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A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80–91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and K m value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.

Amino acid and sucrose contents were analyzed in the chloroplastic, cytosolic, and vacuolar compartments and in the phloem sap of illuminated spinach leaves (Spinacia oleracea L.). The determination of subcellular metabolite distribution was carried out by nonaqueous fractionation of frozen and lyophilized leaf material using a novel three-compartment calculation method. The phloem sap was collected by aphid stylets which had been severed by a laser beam. Subcellular analysis revealed that the amino acids found in leaves are located mainly in the chloroplast stroma and in the cytosol, the sum of their concentrations amounting to 151 and 121 millimolar, respectively, whereas the amino acid concentrations in the vacuole are one order of magnitude lower. The amino acid concentrations in the phloem sap are found to be not very different from the cytosolic concentrations, whereas the sieve tube concentration of sucrose is found to be one order of magnitude higher than in the cytosol. It is concluded that the phloem loading results in a preferential extraction of sucrose from the source cells.

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.

A comparison of barley (Hordeum vulgare L.) leaves was made between the cytosolic content of amino acids and sucrose as determined by subcellular fractionation and the corresponding concentration in phloem sap, which was collected continuously for up to 6 days from severed aphid stylets. Because amino acids were found to be almost absent from the vacuoles, and because the amino acid patterns in the stroma and cytosol are similar, whole leaf contents could be taken as a measure of cytosolic amino acid levels for a comparison of data during a diurnal cycle. The results show that the pattern of amino acids in the phloem sap was very similar to the pattern in the cytosol. Therefore, we concluded that the overall process of transfer of amino acids from the cytosol of the source cells into the sieve tubes, although carrier mediated, may be a passive process and that the translocation of amino acids via the sieve tubes requires the mass flow of sucrose driven by the active sucrose transport involved by the phloem loading.