Susan Dunford, University of Cincinnati

Evidence for Apoplastic Loading of Sieve Elements

Early research on phloem loading focused on the apoplastic pathway (see textbook Figure 4.4). Many studies devoted to testing these predictions have provided solid evidence for apoplastic loading in several species. Some of the more important evidence is:

1. Transport sugars are found in the apoplast. Sucrose is the predominant apoplastic sugar in species that transport mainly sucrose in the phloem, for example, sugar beet (Beta vulgaris) and broad bean (Vicia faba). Treatments and events that alter the rate of translocation from the source leaf also change the flux of sucrose through the apoplast (Geiger et al. 1974) or the apoplastic sucrose level.
2. Sucrose supplied exogenously to a source leaf of sugar beet accumulates in the sieve elements and companion cells of the minor veins, as does sucrose derived from photosynthetic CO₂ fixation (see textbook Figure 11.15). Similar observations have been made in broad bean, pea (Pisum sativum), castor bean (Ricinus communis), and other species.
3. PCMBS (p-chloromercuribenzenesulfonic acid) is a reagent that inhibits the transport of sucrose across plasma membranes but does not enter the symplast. PCMBS inhibits the uptake of sucrose from the apoplast when the sugar is supplied exogenously to sugar beet. Of greater importance, PCMBS also inhibits the export of sucrose synthesized from CO₂ in the mesophyll, implying that short-distance transport in sugar beet normally includes an apoplastic step (Giaquinta 1976) (Web Figure 10.7.A). Assimilate loading is also inhibited by PCMBS in broad bean, pea, and other species.
4. Experiments with transgenic plants further support the interpretation that sucrose moves through the apoplast. (Transgenic organisms carry cloned genes integrated into their DNA by a variety of recombinant-DNA techniques.) Transgenic tomato plants that have a sucrose-cleaving enzyme (invertase) in their apoplast show a very slow growth rate and fail to mobilize the starch in their source leaves during a prolonged dark period (Dickinson et al. 1991). Apparently, hydrolyzing sucrose in the apoplast inhibits phloem loading in source leaves of this species. This result is expected only if the phloem-loading pathway includes an apoplastic step.

Web Figure 10.7.A   Effects of PCMBS on phloem transport from a sugar beet leaf. Export pf labeled photosynthate from a sugar beet leaf decreases upon addition of PCMBS. Photosynthesis was not affected, indicating that PCMBS did not alter the photosynthetic properties of the leaf. (From Fisher 2000, after Giaquinta 1976.)

Some Substances Enter the Phloem by Diffusion

Many substances, such as organic acids and plant hormones, are found in the phloem sap at lower concentrations than carbohydrates. These substances are probably not actively loaded into the sieve element–companion cell complex but enter the sieve elements via other pathways and mechanisms. They may be taken up directly by diffusion across the phospholipid bilayer of the plasma membrane of the sieve element–companion cell complex or by a passive transporter in the plasma membrane of those cells, or they may diffuse into the sieve elements via the symplast.

Once in the sieve elements, these substances are swept along in the translocation stream by bulk flow, the motive force being generated by the active loading of only certain sugars or amino acids. Many substances not normally found in plants, such as herbicides and fungicides, can be transported in the phloem because of their ability to diffuse through membranes at an intermediate rate. In other words, they diffuse through membranes rapidly enough to allow considerable accumulation in the sieve elements, but slowly enough that they are not lost from the sieve elements completely before reaching a sink tissue. In addition, compounds that are weakly acidic tend to be "trapped" within the sieve elements, becoming negatively charged in the basic environment (low H⁺ concentration) of the sieve elements and thus less likely to diffuse out of the cells across the hydrophobic membrane (Kleier 1988). Substances that are not transported in the phloem (such as calcium) may not enter the sieve elements at all.

A recent study has characterized the Arabidopsis gene AUX1 (auxin influx carrier) that encodes an auxin permease that appears to function as an auxin carrier (Swarup et al., 2001, Marchant et al. 2002, see textbook Figure 19.9). AUX1 has been localized in protophloem cells in the root (Swarup et al. 2001). Gene expression studies have shown that AUX1 facilitates IAA loading into the leaf vascular transport system, and IAA unloading in the primary root apex and developing lateral root primordium (Marchant et al. 2002).

The Roles of Sucrose-H⁺ Symporters in the Phloem of Apoplastic Loaders

Sucrose transporters are postulated to load photosynthetically produced sucrose into the sieve elements of apoplastic loaders (see textbook Figure 11.16 and textbook pages 302–304). The carriers are found in plasma membranes of either sieve elements or companion cells. Carriers found in sieve element membranes include SUT1, SUT2, and SUT4, in potato (Solanum tuberosum) and tomato. SUC 2 has been found in the companion cell plasma membranes of Arabidopsis and plantain

As shown in Web Figure 11.7.B, SUC2, one of the major sucrose transporters involved in phloem loading, is found in the companion cells. Mutant Arabidopsis plants containing transferred DNA insertions in the gene encoding SUC2 have been recently identified by reverse genetics (Gottwald et al. 2000). In the homozygous state, these mutations resulted in stunted growth, retarded development, and sterility. The source leaves of mutant plants contained a great excess of starch, and radiolabeled sugar failed to be transported efficiently to roots and inflorescences.

