5.4 - Phloem unloading and sink utilisation

Photoassimilate removal from phloem and delivery to recipient sink cells (phloem unloading) is the final step in photoassimilate transport from source to sink. Within sink cells, cellular metabolism and compartmentation are the end-users of phloem-imported photoassimilates. Combined activities of these sink-located transport and transfer events determine the pattern of photoassimilate partitioning between competing sinks and hence contribute to crop yield.

Phloem unloading describes transport events responsible for assimilate movement from se–cc complexes to recipient sink cells. A distinction must be made between transport across the se–cc complex boundary and subsequent movement to recipient sink cells. The former transport event is termed sieve element unloadingand the latter post-sieve element transport. On reaching the cytoplasm of recipient sink cells, imported photoassimilates can enter metabolic pathways or be compartmented into organelles (e.g. amyloplasts, protein bodies and vacuoles). Metabolic fates for photoassimilates include catabolism in respiratory pathways, biosynthesis (maintenance and growth) and storage as macromolecules (starch and fructans).

Compared with phloem loading, phloem unloading and subsequent sink utilisation of imported photoassimilates operate within a much broader range of configurations:

  1. morphological (e.g. apices, stems, roots, vegetative storage organs, reproductive organs);
  2. anatomical (e.g. provascular differentiating sieve elements, protophloem sieve elements lacking companion cells, metaphloem se–cc complexes);
  3. developmental (e.g. cell division, cell expansion);
  4. metabolic (e.g. storage of soluble compounds/polymers, growth sinks).

A correspondingly large range of strategies for phloem unloading and sink utilisation must be anticipated.

5.4.1 - Cellular pathways of phloem unloading

Most photoassimilates travel along one of three cellular pathways: apoplasmic, symplasmic or a combination of both with symplasmic transport interrupted by an apoplasmic step (Figure 5.19).


Figure 5.18. Scheme describing the cellular pathways of phloem unloading and their relationship with sink types. (a) Apoplasmic unloading showing direct transport of photoassimilates from se—cc complexes to the phloem apoplasm. (b) Symplasmic unloading pathway which may or may not have an apoplasmic barrier between sieve elements and recipient sink cells. (c) Symplasmic unloading with the intervention of an apoplasmic step at (i) the maternal-filial interface of developing seeds and (ii) the vascular parenchyma-sink cell interface. Circled numbers denote different sink types assigned to each pathway. 1, vegetative apex; 2, elongating axis of a dicotyledonous stem; 3, mature axis of a primary dicotyledonous stem i permanent storage; 4, mature axis of a primary monocotyledonous stem i permanent storage; 5, mature primary root; 6, fleshy fruit; 7, developing seed. ab, apoplasmic barrier; apo, apoplasm; gp, ground parenchyma; sc, sink cell; se—cc, sieve element—companion cell complex; vp, vascular parenchyma.

(a) Apoplasmic pathways

Fig 5.31.jpg

Figure 5.19. Fluorescent micrograph of the distribution of a membrane-impermeant fluorescent dye, carboxyfluorscein (CF), imported through the phloem into roots of French bean (Phaseolus vulgaris L.). The green/yellow fluorescence of CF is confined to the se-cc complexes of mature portions of roots as seen by the thin central band of fluorescence away from the root apex. In contrast, dye spreads through the apex itself apparently via the symplasm of young cells. Scale bar = 2 mm

Photoassimilates can move directly across plasma membranes of se–cc complexes to the surrounding apoplasm (Figure 5.19a). Apoplasmic unloading is important along the axial transport pathway of roots and stems where vascular parenchyma and ground tissues serve as reversible storage sinks.

(b) Symplasmic pathways

An entirely symplasmic path of photoassimilate transport from sieve elements to recipient sink cells (Figure 5.18b) operates in a wide range of morphological and metabolic sink types. Terminal growth sinks such as root (Figure 5.19) and shoot apices, as well as vegetative storage sinks such as stems, roots and potato tubers, demonstrate symplasmic unloading.

In most sinks that exhibit symplasmic unloading, photo-assimilates are metabolised into polymeric forms within the recipient sink cells. Sugar cane is a notable exception because it stores sucrose unloaded symplasmically from sieve elements in parenchyma cells of stems. Stem sucrose reaches molar concentrations by this unloading route.

