(a) Delineating the transport path 

Phloem loading is used variously to describe transport events outside, and inside, phloem tissues of leaves. The broader general application is adopted here — that is, phloem loading describes photoassimilate transport from the cytoplasm of photosynthetic mesophyll cells to se–cc complexes of leaf phloem.

Phloem loading commences in mesophyll cells and ends in the leaf vascular system. The se–cc complexes occur in a wide array of vascular bundle sizes. In dicotyledonous leaves, veins undergo repeated branching, forming the extensive minor vein network described in Section 5.2. For example, sugar beet leaves contain 70cm of minor veins cm^–2 of leaf blade, while the major veins contribute only 5.5cm cm^–2 of leaf blade (Geiger 1975). These observations and physiological studies (van Bel 1993) show that the principal site of phloem loading is in the minor vein network of dicotyledonous leaves. In contrast, the major veins transport loaded photoassimilates out of leaves.

Fig 5.22-ann.jpg

Figure 5.13. Transmission electron micrograph through a minor vein of a source leaf of maize (Zea mays L.) This vascular bundle consists of two sieve elements (st), one xylem vessel (v) and five vascular parenchuma cells (vp). These sieve elements are of two types, one thin walled and accompanied by a companion cell, the other thick walled and adjacent to the xylem vessel. Other symbols are: bs, bundle sheath; cc, companion cell; is, intracellular space; st, sieve tube. Scale bar - 4.2 µm (Based on Evert et al. 1978; reproduced with permission of Planta)

Minor veins usually comprise a single xylem element, vascular parenchyma cells and one to two sieve elements surrounded by one to four companion cells (Figure 5.13). The se–cc complex in minor veins bears similarities to that of stems (Figure 5.6). Companion cells have dense cytoplasm containing many mitochondria and are often considerably larger than the sieve elements they accompany. Companion cells are symplasmically connected to the sieve elements by branched plasmodesmata.

Cross-sectional areas of veins in monocotyledonous leaves reveal large and small parallel veins. Photoassimilates are loaded into the small veins and conducted through large veins. Fine transverse veins carry photoassimilates loaded into small veins across to large veins for export.

(b) Cellular pathways — symplasmic versus apoplasmic

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Figure 5.14. Scheme describing symplasmic and apoplasmic pathways of phloem loading. Lines without arrows joining boxes represent symplasmic continuity (i.e. plasmodesmata). Black arrows indicate symplasmic transport (i.e. through plasmdesmata); green arrows indicate apoplasmic transport requiring solutes to cross membranes. vpc, vascular parenchyma cell; se, sieve element; cc, companion cell (Based on van Bel 1993)

Photoassimilates could move intercellularly through interconnecting plasmodesmata from chloroplasts in mesophyll cells to the lumena of sieve elements (symplasmic phloem loading) or across plasma membranes, travelling part of the route through the cell wall continuum (apoplasmic phloem loading). These fundamentally different pathways are shown schematically in Figure 5.14. Debate persists over which cellular pathway of phloem loading prevails because experiments on transport from mesophyll cells to sieve elements are difficult.

Extraordinarily, the cellular pathway of phloem loading reflects evolutionary relationships. Species from ancient plant groups display symplasmic loading, while species of more modern plant groups appears to exhibit apoplasmic phloem loading (van Bel 1993). Evidence for respective routes of loading follows.

A symplasmic pathway depends upon development of extensive plasmodesmal interconnections between adjoining cells, forming a cytoplasmic continuum from mesophyll to se–cc complexes (Figure 5.14). Such symplastic continuity is found in leaves of plant families containing trees and shrubs as well as cucurbits such as squash (van Bel 1993). An abundance of plasmodesmal interconnections demonstrates potential for symplasmic transport but does not establish whether such transport actually occurs. Membrane-impermeant fluorescent dyes microinjected into mesophyll cells are transported to se–cc complexes, demonstrating that plasmodesmata can provide a route for photoassimilate transport. Furthermore, when leaves were fed¹⁴CO₂ and treated with inhibitors that block sugar transport across plasma membranes, transport of ¹⁴C-labelled photo-assimilates continued unaffected along the enforced symplasmic unloading route (Figure 5.15; van Bel 1993). In this case, sugar levels are higher in the mesophyll than in the phloem and ions and molecules diffuse through plasmodesmata at each interface, without a concentrating step (Turgeon 2010). Therefore, this is a passive symplasmic phloem loading.