Web Figure 11.7.B   This micrograph shows a single companion cell from broad-leaved plantain (Plantago major) stained with two fluorescent dyes. One of the dyes (green) is (indirectly) linked to an antibody that is specific for the PmSUC2 sucrose–H⁺ symporter. The second dye (blue) binds to DNA. Since the two dyes are found on a single phloem cell, which is always adjacent to a sieve element, the sucrose symporter is concluded to be located in the companion cell membrane in this species. (From Stadler et al. 1995, courtesy of N. Sauer.) Interestingly, expression of the SUC2 transporter begins in the tip and proceeds to the base in developing leaves during a sink-to-source transition, the same pattern shown by photosynthate export capacity.

Work with SUT1 has shown that the messenger RNAs for symporters found in the sieve element membrane are synthesized in the companion cells (Kuhn et al. 1997; Vaughn et al. 2002). This finding agrees with the fact that sieve elements lack nuclei. The symporter protein is probably also synthesized in the companion cells, since ribosomes do not appear to persist in mature sieve elements.

All sucrose transporters fall into one of three large subfamilies; the roles played by the many of the carriers are still being investigated. SUT1 and SUC2 appear to be the major sucrose transporters in phloem loading, into either companion cells or sieve elements. Additional functions of these and the other sucrose transporters include sucrose retrieval from the apoplast following leakage from the transport stream, transport of sucrose from the vacuole to the cytoplasm, sucrose uptake into germinating pollen, and sucrose release into the apoplast of developing seeds. (See Phloem Unloading, p. 305, in the textbook.)

Studies Supporting the Polymer-Trapping Model of Symplastic Loading

Many studies support the polymer-trapping model. For instance, the enzymes required to synthesize stachyose from sucrose are localized in intermediary cells. In melon, raffinose and stachyose are present in high concentrations in intermediary cells, but not in mesophyll cells. Down-regulation of raffinose and stachyose synthesis in purple mullein (Verbascum phoeniceum) results in the accumulation of carbohydrates in the leaves, reduced capacity to export sugar in prolonged darkness, and, in severely down-regulated plants, in impaired growth (McCaskill and Turgeon 2007). However, mass flow via plasmodesmata has also been proposed as a mechanism for symplastic entry of solutes into the phloem.

Relationships between Loading Characteristics and Loading Mechanisms

What characteristic—type of companion cell, transport sugars, or abundance of plasmodesmata—is the best predictor of symplastic loading? The number of plasmodesmata linking the sieve element–companion cell complex and surrounding cells has often been considered to be diagnostic for loading type. However, some species that had been classified as symplastic loaders on the basis of plasmodesmatal frequencies have now been shown to load apoplastically. Plants with abundant plasmodesmata in the minor vein phloem also tend to possess abundant plasmodesmata between mesophyll cells, so plasmodesmatal frequencies may be related to some other plant characteristic.

The type of transport sugar (sucrose alone or sucrose plus larger oligosaccharides) can differentiate between polymer trapping and the other loading strategies. Autoradiographs of leaf discs fed ¹⁴C-sucrose can then be used to distinguish between apoplastic loading and passive symplastic loading. In species which concentrate sugars in the phloem (apoplastic loading and polymer trapping), the veins should be clearly visible on the autoradiographs. In species which move sugars from the mesophyll into the phloem by means of diffusion down a concentration gradient, veins should not be visible on the autoradiographs. Web Figure 11.7.C confirms these expectations in the three loading types.

A number of species have more than one type of companion cell in their minor veins. For example, coleus has both intermediary cells and ordinary companion cells. It has been suggested that active and passive loading may coexist in some species, simultaneously or at different times, in different sieve elements in the same vein. However, symplastic and apoplastic pathways do not exist in the same sieve element-companion cell complex, due to restrictions imposed by anatomical features, such as plasmodesmatal frequencies.

Web Figure 11.7.C   Autoradiographs of leaf discs from species which load by polymer trapping (left, twining snapdragon, Asarina scandens), from the apoplast (middle, celery, Apium graveolens), and by passive diffusion through the symplast (right, cherry laurel, Prunus laurocerasus). The leaves were treated in a fashion similar to sugar beet (textbook Figure 11.15), except that the labeling period was one hour. Label (black) accumulates only in the species which concentrate sugars in the minor veins, by polymer trapping (left) and by active loading from the apoplast (middle). (Rennie and Turgeon 2009)