(c) Symplasmic pathway interrupted by an apoplasmic step 

Symplasmic discontinuities exist at interfaces between tissues of differing genomes including biotrophic associations (e.g. mycorrhizas and mistletoes) and developing seeds (Figure 5.18c). In addition, within tissues of the same genome, plasmodesmata can close permanently or reversibly at points along the post-sieve-element pathway. This necessitates photoassimilate exchange between symplasmic and apoplasmic compartments (Figure 5.18c). For instance, photoassimilate exchange between apoplasm and symplasm has been detected in sinks that store high solute concentrations and have unrestricted apoplasmic transport between vascular and storage tissues. Developing seeds, particularly of cereals and large-seeded grain legumes (Patrick and Offler 1995), are another model for symplasmic/apoplasmic pathways.

The apoplasmic space between maternal (seed coat) and filial (embryo plus endosperm) tissues in seeds prevents symplasmic continuity in the unloading pathway (Patrick and Offler 1995). In these organs, photoassimilates are effluxed across membranes of maternal tissues and subsequently taken up across the membranes of filial tissues (Figure 5.18c). Photoassimilates are unloaded from sieve elements and transported symplasmically to effluxing cells where they are released to the seed apoplasm. Influx from the seed apoplasm by the filial generation is restricted to specialised cells located at the maternal–filial interface. The final transport of photoassimilates to the filial storage cells largely follows a symplasmic route.

(d) Pathway linkage with sink function and pathway switching

The symplasm is the most frequently engaged cellular pathway of phloem unloading. Even where an apoplasmic step intervenes (e.g. developing seeds), photoassimilates travel predominantly through the sink symplasm (Figure 5.18c). Symplastic routes do not involve membrane transport and therefore offer lower resistances than apoplasmic routes.

Apoplasmic pathways are restricted to circumstances where (1) symplasmic transport compromises phloem translocation and (2) photoassimilate transport is between genetically distinct (e.g. maternal–filial) tissues. Phloem translocation would be compromised when solutes accumulate to high concentrations in sink cells were it not avoided by symplasmic isolation of phloem from sinks. This is exemplified by the switch to an apoplasmic step during development of tomato fruit. In young fruit, imported sugars are converted into glucose or fructose to support cell division and excess photoassimilate is accumulated as starch. At this stage, phloem unloading of photoassimilates follows a symplasmic route (Figure 5.18b). However, once sugars commence accumulating during cell expansion, apoplasmic transport is engaged (Figure 5.18c). The apoplasmic path isolates pressure-driven phloem import from rising osmotic pressures (P) occurring in fruit storage parenchyma cells (Patrick and Offler 1996).

Radial photoassimilate unloading in mature roots and stems may switch between apoplasmic or symplasmic routes depending upon the prevailing source/sink ratio of the plant. At low source/sink ratios, photoassimilates remobilised from axial stores are loaded into the phloem for transport to growth sinks (Wardlaw 1990). Under these conditions, symplasmic unloading into axial stores might be blocked by plasmodesmal closure while photoassimilates are absorbed by se–cc complexes from the surrounding apoplasm. This would prevent futile unloading while stores are drawn upon. In contrast, net flow of photoassimilates into axial storage pools at high source/sink ratios would be facilitated by plasmodesmal opening.

5.4.2 - Mechanisms of phloem unloading

(a) Apoplasmic transport


Figure 5.20. Mechanistic model for plasma membrane transport of sucrose from the coat and into the cotyledons of a developing legume seed. Plasma membrane ATPases vectorially pump protons to the seed apoplasm from both the opposing seed coat and cotyledon cells. The proton gradient is coupled to drive sucrose efflux from the seed coats through a sucrose/proton antiporter and sucrose influx into the cotyledons by a sucrose/proton symporter.

Se–cc complexes contain high sugar concentrations (Section 5.2.3(b)). Thus, a considerable transmembrane concentration gradient exists to drive a passive leakage of sugars to phloem apoplasm. Sugars leaked to phloem apoplasm are often retrieved by an active sucrose/proton symport mechanism (Figure 5.20). Thus, net efflux of sugars from se–cc complexes is determined by the balance between a passive leakage and sucrose/proton retrieval.

