose and revealed saturation kinetics (K_m = 28 mM). Comparable results have been obtained with tonoplast vesicles by Greutert and Keller (1993). Experimental data presented here provide circumstantial evidence for symplastic unloading of stachyose in the tubers of Stachys sieboldii and demonstrate that the stachyose concentration in the cytoplasm of storage parenchyma cells is kept low by active stachyose transport into the vacuoles. The results suggest a stachyose/H⁺-antiport system.     

Intercellular Symplastic Connection and Isolation of the Unloading Zone in Flesh of the Developing Grape Berry

The uhrastructure and intercellular connection of the sugar unloading zone (i. e. the phloem in the dorsal vascular bundle and the phloem-surrounding the assimilate sink-cells) of grape ( Vitis vinifera x V. labrusca cv. Jingchao) berry was observed via transmission electron microscopy. The results showed that during the early developmental stages of grape berry, numerous plasmodesmata were found in the phloem between sieve element (SE) and companion cell (CC), between SE/CC complexes, between SE/CC complex and phloem parenchyma cell and in between phloem parenchyma cells, which made the phloem a symplastic integration, facilitating sugar unloading from sieve elements into both companion cells and phloem parenchyma cells via a symplastic pathway. On the contrary, there was almost no plasmodesma between phloem and its surrounding flesh photoassimilate sink-cells, neither in between the flesh photoassimilate sink-cells giving rise to a symplastic isolation both between phloem and its surrounding flesh photoassimilate sink-cells, as well as among the flesh photoassimilate sink-cells. This indicated that both the sugar unloading from phloem and pestphloem transport of sugars should be mainly via an apoplastic pathway. Dining the ripening stage, most of the plasmodesmata between SE/CC complex and the surrounding phloem parenchyma cells were shown to be blocked by the electron-opaque globules, and a phenomenon of plasmolysis was found in a number of companion cells, indicating a symplastic isolation between SE/CC complex and its surrounding parenchynm cells during this phase. The symplastic isolation between the whole phloem and its surrounding photoassimilate sink-cells during the early developmental stages shifted to a symplastic isolation within the phloem during the ripening phase, and thus the symplastic pathway of sugar unloading from SE/CC complex during the early development stages should be replaced by a dominant apoplastic unloading pathway from SE/CC complex in concordance.     

Translocation velocity and specific mass transfer in the sieve tubes of Fraxinus americana L.

Summary The ratios of sugar concentrations (sucrose/stachyose, raffinose/stachyose and sucrose/raffinose) in the sieve-tube exudate of Fraxinus americana L. undergo slight diurnal fluctuations. These ratio waves have been found to move down along the stem of trees at a velocity of 30–70 cm/h. They are, in contrast to the concentration waves of Huber et al. (1937), independent of the absolute exudate concentration and thus unaffected by hydrodynamic pressures changes in the xylem. The velocity is of the order of magnitude required by the mass transfer equation and thus indicates that sieve tube exudate is a moving solution. Furthermore, the phenomenon enabled us for the first time to follow phloem transport over distances of 12 meters.     

Synthesis of α-galactooligosaccharides with α-galactosidase from<Emphasis Type="Italic"> Lactobacillus reuteri</Emphasis> of canine origin

Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, -galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 °C (K_M 0.55 mM, V_max 0.80 mol min^–1 mg^–1 of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6 -galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.     

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