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.

Willow (Salix babylonica L.) is representative of a large group of plants that have extensive plasmodesmatal connections between minor vein phloem and adjoining cells. Because plasmodesmata provide a diffusion pathway for small molecules, it is unclear how sucrose could be loaded from the mesophyll into the phloem against a concentration gradient. In the studies reported here, the minor vein phloem of willow leaves plasmolyzed in approximately the same concentration of osmoticum as the mesophyll. Sucrose concentrations in mesophyll cells were greater than those reported in the literature for aphid stylet exudate from willow stems. Calculated turgor pressures in the mesophyll and minor vein phloem were greater than turgor reported in the literature for sieve elements in the stems of willow. Images of minor veins were not obtained in autoradiographs when attached leaves, or leaf pieces, were provided with 14 CO 2 or [ 14 C]sucrose. Therefore, no evidence could be found for accumulation of sucrose against a concentration gradient in the minor vein phloem of willow. In these leaves, the mesophyll apparently acts as the ''source'' for long distance transport of sugar. The mechanism of translocation in willow, and the evolution of phloem loading, are discussed.

Plant viruses encode movement proteins that are essential for infection of the host but are not required for viral replication or encapsidation. Squash leaf curl virus (SqLCV), a bipartite geminivirus with a single-stranded DNA genome, encodes two movement proteins, BR1 and BL1, that have been implicated in separate functions in viral movement. To further elucidate these functions, we have investigated the nucleic acid binding properties and cellular localization of BR1 and BL1. In this study, we showed that BR1 binds strongly to single-stranded nucleic acids, with a higher affinity for single-stranded DNA than RNA, and is localized to the nucleus of SqLCV-infected plant cells. In contrast, BL1 binds only weakly to single-stranded nucleic acids and not at all to double-stranded DNA. The nuclear localization of BR1 and the previously demonstrated plasma membrane localization of BL1 were also observed when these proteins were expressed from baculovirus vectors in Spodoptera frugiperda insect cells. The biochemical properties and cellular locations of BR1 and BL1 suggest a model for SqLCV movement whereby BR1 is involved in the shuttling of the genome in and/or out of the nucleus and BL1 acts at the plasma membrane/cell wall to facilitate viral movement across cell boundaries.

Plant viruses encode movement proteins that are essential for infection of the host but are not required for viral replication or encapsidation. Squash leaf curl virus (SqLCV), a bipartite geminivirus with a single-stranded DNA genome, encodes two movement proteins, BR1 and BL1, that have been implicated in separate functions in viral movement. To further elucidate these functions, we have investigated the nucleic acid binding properties and cellular localization of BR1 and BL1. In this study, we showed that BR1 binds strongly to single-stranded nucleic acids, with a higher affinity for single-stranded DNA than RNA, and is localized to the nucleus of SqLCV-infected plant cells. In contrast, BL1 binds only weakly to single-stranded nucleic acids and not at all to double-stranded DNA. The nuclear localization of BR1 and the previously demonstrated plasma membrane localization of BL1 were also observed when these proteins were expressed from baculovirus vectors in Spodoptera frugiperda insect cells. The biochemical properties and cellular locations of BR1 and BL1 suggest a model for SqLCV movement whereby BR1 is involved in the shuttling of the genome in and/or out of the nucleus and BL1 acts at the plasma membrane/cell wall to facilitate viral movement across cell boundaries.

(R.T.); and Electron Microscopy Services and Consultants, Phoenix, Arizona 85022 (R.M.)The incidence of plasmodesmata in the minor vein phloem of leaves varies widely between species. On this basis, two pathways of phloem loading have been proposed: symplastic where frequencies are high, and apoplastic where they are low. However, putative symplastic-loading species fall into at least two categories. In one, the plants translocate raffinose-family oligosaccharides (RFOs). In the other, the primary sugar in the phloem sap is sucrose (Suc). While a thermodynamically feasible mechanism of symplastic loading has been postulated for species that transport RFOs, no such mechanism is known for Suc transporters. We used p-chloromercuribenzenesulfonic acid inhibition of apoplastic loading to distinguish between the two pathways in three species that have abundant minor vein plasmodesmata and are therefore putative symplastic loaders. Clethra barbinervis and Liquidambar styraciflua transport Suc, while Catalpa speciosa transports RFOs. The results indicate that, contrary to the hypothesis that all species with abundant minor vein plasmodesmata load symplastically, C. barbinervis and L. styraciflua load from the apoplast. C. speciosa, being an RFO transporter, loads from the symplast, as expected. Data from these three species, and from the literature, also indicate that plants with abundant plasmodesmata in the minor vein phloem have abundant plasmodesmata between mesophyll cells. Thus, plasmodesmatal frequencies in the minor veins may be a reflection of overall frequencies in the lamina and may have limited relevance to phloem loading. We suggest that symplastic loading is restricted to plants that translocate oligosaccharides larger than Suc, such as RFOs, and that other plants, no matter how many plasmodesmata they have in the minor vein phloem, load via the apoplast.Phloem loading is an energy-requiring process that elevates the solute content of sieve elements (SEs) and companion cells (CCs) to levels far above those of surrounding cells (Geiger et al., 1973;van Bel, 1993;Grusak et al., 1996;Turgeon, 1996Turgeon, , 2000Beebe and Russin, 1999;Hellmann et al., 2000;Komor, 2000;Lalonde et al., 2003). The pressure gradient between source and sink organs drives long-distance transport. It has been suggested that there are two loading strategies-apoplastic and symplastic. This hypothesis derived originally from the observation that there is great variation in the number of plasmodesmata in the phloem of minor veins (Turgeon et al., 1975;Gamalei, 1989Gamalei, , 1990Gamalei, , 1991Gamalei, , 2000van Bel, 1993). Gamalei has classified over 1,000 species on this basis and finds that it is a relatively consistent feature at the family level. He terms those with the highest number of minor vein plasmodesmata as type 1.The physical basis for phloem loading via the apoplast is reasonably well understood; Suc enters the cell wall space and is driven across the plasma membranes of CCs and/or SEs by cotransport with protons (Lalonde ...