Passive unloading (Ep) of sucrose from se-cc complexes to the phloem apoplasm (Equation 5.6) is determined by the permeability coefficient (P) of se–cc complex plasma membranes and the transmembrane sucrose concentration (C) gradient between sieve element lumena (se) and surrounding phloem apoplasm (apo).

 \[E_p=P(C_{se}-C_{apo}) \tag{5.6} \]

 Sinks containing extracellular invertase (e.g. developing tomato fruit, sugar beet tap roots, maize seeds) can hydrolyse sucrose, lowering Capo thereby enhancing sucrose unloading from se–cc complexes. Furthermore, hydrolysis of sucrose renders it unavailable for se–cc complex retrieval by sucrose/proton symport. The resulting hexoses can act as signals to promote cell division in many sinks such as developing seed of Vicia faba.

(b) Symplasmic transport


Figure 5.21. Externally supplied solutes have a marked effect on sucrose import into root tips of hydroponically grown pea seedlings. This was tested by immersing root tips in (a) sucrose or (b) mannitol solutions ranging up to 350 mM. Cotyledons, which supply these young roots with carbon, were fed 14C sucrose and 14C arriving in different root parts was measured (Bq per root segment). (a) Import of 14C into root tips was diminished when they were exposed to external sucrose concentrations of less than 100 mM but promoted by sucrose concentrations of 150 to 350 mM; Two effects operate. At low concentrations, sucrose might enter root tip cells and suppress phloem import by a feedback mechanism. At higher concentrations, sucrose might act mainly as an osmoticum (see (b)). Mannitol is not taken up or metabolised quickly and can therefore help answer these questions. (b) Import through the phloem was stimulated by exposing root tips to the slowly permeable sugar mannitol, at concentrations of from 13 to 350 mM. This demonstrates an osmotic dependence of import through the phloem pathway, presumably through progressively decreasing P of root tip cells as external solute concentrations rise (Based on Schultz 1994)

Symplasmic transport is mediated by cytoplasmic streaming in series with intercellular transport via plasmodesmata. Plasmodesmal transport is usually the overriding resistance determining transport rates between cells.

Root tips offer a useful experimental model to explore post-sieve-element symplasmic transport because of morphological simplicity and accessibility. Exposing pea root tips to low sucrose concentrations (<100mM) slowed photoassimilate accumulation (Figure 5.21a) by raising intracellular sucrose concentrations. This response to concentration gradients is consistent with a diffusion component to phloem unloading (Equation 5.7). When roots were bathed in much higher concentrations of either sucrose (Figure 5.22a) or a slowly permeating solute, mannitol (Figure 5.33b), turgor pressure (P) of sieve elements and surrounding tissues decreased and 14C import rose. This is consistent with a hydraulically driven (bulk) flow of photoassimilates into the root apex. Thus, photoassimilate movement from phloem through a symplasmic path can be mediated by diffusion and/or bulk flow. The relative contribution of each transport mechanism depends on the magnitude of concentration and pressure gradients (Equations 5.6 and 5.8).

Physical laws can be used to model diffusion and bulk flow of sucrose through a symplasmic route. Sucrose diffuses through symplasm at a rate (Rd) defined by the product of plasmodesmal number in the path (n), plasmodesmal conductivity to diffusion (Kd) and sucrose concentration difference (ΔC) between sieve elements and sink cell cytoplasm. That is:

 \[R_d=n \cdot K_d \cdot \Delta C \tag{5.7} \]

 Transport by bulk flow (Rf) is determined by the product of flow speed (S), cross-sectional area of the plasmodesmal flow path (A) and concentration (C) of sucrose transported (Equation 5.2). Flow speed (S), in turn, is a product of hydraulic conductivity (Lp) of a plasmodesma and turgor pressure difference (ΔP) between se–cc complexes and recipient sink cells (Equation 5.8). Flow over the entire pathway considers the number of interconnecting plasmodesmata (n). Thus, bulk flow rate (Rf) is given by:

 \[R_f=n \cdot L_p \cdot \Delta P \cdot A \cdot C \tag{5.8} \]

 Equations 5.7 and 5.8 predict that sink control of symplasmic photoassimilate transport resides in plasmodesmal conductivity and/or sucrose metabolism/compartmentation.

