Protoplasma (2011) 248:173–180 DOI 10.1007/s00709-010-0237-2
ORIGINAL ARTICLE
Amborella trichopoda, plasmodesmata, and the evolution of phloem loading Robert Turgeon & Richard Medville
Received: 6 July 2010 / Accepted: 26 October 2010 / Published online: 16 November 2010 # Springer-Verlag 2010
Abstract 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.
Handling Editor: Karl Oparka R. Turgeon (*) Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA e-mail:
[email protected] R. Medville Electron Microscopy Consultants, LLC, Colorado Springs, CO 80920, USA
Keywords Amborella trichopoda . Intermediary cell . Phloem loading . Plasmodesmata . Raffinose . Stachyose
Introduction Phloem loading, the process in which photoassimilates and other nutrients are transported from the mesophyll into the minor vein phloem, is a major determinant of carbon partitioning and nutrient distribution in general (Schobert et al. 2004; Schulz 2005; Turgeon and Ayre 2005; Turgeon 2006, 2010; Slewinski and Braun 2010; Turgeon and Wolf 2009; Dinant and Lemoine 2010). Three loading strategies— one passive and two active—are known, but it is not clear what selective advantages they bestow. As a first step in resolving this issue, it is important to determine in which groups, and under what circumstances, the different strategies have evolved. Passive loading is driven by high concentrations of photoassimilates in the cytosol of mesophyll cells (Turgeon and Medville 1998; Reidel et al. 2009; Rennie and Turgeon 2009). These compounds simply diffuse through plasmodesmata into the phloem and without any further elevation in concentration establish enough hydrostatic pressure in the sieve elements, by osmosis, to drive long distance transport. However, it is more common, especially in herbaceous species, for photoassimilates to accumulate actively, against a thermodynamic gradient, in the minor vein phloem. Two strategies of active phloem loading have been described. In one, the loading pathway involves an apoplastic step (Sauer 2007; Dinant and Lemoine 2010). Sucrose migrates to the veins, probably within the symplast, and in the vicinity of the minor vein phloem it effluxes into the apoplast. The sucrose is then retrieved from the apoplast by the
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companion cells and/or sieve elements. Plasma membrane sucrose transporters mediate the process, and the proton motive force provides the energy. Apoplastic phloem loading is widespread in the angiosperms (Gamalei 1989, 1991; Turgeon et al. 2001), and considerable progress has been made in identifying the transporters involved (Sauer 2007; Slewinski and Braun 2010; Dinant and Lemoine 2010). A second, species-specific, active loading pathway is symplastic. The mechanism is restricted to species in which raffinose and stachyose are abundant sugars in the phloem sap (Schulz 2005; McCaskill and Turgeon 2007; Zhang and Turgeon 2009). These plants are also characterized by the presence of a specialized form of minor vein companion cell, known as the “intermediary cell” (Turgeon et al. 1993; Flora and Madore 1996; Sprenger and Keller 2000; Hoffmann-Thoma et al. 2001; Knop et al. 2001; Turgeon and Ayre 2005). Intermediary cells have only been recognized in species that translocate abundant raffinose and stachyose. Intermediary cells are especially large companion cells with respect to their adjacent sieve elements. They are connected to bundle sheath cells by large numbers of highly branched plasmodesmata. The plasmodesmatal branches on the intermediary cell side of the common wall are more abundant than those on the bundle sheath side. The tight correlation between the presence of intermediary cells and raffinose and stachyose transport is the basis of the “polymer trap” model of symplastic phloem loading (Turgeon et al. 1993). The essence of this model is that sucrose diffuses from the bundle sheath into intermediary cells through the numerous plasmodesmata that connect the two cell types. In the intermediary cells, it is converted into raffinose and stachyose, thus keeping the concentration of sucrose low and permitting continued diffusion. It is hypothesized that raffinose and stachyose are prevented from diffusing in the opposite direction, back into the bundle sheath, by the narrowness of these specialized plasmodesmatal channels. Thus, raffinose and stachyose are trapped in the minor vein phloem and are transported to sink tissues. Gamalei (1989, 1991) studied the evolution of phloem loading by conducting a large-scale survey of minor vein anatomy at the electron microscope level and projecting his results onto the Takhtajan phylogenetic system. He assumed loading to be either symplastic or apoplastic in plants with abundant or few minor vein plasmodesmata, respectively. No distinction was made between passive and active modes of symplastic loading, in other words between plants with intermediary cells and those that simply have large numbers of plasmodesmata. On the basis of this survey, Gamalei (1989, 1991) and van Bel and Gamalei (1992) concluded that symplastic loading is characteristic
