The complexity of the phloem loading pathways, as discussed above, is repeated in sink organs, where a number of phloem unloading pathways may be operational (Oparka et al., 1992). The first step in the phloem unloading pathway is the exit of assimilates from the sieve element-companion cell complex. Solutes can leave through the symplast via plasmodesmata and this would represent the pathway of least resistance. Evidence for symplastic sieve element unloading is widespread, for example, in importing leaves of sugar beet (Gougler Schmalstig and Geiger, 1985) and tobacco (Turgeon, 1987), developing potato tubers (Oparka and Prior, 1986), maternal tissues of developing seeds (both legumes and cereals) (Thorne, 1985) and pea roots (Dick and ap Rees, 1975). The alternative exit out of the sieve element- companion cell complex into the apoplast has not been so evident and has proved difficult to demonstrate conclusively. The model of phloem unloading proposed for sugar beet taproots (Fieuw and Willenbrink, 1990) assumes that sucrose is released first from the SE-CC complex into the apoplast where a cell wall bound invertase causes its hydrolysis. Their assumption is based on uptake studies which show that glucose is transported preferentially, the presence of an active acid invertase and absence of symplastic connections between sieve elements and sink cells. However, the cellular site of unloading has yet to be identified.
The examples given above are of unloading either symplastically or apoplastically from the SE-CC complex. However, as explained by Oparka and van Bel (1992) four possible pathways of phloem unloading (i.e. the entire pathway from the SE-CC complex to the cells of the sink organ) may exist: 1)
symplastic phloem unloading in conjuntion with symplastic SE unloading; 2) apoplastic phloem unloading in conjunction with apoplastic SE unloading; 3) apoplastic phloem unloading but symplastic SE unloading; and 4) apoplastic phloem unloading in which symplastic continuity is interrupted at some distance away from the SE-CC complex. Unequivocal evidence supporting a particular pathway is only available for a few plant species.
The phloem unloading pathway is perhaps best understood in developing seeds where the embryo is symplastically isolated from the maternal tissue and assimilates have to pass into the apoplast prior to uptake by the seed (Thorne, 1985). The pathway of photoassimilate movement in developing seeds of Vida
faba has been studied in detail (Offler et al., 1989; Offler and Patrick, 1993). The proposed pathway is one in which photoassimilate moves away from the SE- CC complex radially and laterally in the symplast to thin-walled parenchyma/transfer cells which form the main site for membrane exchange to the seed apoplast. These transfer cells, bordering the entire inner surface of the seed coat, have 43% of their cell wall length exhibiting ingrowths which results in a 58% increase in plasma membrane surface area. This characteristic of the transfer cells, along with an increase in the number of mitochondria and ribosomes found within the cytoplasm, are thought to have developed to facilitate exchange to the seed apoplast. It is thought that a similar pathway of transfer occurs in the seed coats of most grain legumes.
Information on the phloem unloading pathways in vegetative sinks is not so detailed. In potato tubers, evidence of symplastic continuity between the SE- CC complex and surrounding parenchyma cells and the inhibition of tracer efflux by induced plasmolysis suggest that the phloem unloading pathway is symplastic (Oparka, 1986: Oparka and Prior, 1987). However, when apoplastic yeast invertase was expressed in potato tubers, the resulting accumulation of glucose and fructose and decrease in the level of sucrose indicates that
movement into the apoplast occurs in growing potato tubers (U. Sonnewald pers. comm, to H. V. Davies). At what stage in the phloem unloading pathway this takes place is not clear, therefore the possibility of either a symplastic or apoplastic route of phloem unloading in potato tubers still exists. As yet, no clear pattern associating specific vegetative sink organs with a particular route of phloem unloading has been identified.
According to the pressure-flow hypothesis (Munch, 1930), as described above (1.2.2), the movement of solutes will be driven by a pressure gradient between source and sink cells. Thus sink cells would need to maintain a low sucrose concentration in comparison to the SE-CC complex to ensure the continued transport of sucrose into the sink organ. Several mechanisms have been proposed to allow the continued import by the sink organ. When apoplastic SE unloading occurs, hydrolysis of sucrose by apoplastic acid invertase would cause a decrease in the apoplastic sucrose level, prevent reloading and enhance SE unloading (Eschrich, 1980, 1986). However, the hydrolysis of sucrose prior to uptake by parenchyma cells is not a prerequisite for the continuance of SE unloading (Gougler Schmalstig and Hitz, 1987; Lingle, 1989). A gradient may also be maintained by compartmentation of sucrose into the vacuoles of storage cells or by the conversion of sucrose to starch (Ho, 1988).
This traditional view of phloem unloading being enhanced by low apoplastic sucrose concentrations needs reviewing in the light of results with developing legume seeds (Wolswinkel, 1992). No inhibition of sucrose unloading from the testa or sucrose transport into the empty seed occurred when solutions of high sucrose concentration were present in empty ovules (Patrick, 1984; Wolswinkel and Amerlaan, 1984). Phloem unloading in legume seeds, rather than being inhibited by high apoplastic solute concentrations, appears to be stimulated. The suggestion is that a high concentration of osmotically active solutes in the sink apoplast will result in an osmotic efflux of water from the sieve elements
vacuole
Fig. 1.2 Sites of sucrose cleavage shown in a storage parenchyma cell from
a young potato tuber with possible enzymes involved:- 1) cell wall acid invertase, 2) vacuolar acid invertase, 3) alkaline invertase, and 4) sucrose synthase.
and other cells in the seed-coat, further reducing the turgor pressure at the sink end of the phloem pathway and thus promoting phloem transport into developing sinks (Wolswinkel, 1992). Restricted xylem connections between developing seeds and transpiring leaves is thought to prevent the movement of apoplastic solutes back along the water potential gradient (Wolswinkel, 1992). While no generalizations on the most common phloem unloading pathway can be made, the form of carbon most likely to be delivered and taken up by sink organs is sucrose. It appears likely from the studies of uptake with fluorosucrose (Gougler Schmalstig and Hitz, 1987; Lemoine et al., 1988) and the use of [^CJfructosyl sucrose (Lingle, 1989; Thom and Maretzki, 1992) that while hydrolysis in the apoplast may occur it is not a prerequisite of uptake and that sucrose is taken up intact by maize endosperm cells and by parenchyma cells in sugarcane internode tissue. Similarly, translocated sucrose in sugar beet enters the storage parenchyma cells in mature taproots without apoplastic hydrolysis (Giaquinta, 1977,1979).
Sucrose thus delivered to the storage cells of sink organs may be cleaved, prior to storage or utilisation in cell metabolism, by four possible routes involving the following enzymes: 1) cell wall acid invertase, 2) vacuolar acid invertase, 3) alkaline/neutral invertase or 4) sucrose synthase (see Figure 1.2).
1.3 Properties of the enzymes involved in sucrose cleavage