Sucrose metabolism within sink cells influences cytoplasmic sucrose concentration and Πsink. The difference between Πsink and Πapo determines P (Section 4.3). Sucrose metabolism and compartmentation can affect sucrose concentration gradients and ΔP, both driving forces for symplasmic transport from se–cc complexes to sink cells (Equations 5.6 and 5.8).

Fig 5.34.jpg

Figure 5.22. Cellular distribution of the apoplasmic tracer fluorescent dye 3-hydroxy—5,8,10—pyren—etrisulphonate (PTS) imported through the xylem in stem explants of sugar cane. (Left) Fluorescent micrograph of a longitudinal section of a stem with PTS (green fluorescence) localised to the vascular bundle. (Right) Fluorescent micrograph of a transverse section showing PTS confined to the vascular bundle. Retention of PTS in vascular bundles demonstrates that a barrier to lateral dye movement must be located in the walls of bundle sheath cells (bs). (Based on Jacobsen et al. 1992)

Transgenic plants which under- or over-express key sugar metabolising enzymes have allowed definitive experiments to be carried out on the role of sucrose metabolism in symplasmic phloem unloading. For example, reduction of sucrose synthase activity (Section 5.4.4) in tubers of transformed potato to 5–30% of wild-type levels depressed dry weight of tubers and starch biosynthesis (Table 5.3). Tubers of transformed plants had very high hexose levels (hence high P) which might contribute to downregulation of photoassimilate import. As a corollary, plants with enhanced starch biosynthesis through overexpression of the key starch synthesising enzyme, ADP-glucose pyrophosphorylase (Section 5.4.5), also had higher rates of photoassimilate import.

For sinks that store sugars to high concentrations (e.g. sugar cane stems), gradients in Π, and hence P, between se–cc complexes and sink storage cells could become too small to sustain transport. Instead, P in the apoplasm of storage tissues increases as sucrose (hence Π) in the storage cell sap rises. This maintains a lower P in storage cells than in sieve elements and sustains transport. High sucrose concentrations in the apoplasm of storage cells is achieved through an apoplasmic barrier which isolates storage parenchyma cells from sieve elements (Figure 5.22).

(c) Symplasmic transport interrupted by an apoplasmic step

Fig 5.35_0.jpg

Figure 5.23. Experimental systems used to determine sucrose fluxes in (a) attached caryopses of wheat and (b) coats of developing legume seed. In (a), sucrose effluxed from the maternal tissues was collected by infusing the endosperm cavity of an attached wheat grain with solutions delivered and retrieved through micro-capillaries (Wang and Fisher 1994). In (b), embryos are surgically removed from the coats which may be (i) attached to or (ii) detached from the pod wall. The space vacated by the embryo is filled with a wash solution that is changed at frequent intervals. The wash solution is used to deliver treatments to the seed coat and as a trap to collect the effluxed sucrose.

Phloem unloading in legume seed pods is one case of symplasmic and apoplasmic transport operating in series; the pathway is described in Section 5.4.2(c). Whether sucrose efflux requires energy remains unknown since concentration gradients between seed coats and apoplasm might be steep enough to drive facilitated diffusion. Indeed, using an elegant infusion technique (Figure 5.23a), Wang and Fisher (1994) concluded that efflux from the nucellar projection cells of wheat grain was unlikely to be energy dependent. In contrast, sucrose efflux from coats (maternal tissue) of surgically modified legume seeds (Figure 5.23b) is inhibited by about 50% in the presence of PCMBS, a membrane transport inhibitor. Efflux from legume seed coat cells exhibits charac-teristics of a sucrose/proton antiport. Sucrose uptake by filial tissues is mediated by sucrose/proton symport (Figure 5.23).

A fascinating aspect of phloem unloading in legume seed pods is how photoassimilate demand by filial tissues is integrated with supply from maternal tissues, itself an integration of photoassimilate efflux and import from phloem. One variable that could regulate rates of photoassimilate transport through seed coat symplasm and efflux into apoplasm of the maternal–filial interface is P of seed coat cells (Psc): this would sense depletion of apoplasmic sucrose through uptake by cotyledons, producing a signal in the form of a ΔPsc (Figure 5.24). Specifically, Psc is determined by ΔΠ between the seed coat (Πsc) and seed apoplasm (Πapo), which fluctuates according to photoassimilate withdrawal by cotyledons.