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of ancestral families, especially in the tropics, and that apoplastic loading evolved later as the angiosperms became more herbaceous and moved into colder and drier regions of the world. In a subsequent analysis, based on recent phylogenetic treatments (Turgeon et al. 2001), symplastic loading was examined in more detail and it was found that the polymer trap mechanism, identified by the presence of intermediary cells and the transport of raffinose and stachyose, is not ancestral; rather, it has evolved multiple times in more recent clades. However, due to a paucity of data, there remains a considerable amount of ambiguity concerning the evolution of loading strategies in early angiosperm history (Turgeon et al. 2001). The ancestral form of phloem loading in the angiosperms remains unknown. To further our understanding of the evolution of phloem loading, we examined the ultrastructure of minor veins and the nature of the translocated sugars in mature leaves of Amborella trichopoda. A. trichopoda is a premontane shrub that grows in cloud forest habitats in New Caledonia. There is a great deal of interest in this plant, the sole member of the Amborellaceae, since most, but not all, phylogenetic analyses indicate that it is sister to all other extant angiosperms (Graham and Iles 2009 and references therein). Our results indicate that the minor veins of A. trichopoda are large, with numerous companion cells characteristic of apoplastic loaders. Intermediary cells surround the veins on the abaxial side. Sucrose, raffinose, and stachyose are transported. Therefore, A. trichopoda exhibits an unusual combination of apoplastic and symplastic (polymer trapping) mechanisms of phloem loading.
Materials and methods Plant material A. trichopoda was obtained from the University of California at Santa Cruz and grown in potting mix in a humid greenhouse under shaded conditions. Microscopy Tissue was fixed in 2.5% glutaraldehyde plus 2% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA, USA) in 100 mM sodium cacodylate buffer, pH 6.8 at room temperature for 3 h. Fixed tissue was washed in buffer and post-fixed in 1% OsO4 in the same buffer at room temperature for 2 h, or at 4°C overnight. Tissue was then dehydrated in a graded acetone series over a 3-h period and embedded in Spurr resin (Electron Microscopy Sciences). Given the fibrous nature of the material, embedding times were extended. The tissue was subjected to a graded
A. trichopoda, plasmodesmata, and the evolution of phloem loading
series of acetone/resin mixtures over a total of 18 h followed by three changes of pure resin over a total of 12 h to 2 days before hardening at 70°C overnight. Semi-thin (0.5 μm) sections for light microscopy were stained with 0.5% toluidine blue (w/v) in 0.1% sodium borate (w/v) at 80°C. Thin sections for electron microscopy were stained with uranyl acetate and lead citrate and photographed at 60 kV with a Philips EM-300 transmission electron microscope. Carbohydrate synthesis and transport Mature leaves were enclosed in a gas-tight cuvette and exposed under light from a 1,000-W incandescent metalhalide lamp (300 μmolphotonsm−2 s−1) for 30 min to 14 CO 2 (1.0 MBq) generated from Na2 14 CO 3 (6.6 × 105 MBqmmol) by the addition of excess 80% lactic acid. Transport and sink tissues (the petioles of labeled leaves and sink leaves at the shoot apex) were covered with aluminum foil during the labeling period to prevent photosynthesis in case 14CO2 leaked from the cuvette. Following an additional 6-h chase period in room air, the source and combined transport and sink tissues were separately frozen in liquid nitrogen. Soluble carbohydrates were extracted in a mixture of methanol, chloroform, and water (12:5:3; v/v/v), and the aqueous phase was separated by the addition of water (0.6 volumes). The neutral fraction was obtained by passing the aqueous phase through anion and cation exchange resins (Haritatos et al. 2000). The neutral fraction was then dried, resuspended in a small volume of water, and analyzed by twodimensional thin layer chromatography and autoradiography (Turgeon et al. 2001). Spots on the chromatograms were scraped from the plates and counted in Ecoscint scintillation solution (National Diagnostics, Atlanta, GA, USA).