Symplasmic phloem loading may also be an active process occurring in some herbaceous eudicots. This model of phloem loading, called polymer trap mechanism, depends on sucrose being biochemically converted to raffinose oligosaccharides (RFOs) in specialized CCs (intermediary cells - ICs) (Turgeon 2010). The biochemical synthesis of RFOs from sucrose requires metabolic energy. The synthesized RFOs exceed size exclusion limits of plasmodesmata linking mesophyll cells with ICs and therefore are trapped and accumulate to high concentrations in SE/IC complexes of minor veins for long distance transport (Turgeon 2010).

Plant species that load phloem from the leaf apoplasm are characterised by a low abundance of plasmodesmata between se–cc complexes and abutting vascular cells. However, as for symplasmic loaders, mesophyll cells of these species are interconnected by abundant plasmodesmata (Figure 5.23). Herbaceous and many crop species belong to this group of phloem loaders, including grasses (van Bel 1993). Conventional physiological observations are consistent with phloem loading in leaves of these species including a membrane transport event located somewhere between mesophyll cells and the se–cc complexes of minor veins (Figure 5.15).

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Figure 5.15. Testing whether photoassimilates move from mesophyll cells to se-cc complexes through (a) an entirely symplasmic route or (b) a route with an apoplasmic step. The approach is to use PCMBS as an inhibitor of membrane transport. PCMBS does not cross membranes but binds to the apoplasmic face of plasma membranes. Therefore, it blocks apoplasmic transport while symplasmic phloem loading is unaffected. PCMBS was introduced into the leaf apoplasm through the transpiration stream of excised leaves. Leaves were then exposed in a closed illuminated chamber to ¹⁴CO₂. The ¹⁴C photoassimilate exported from labelled leaf blades was used to monitor phloem loading. PCMBS only reduced photoassimilate export (i.e. phloem loading) from those leaves with few plasmodesmata interconnecting se-cc complexes with surrounding cells. Thus, photoassimilate flow included a membrane transport step from the leaf apoplasm in certain plant species while others loaded via a symplasmic route. cc, companion cell; mc, mesophyll cell; msc, mesophyll sheath cell; PCMBS (para-chloromercuriben-zenesulphonic acid, also abbreviated to P); se, sieve element; vp, vascular parenchyma (Based on van Bel 1993)

Molecular biology has brought new insights to phloem loading. For instance, existence of an apoplasmic step demonstrated with PCMBS (Figure 5.15) has been elegantly confirmed using molecular biology to control activity of the sucrose/proton symporter responsible for sucrose uptake from phloem apoplasm into se–cc complexes. Specifically, potato plants were transformed with an antisense copy of the gene encoding the sucrose/proton symporter, producing a phenotype with low levels of the symporter in plasma membranes of se–cc complexes (Frommer et al. 1996). Excised leaves of transformed plants exported significantly less photoassimilates than wild-type plants, corroborating the inhibitory effect of PCMBS on apoplasmic phloem loading (Figure 5.15). This provides compelling evidence that passage of photoassimilates from mesophyll cells to se–cc complexes in potato leaves includes an apoplasmic step.

Vascular parenchyma cells are the most probable site for photoassimilate exchange to phloem apoplasm (van Bel 1993), ensuring direct delivery for loading into se–cc complexes. Furthermore, plasma membranes of se–cc complexes in minor veins have increased surface areas to support photoassimilate transfer from phloem apoplasm. Notably, the surface area of se–cc complexes in sugar beet leaves is surprisingly large—0.88 cm^–2of leaf blade surface. By implication, these large membrane surfaces are involved in phloem loading. Further support comes from cytochemical studies, demonstrating a great abundance of proteins associated with energy-coupled sucrose transport (Section 5.3.3(b)).

