Phloem loading. A reevaluation of the relationship between plasmodesmatal frequencies and loading strategies

Abstract

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.

Figure 1.

Radiolabel in the neutral fraction of extracts following exposure of the laminas of mature leaves to ¹⁴CO₂. Extracts were made 1.5 h after the beginning of the exposure period either from the radiolabeled lamina itself (A, C, and E) or from sink tissue (B, D, and F). Data are expressed as the percentage of radioactivity in the neutral fraction. A and B, C. barbinervis; C and D, L. styraciflua; E and F, C. speciosa.

Figure 2.

Electron micrograph of a C. barbinervis minor vein. Two SEs are flanked by two CCs that directly abut bundle sheath (BS) cells on the other side. The cell between the two CCs may be phloem parenchyma, but the distinction between these cell types is often difficult to make. Bar = 3 μm. Plasmodesmata between BS cells and CCs are shown in the inset. They are H shaped and equally branched on both sides. Inset bar = 0.15 μm.

Figure 3.

Exudation of ¹⁴C from leaves from C. barbinervis (A), L. styraciflua (B), and C. speciosa (C). Cut leaves were allowed to transpire either water or PCMBS for 30 min (C. barbinervis and C. speciosa) or 60 min (L. styraciflua) and were then exposed to ¹⁴CO₂ for 1 h. Following another 1-h period in the dark to close the stomata, the petioles were recut and submerged in a solution of EDTA to facilitate exudation. Zero time refers to the beginning of the exudation period, 2 h after initial exposure to ¹⁴CO₂. Exudation amounts are cumulative and are expressed as a percentage of the highest number of counts in replicate 8-h samples. Each experiment was replicated six times.

Figure 4.

Electron micrograph of an L. styraciflua minor vein. A, Low magnification micrograph illustrating the arrangement of cell types. The adaxial surface of the leaf is toward the lower left of the figure. SEs and CCs are surrounded by phloem parenchyma (PP) cells. Arrows indicate clusters of plasmodesmata between PP cells and CCs. Bar = 3 μm. B, High magnification of the cluster of plasmodesmata delimited by the rectangle in A. The plasmodesmata are equally branched on both sides. Bar = 0.3 μm.

Figure 5.

Electron micrograph of a portion of a C. speciosa minor vein. A, Low magnification micrograph illustrating an intermediary cell flanked by a BS cell and an SE. Intermediary cells have dense cytoplasm, small vacuoles (V), and numerous mitochondria (M). Bar = 1.0 μm. Plasmodesmata between BS cells and CCs are shown in the inset. They are highly branched on the intermediary cell side (short arrow) but much less so on the bundle sheath side (long arrow). Inset bar = 0.3 μm.

Figure 6.

[¹⁴C]Suc uptake by C. speciosa leaf discs incubated in different concentrations of Suc either with or without PCMBS for 1 h. PCMBS reduces uptake to a linear function of Suc concentration, indicating that carrier-mediated uptake has been inhibited. Where error bars are not shown, they are smaller than the data points.

Figure 7.

Frequency of plasmodesmata between mesophyll cells. A, Data arranged according to minor vein type (Gamalei, 1989). Type 1 species have the most abundant plasmodesmata in the phloem of minor veins, type 2b the least. B, Data arranged according to growth habit. The data in A and B are from the following: 1 and 2, this study; 3, Fisher (1990); 4, Goggin et al. (2001); 5, Russin and Evert (1985); 6, Haritatos et al. (2000); 7, Beebe and Evert (1992); 8, McCauley and Evert (1989); 9, Warmbrodt and VanDerWoude (1990); 10, Evert and Mierzwa (1986); 11, Wimmers and Turgeon (1991); 12, Bourquin et al. (1990); 13, Fisher (1991); 14, this study; 15, Fisher (1986).