Results Veins have two types of phloem The minor veins of A. trichopoda leaves are relatively large. Most minor veins are composed of 30 or more cells and include a complex phloem (Fig. 1a). A schematic representation of vein architecture is provided in Fig. 2. The extreme terminals of the veins are smaller but do not contain phloem (not shown). Veins are collateral, i.e., the phloem is present only on the abaxial side, and they have a concentric appearance. Veins are surrounded by a bundle sheath of large cells that, in young leaves, are similar to mesophyll cells in appearance. Bundle sheath cells have large chloroplasts that store starch. After the leaves reach full size, a heavy, lignified secondary wall is deposited over the primary wall of the
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bundle sheath toward the inside of the vein and along the lateral walls adjacent to other bundle sheath cells (Figs. 1a and 2). This deposition is gradual and does not occur simultaneously in all veins, so that in younger, fully grown leaves some bundle sheath cells have such a wall and others do not (Figs. 1a and 2). Gaps (secondary pit fields) in the thickened walls allow intercellular communication between adjacent bundle sheath cells and between the bundle sheath and cells within the vein. These secondary pit fields occupy a larger fraction of the wall than is apparent in the light microscope because they are often obscured by wall material either above or below them in sections (Fig. 1a). The percentage of wall length occupied by secondary pit fields, as measured in electron micrographs, is 20.8±4.7% (SE). Centripetal to the bundle sheath, the first, bounding layer of the vein is generally one cell thick (Fig. 1a, b). These are large cells in the context of the vein (5–10 μm in diameter), especially on the abaxial side, but are much smaller than bundle sheath cells. They may be partly or entirely absent in the smallest veins, such that elements of the xylem come in direct contact with the bundle sheath. However, this bounding layer of cells is otherwise ubiquitous. The large cells in the abaxial half of the bounding layer have dense cytoplasm and small vacuoles and are clearly more highly differentiated than those on the adaxial side (Fig. 1b). As discussed below, these are intermediary cells. In the median, abaxial position of the vein, several small parenchyma cells with diminutive, but well-differentiated chloroplasts and central vacuoles interrupt the arc of intermediary cells (Fig. 1b). In fully mature leaves, the vacuoles of these parenchyma cells contain densely staining material (not shown). Adaxial to the intermediary cells lies an arc of sieve elements and “ordinary” companion cells (Figs. 1b, c, i and 2). The companion cells are ordinary in the sense that they are not specialized by the presence of asymmetrically branched plasmodesmata, or transfer cell wall ingrowths (Turgeon and Ayre 2005). Both ordinary companion cells and sieve elements are smaller in transverse section than the intermediary cells. Xylem occupies the central, adaxial region of the veins (Figs. 1b and 2). Between the xylem and the arc of phloem, a continuous layer of vascular parenchyma cells is found, one or two cells deep and continuous with the bounding layer of parenchyma cells adaxial to the intermediary cells. Ultrastructure: companion cell types indicate two modes of phloem loading Sieve elements are commonly arranged in the phloem arc as a single, loosely arranged, lateral file of cells that often abut one another (Fig. 1b). Mature sieve elements are approximately 1.5 μm in diameter and are structurally specialized
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by reduction of protoplasmic contents in the same manner described for other angiosperms (Van Bel 2003): the cytoplasm is parietal and consists primarily of smooth endoplasmic reticulum (the sieve element reticulum), Stype plastids, and mitochondria (Fig. 1c, e, h, i). The nucleus and tonoplast are absent. Ordinary companion cells, of approximately the same width as the sieve elements, have small plastids and may have very small starch grains (Fig. 1i). In some cases, these may be phloem parenchyma cells, rather than companion