Leaf anatomies in some plant species suggest a potential for simultaneous phloem loading through apoplasmic and symplasmic pathways (van Bel 1993). Whether these pathways connect the same sieve element, different sieve elements in the same minor vein order or sieve elements in different vein orders is still unknown.

(a) General characteristics

Any hypothesis of phloem loading must account for the following characteristics:

1. Elevated solute concentration in se–cc complexes. Estimated solute concentrations in sap of se–cc complexes is much higher than concentrations in sap of surrounding cell types, irrespective of whether phloem loading is by an apoplasmic or a symplasmic route.
2. Selective loading of solutes into se–cc complexes. Chemical analysis of phloem sap by techniques shown above in Section 5.2 reveals relative solute concentrations different from those in surrounding cells. Phloem loading is therefore a selective process.

(b) Symplasmic loading 

The above-described characteristics have been used to argue against loading of se–cc complexes through a symplasmic route on the grounds that plasmodesmata lack mechanisms for concentrating and selecting solutes. However, a contribution of plasmodesmata to concentrating and selecting solutes cannot be precluded from our current knowledge of plasmodesmal structure and function.

Plants that load se–cc complexes through a symplasmic route translocate 20–80% of sugars in the form of raffinose-related compounds such as raffinose, stachyose and verbascose (Section 5.2.3(c)). Grusak et al. (1996) proposed a model for symplasmic phloem loading that accounts for the general characteristics stated above. According to this model (Figure 5.16), sucrose diffuses from mesophyll and bundle sheath cells into intermediary (companion) cells through plasmodesmata. Within companion cells, sucrose is thought to be enzymatically converted to oligosaccharides (raffinose or stachyose) maintaining a diffusion gradient for sucrose from mesophyll cells into se–cc complexes. The molecular-size-exclusion limit of plasmodesmata interconnecting mesophyll and companion cells is such that it prevents back diffusion of stachyose and raffinose molecules, which are larger than sucrose. These oligosaccharides are able to diffuse through plasmodesmata with larger diameters linking companion cells with sieve elements (van Bel 1993). This model accounts for selective loading of sugars to achieve high photoassimilate concentrations in phloem elements.

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Figure 5.16. Model of the ‘polymerisation trap mechanism’ to explain symplasinic phloem loading against a solute concentration gradient. Sucrose moves through a symplasmic path from photosynthetic cells into intermediary (companion) cells of the minor veins. Sucrose movement is by diffusion down a concentration gradient maintained by the polymerisation of sucrose into oligosaccharides (raffinose and stachyose) in intermediary cells. Diffusion of these oligosaccharides into mesophyll cells is prevented, as their size exceeds the molecular exclusion limit of plasmodesmata joining mesophyll and intermediary cells. However, the larger-diametered plasmodesmata linking intermediary cells with sieve elements permit oligosaccharides to be loaded into sieve elements for export from the leaf. [], glucose; Δ, fructose; • galactinol (Based on Grusak et al. 1996)

(c) Apoplasmic loading 

Phloem loading with an apoplasmic step is an attractive model, explaining both how solutes become concentrated in se–cc complexes (energy-coupled membrane transport) and how they could be selected by specific membrane transporters (see van Bel 1993). Identifying transport mechanisms responsible for photoassimilate transport to and from the leaf apoplasm has proved challenging.

Based on estimates of sucrose fluxes and high sucrose concentrations in phloem sap, there is little doubt that sucrose loading into phloem is energy dependent. The demonstration that PCMBS blocks loading of photoassimilates in whole leaves of certain species (Section 5.3.2(b)) points to carrier-mediated transport across plasma membranes. Genes encoding sucrose porters have been cloned from leaf tissue (Frommer et al. 1996) and shown to be specifically expressed in leaf phloem. Complementation studies in yeast defective in sucrose transport suggest that the phloem-located sucrose porter catalyses sucrose/proton symport in a similar way to that illustrated in Figure 5.32. Antisense transformants of potato with low abundance of this symporter have impaired sucrose transport (Section 5.32(b)).

In contrast to photoassimilate uptake from phloem apoplasm, very little is known about the mechanism of sugar efflux into the apoplasm until very recently. Estimates of photoassimilate flux to phloem apoplasm, based on rates of sucrose export from leaves, suggest that this transport event must be facilitated by other transport processes (van Bel 1993). This is now confirmed by the recent cloning of sucrose efflux protein that sheds a light on the molecular mechanisms of phloem loading (Chen et al. 2012).  

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Figure 5.17. Time-course of photoassimilate export from source leaves of tomato plants. Control plants, in which fruits were a major sink for photoassimilates, were maintained at 20°C. Treatments involved (1) removing fruit or (2) exposing plants with fruits to 30°C. The proportion of ¹⁴C label remaining in source leaves after a radioactive pulse was monitored through time to show that (1) presence of major sinks or (2) more rapid metabolism accelerated ¹⁴C export from source leaves (Based on Moorby and Jarman 1975).

(a) Sink effects on export

The response of photoassimilate export to changes in sink demand depends upon whether photoassimilate flow is source or sink limited (Wardlaw 1990). A source-limited system does not respond rapidly to an increase in sink demand, depending more on the capacity of leaves to increase the size of the transport pool. In contrast, alterations in sink demand in a sink-limited system elicit immediate effects on photoassimilate export. Figure 5.17 shows how the presence of fruits accelerates ¹⁴C export, especially at high temperatures. For leaves that load the se–cc complexes from apoplasmic pools, changes in sink demand probably influence photoassimilate export by altering membrane transport properties. These changes in membrane transport entrain a flow of adjustments in biochemical partitioning within the leaf through substrate feedback (see below).

(b) Sink effects on membrane transport

Changes in the turgor pressure of phloem sap or altered phytohormone levels could serve as signals for sink demand.

Changes in the pressure of sink phloem sap are rapidly transmitted through sieve tubes to sources. Phloem loading in source tissues responds to this pressure signal by changes in solute transport rates mediated by membrane-associated porters (van Bel 1993). This is a proposed mechanism for phloem loading which would respond rapidly (within minutes) to changes in sink demand.

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Phytohormone levels in leaves respond to changes in the source/sink ratio. For instance, gibberellin levels in leaves proximal to developing inflorescences increase at fruit set. In contrast, abscisic acid levels in soybean and grape leaves are inversely related to alterations in sink demand (Brenner 1987). Therefore, changes in leaf phytohormone levels could serve to signal shifts in sink demand for photoassimilates. In this context, direct application of auxin and gibberellic acid to source leaves results in a rapid enhancement of photoassimilate export (Table 5.2). Gibberellic acid did not stimulate leaf photosynthesis or alter photoassimilate partitioning, appearing instead to upregulate phloem loading. This was confirmed by faster ¹⁴C loading into isolated phloem strands (Table 5.2).

(c) Sink influences on biochemical partitioning within source leaves  

A substrate feedback response is elicited if the rate of photo-assimilate export from chloroplasts is limited by sink demand. If sucrose export from source pools is accelerated by phloem loading, substrate feedback inhibition of photoassimilate delivery is alleviated. A cascade of adjustments in the activity of key regulatory enzymes follows (see Section 2.3) with the final outcome of an increased flow of sucrose into transport pools. Conversely, if photoassimilate flow is limited by photosynthetic rate, the activity of enzymes responsible for sucrose biosynthesis is not subject to feedback inhibition by substrates. As a consequence, responses to increased sink demand can only be mediated by increases in photosynthetic enzyme activity.