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cells, since the two cell types are not easily distinguished from one another. Although quantitative analyses were not performed, it is clear that symplastic connections between ordinary companion cells and sieve elements are not as common as those between intermediary cells and sieve elements. As indicated above, most of the large cells on the abaxial side of the vein, just inside the bundle sheath, are intermediary cells. They have the ultrastructural features of this cell type found in other plants that translocate
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Fig. 1 Transverse light (a) and electron (b–i) micrograph sections of Amborella trichopoda leaves. a Mature leaf in transverse section with a minor vein in the center of the image. Thick secondary walls are evident in many, but not all, of the bundle sheath cells (B) that surround the vein. A secondary pit fields is evident (arrow). Secondary pit fields are more extensive than they appear in light micrographs and occupy approximately 20% of the vein perimeter once the secondary walls enclose the entire vein. Bar=30 μm. This vein is drawn in Fig. 2 to illustrate the locations of the different cell types. b Low magnification electron micrograph of a minor vein in a young, fully expanded leaf. Secondary walls are not yet present in bundle sheath cells. Intermediary cells (IC) form the abaxial border of the vein and are in close contact with bundle sheath cells. Sieve elements (asterisks), companion cells (CC), and phloem parenchyma cells (P) are found just inside the intermediary cells. A continuous layer of larger parenchyma (P) cells lies between the phloem and the xylem (X). A cluster of parenchyma cells also interrupts the file of intermediary cells in the median abaxial position, providing a link between the bundle sheath and the internal phloem. Bar=10 μm. c Cells at the abaxial boundary of a minor vein showing the relationships between the bundle sheath, ICs, and sieve elements (SE). This leaf is older than the one illustrated in b and the bundle sheath cells have thick secondary walls (arrows). A secondary pit fields in the wall is evident. Bar=2.0 μm. d An intermediary cell with plasmodesmata (arrows) linking it to bundle sheath cells and another intermediary cell. The plasmodesmata are more branched on the intermediary cell than the bundle sheath cells. Note the absence of secondary walls in the bundle sheath cells in this vein and the relatively dispersed arrangement of plasmodesmata around intermediary cells compared to the IC–B plasmodesmata in e and f. Bar=2.0 μm. e An older, mature leaf in which bundle sheath cells have developed thick secondary walls (arrows). Plasmodesmata are clustered densely along the primary wall in the secondary pit field. Bar=3.0 μm. f Higher magnification view of the plasmodesmata between the intermediary cell and bundle sheath cell in e. Bar=2.0 μm. g Plasmodesmata between bundle sheath cells and intermediary cells are highly branched. Bar=0.3 μm. h One intermediary cell is linked to two sieve elements by plasmodesmata (arrows). Bar=1.0 μm. i Two ordinary companion cells with adjacent sieve elements in the interior of a minor vein. Bar=2.0 μm
raffinose and stachyose. These features include numerous small vacuoles, dense cytoplasm with plentiful mitochondria, and proplastids (Fig. 1b–h). As in the intermediary cells of other species, abundant plasmodesmata connect them to bundle sheath cells (Fig. 1d–g). These plasmodesmata are highly branched, more so on the intermediary cell side (Fig. 1g). In leaves that have not yet fully matured, and in which the bundle sheath cells have not deposited a secondary wall, the plasmodesmata are clustered in the common wall (Fig. 1d). After the secondary walls have been laid down, the plasmodesmata occupy the secondary pit fields in the walls (Fig. 1f). Within the secondary pit fields between the bundle sheath cells and intermediary cells, plasmodesmata are noticeably denser than before the secondary wall was produced (compare Fig. 1d and f). Thus, it appears that at least some of the plasmodesmata in the secondary pit fields were formed across already existing primary walls (Volk et al. 1996).
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B
B B
B B
X B IC
P
IC
IC IC B
SEs & CCs
*
*
IC
IC
IC
B
IC
B
B Fig. 2 Drawing of the Amborella trichopoda vein in Fig. 1a, illustrating the spatial arrangement of the cell types. Secondary walls of bundle sheath cells are shaded. Abbreviations as in Fig. 1
Sieve elements do not form symplastically isolated pairs with associated ordinary companion cells in the internal phloem arc, or with intermediary cells. Instead, a single sieve element may be symplastically linked to more than one companion cell or intermediary cell, or to both types of cells (not shown). A single companion cell or intermediary cell may be symplastically linked to more than one sieve element (Fig. 1h). Intermediary cells are linked to one another (Fig. 1d, e). Companion cells may be also linked to one another and, less commonly, to an intermediary cell (not shown). The resulting impression is one of a cluster of symplastically interconnected elements. Certain features of intermediary cell structure are consistent with a role for these cells in symplastic phloem loading. First, in small veins with no sieve elements, and presumably no phloem loading, few plasmodesmata traverse the secondary pit fields. Second, in larger veins in which all the cell types are represented, the number of bundle sheath plasmodesmata differs in the abaxial and adaxial halves of the vein. In the abaxial half, these plasmodesmata link to intermediary cells and are abundant, whereas in the adaxial half, the cells that abut the bundle sheath are not differentiated as intermediary cells and the plasmodesmata are far less frequently seen. Sucrose, raffinose, and stachyose are translocated Mature A. trichopoda leaves provided with 14CO2 synthesized radiolabeled sucrose, galactinol, raffinose, and stachyose in the lamina (Fig. 3). In the petioles and young
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80 60 40 20 0 Fig. 3 Radiolabeled sugars in Amborella trichopoda, 6 h after exposure of the lamina to 14CO2 expressed as a percentage of the total. In the lamina, radiolabeled sucrose dominates, followed by galactinol. In sink tissues (petiole and young leaves), the relative proportion of radiolabeled raffinose and stachyose is higher and some verbascose is detected. Radiolabeled galactinol is not as prevalent as in the lamina
leaves, which contained sugars translocated from the lamina, the same radiolabeled sugars were present and there was also a small amount of label in verbascose, the next larger sugar in the raffinose–stachyose series. The proportions of radiolabeled sucrose and galactinol were higher in the lamina than in the petiole. Little galactinol was translocated.
Discussion It has been difficult to determine the ancestral mode of phloem loading in flowering plants. With reference to the Takhtajan system, Gamalei (1989) suggested that symplastic phloem loading is ancestral. Gamalei’s data also indicate that symplastic loading is most common in the tropics and in woody plants. According to this scheme, apoplastic loading arose with the evolution of the herbaceous life form in colder and drier parts of the world (Gamalei 1989; van Bel and Gamalei 1992). A problem with this interpretation is that no distinction was made between the two types of symplastic loading, one passive and the other resulting in active accumulation of high concentrations of photoassimilate in the phloem in the form of raffinose and stachyose, so-called polymer trapping. This difficulty was addressed by treating the families of polymer trapping species separately from other species that have numerous plasmodesmata (Turgeon et al. 2001). When the revised data were projected onto the phylogenetic tree of the angiosperms developed by the Angiosperm Phylogeny Group, the presence of intermediary cells was resolved as a derived trait with at least four and perhaps six independent points of origin (Turgeon et al. 2001). The presence of abundant plasmodesmata in the minor vein phloem (in the absence of intermediary cells) remained as
the ancestral condition. The structural data are therefore most consistent with the hypothesis that loading was initially passive in the flowering plants and that active loading, either by polymer trapping or transporter-mediated retrieval from the apoplast, evolved later. However, there are two problems that stand in the way of this interpretation. First, the resulting tree contained many ambiguities in the more basal groups, due to the paucity of information available for these families (Turgeon et al. 2001). Second, there is an implicit assumption in these analyses that structural data are reliable indicators of loading mechanisms. This is not necessarily true. For example, some species that have an abundance of plasmodesmata in the minor vein phloem nevertheless load apoplastically (Goggin et al. 2001; Turgeon and Medville 2004). Therefore, to gain a better understanding of the evolution of this important function, it is necessary to conduct more comprehensive studies of anatomy and transport physiology. In this spirit, we conducted a structural and physiological assessment of phloem loading in A. trichopoda, which according to most analyses is sister to all other extant angiosperms. There is no evidence for passive photoassimilate loading in A. trichopoda. Passive loading requires extensive symplastic continuity from the mesophyll cells to the sieve elements. While there is such a pathway, it leads through intermediary cells, which are specialized for the synthesis of raffinose and stachyose and the active accumulation of these sugars against a steep concentration gradient. While there is no evidence for passive loading, the data strongly suggest that A. trichopoda employs both available forms of active loading: polymer trapping and retrieval from the apoplast, so-called mixed loading (van Bel 1993; Knop et al. 2004; Turgeon and Ayre 2005). The presence of intermediary cells in A. trichopoda, with their abundant and distinctively branched plasmodesmata, is a compelling indicator of polymer trapping. Intermediary cells occur only in plants that transport raffinose and stachyose, as does A. trichopoda. The converse is also true: all plants known to translocate abundant raffinose and stachyose have characteristic intermediary cells in the minor veins (Turgeon et al. 1993). The data on A. trichopoda extend this correlation to a basal angiosperm. This marks an additional independent origin of the trait in the flowering plants. Apoplastic loading in A. trichopoda leaves can be reasonably inferred from the presence of ordinary companion cells in the interior of the veins. Relatively few plasmodesmata connect these companion cells to surrounding cells other than their associated sieve elements. This positioning of ordinary companion cells and sieve elements in the interior of veins is common in other species, for example Arabidopsis thaliana (Haritatos et al. 2000) and Beta vulgaris (Esau 1967) that load apoplastically.
A. trichopoda, plasmodesmata, and the evolution of phloem loading
In addition to the anatomical evidence, the relative proportions of radiolabeled transport sugars following exposure of leaves to 14CO2 also suggest the involvement of apoplastic loading as well as polymer trapping. Since sucrose in intermediary cells is converted to raffinose and stachyose, the proportion of sucrose in phloem sap can serve as a rough estimate of the degree of apoplastic loading. This proportion is very low in Catalpa speciosa (Turgeon and Medville 2004), in which almost all the radiolabeled sugar transported following exposure of the lamina to 14CO2 is raffinose and stachyose, indicating that apoplastic loading is minimal. In contrast, Asarina spp. transport a considerable amount of sucrose in addition to raffinose and stachyose (Turgeon et al. 1993; Voitsekhovskaja et al. 2006). In Asarina spp., the ordinary companion cells are enlarged and specialized as transfer cells, with extensive wall ingrowths that increase uptake capacity from the apoplast (Turgeon et al. 1993; Voitsekhovskaja et al. 2006). This structural feature correlates well with radiolabeling data and indicates that phloem loading is mixed in Asarina. Since approximately one third of the radiolabel in the transported sugar in A. trichopoda is in the form of sucrose, it seems likely that loading is mixed, with considerable involvement of ordinary companion cells. It is useful to compare details of vein architecture and cell structure in A. trichopoda with those of other species with intermediary cells since common features are likely to be central, and perhaps indispensable, to the loading mechanism. The minor veins of A. trichopoda are more complex than in other species with intermediary cells. The most common pattern in other plants, such as members of the Lamiales (Fisher 1986), is for the minor veins to be composed of relatively few cells, with only two lateral files of intermediary cells subtended on the abaxial side by a single file of ordinary companion cell. One possible explanation for the more complex veins in A. trichopoda is that loading was apoplastic in an ancestor species and the polymer trapping mechanism evolved later by the specialization of the abaxial cells in the bounding layer of parenchymatic elements as intermediary cells. However, other interpretations are possible. Certain features of carbohydrate synthesis and transport in A. trichopoda also reflect patterns noted in other species with intermediary cells. Following exposure of a mature leaf to 14CO2, radiolabeled galactinol is not as well represented in the transport stream as it is in the lamina, even though it is an integral component of the pathway leading to raffinose and stachyose synthesis and has the same access to sieve elements as do these sugars (Ayre et al. 2003). It is also the general case in plants with intermediary cells that the proportion of radiolabeled raffinose and stachyose is considerably higher in the transport stream
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than in the lamina as a whole. This is due to the fact that raffinose and stachyose are synthesized in the intermediary cells, which constitute only a small percentage of the volume of the leaf (Turgeon and Gowan 1992; Turgeon et al. 1993; Bachmann and Keller 1995; Haritatos et al. 1996; Sprenger and Keller 2000). Thus, the proportion of label in raffinose and stachyose is low in the lamina but high where these sugars are carried away in the transport stream. The fact that this relationship is also seen in A. trichopoda suggests that the raffinose/stachyose synthesis pathway is in the intermediary cells in this species. The absence of passive loading in A. trichopoda does not support the hypothesis that this transport strategy is ancestral in the angiosperms. Our data are more consistent with either apoplastic loading or polymer trapping as the original mechanism. However, it is important to remember that A. trichopoda has undoubtedly evolved many characteristics since it diverged from other flowering plants (Feild et al. 2000) and that the phloem loading mechanisms it employs may be examples of such modern innovations. Acknowledgment This work was supported by the U.S.–Israel Binational Agricultural Research and Development Fund (grant no. IS-3884-06). Conflict of interest The authors declare that they have no conflict of interest.
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