A pressure difference (ΔP) between the points of photo-assimilate arrival (sieve tubes) and efflux (seed coats) drives bulk flow of photoassimilates through the seed coat symplasm. Turgor pressure of seed coat efflux cells is maintained homeostatically at a set point (Pset) by P-dependent efflux into the seed apoplasm. Changes in apoplasmic assimilate concentrations and hence Π are sensed immediately as deviations of Psc from Pset. A rise in Psc produced by photoassimilate depletion around filial tissues elicits an error signal, activating P-dependent solute efflux (Figure 5.25b) and thereby raising photoassimilate concentrations in the apoplasm to meet demand by cotyledons (Figure 5.24c). Long-term increases of sucrose influx by cotyledons, for example over hours, are accompanied by adjustments in Pset (light to dark arrows in Figure 5.24b) which elicit commensurate increases in phloem import rates (light to dark arrows in Figure 5.24a).


Figure 5.24. A turgor-homeostat model describing the integration of photoassimilate transport to developing legume seeds. Photoassimilate import through phloem (a) and efflux from seed coat to seed apoplasm (b) is mediated by a turgor (P)-dependent efflux mechanism and uptake of sugars by cotyledons (c). Metabolic activity in growing seeds influences sucrose concentrations within cotyledon cells, possibly feeding back on activities of symporters located in plasma membranes of the cotyledon dermal cell complex. Graph (c) denotes increased influx (R) from light to dark curve as sugar demand increases. The apoplasmic solute pool size is small and turns over in less than one hour (Patrick and Offler 1995). Thus, faster photoassimilate withdrawal from the seed apoplasm by cotyledons will rapidly lower apoplasmic osmotic concentration. Since the osmotic difference between seed apoplasm (Πapo) and seed coat (Πsc) is only 0.1-0.2 MPa, a small decrease in osmolality of the apoplasm will elicit a significant increase in seed coat P (Psc).A shift in Psc above the turgor set point (Pset) results in an error signal (see model) which in turn induces an immediate compensatory increase in photoassimilate efflux to the apoplasm (light curve in graph b). Increased photoassimilate efflux acts to maintain a constant Πapo in spite of enhanced flux through the apoplasm (graph c). Consequently, the increased potential for photoassimilate uptake by cotyledons can be fully realised (dark curve in graph c). In the short term (minutes), the turgor-homeostat ensures that Psc is maintained and hence phloem import, which is driven by the turgor difference between sieve tubes and unloading cells (PstPsc) is also maintained. Under conditions where cotyledon demand is sustained, Pset in the seed coat adjusts downwards. This results from decreases in Πsc, while Πapo is homeostatically maintained. The decrease in Pset of efflux cells serves to enhance the pressure difference between these cells and the importing sieve elements. As a result, the rate of phloem import into seed coats (Rimport) is increased (graph a, light to dark curve). This new rate of import is commensurate with accelerated sucrose efllux from seed coats to the apoplasm (graph b, light to dark curve) and, ultimately, cotyledons.

5.4.3 - Sugar metabolism and compartmentation in sinks

The fate of imported photoassimilates depends on sink cell function. In broad terms, imported photoassimilates are primarily used to provide carbon skeletons or signals for growth or storage. Some photoassimilates provide energy for maintenance. Relative flows of photoassimilates to these fates change during cell development and sometimes over shorter time scales depending upon a plant’s physiological state.

(a) Cell maintenance

Irrespective of sink function, a portion of imported sugars is respired to provide energy (ATP) for maintenance of cell function and structure. Most of this energy is required for continual turnover of cellular constituents such as enzymes and mem-branes. Rates of synthesis and degradation of individual macromolecules vary widely, as does the energy invested in different molecular configurations, so sugar demand for maintenance respiration could differ substantially between tissues.

(b) Cell growth

In growing organs, photoassimilates become substrates for synthesis of new cell material either directly or after biochemical conversions. Other fates for sugars include catabolism in energy-generating pathways which support growth (growth respiration) and storage in vacuolar pools. Stored sugars make an osmotic contribution to growing cells and can act as energy stores in species such as sugar cane. In roots of young barley plants, 40% and 55% of imported sugars are respired and used in structural growth, respectively. Stored sugars turn over each 30 min but account for only 1% of root weight.

(c) Reserve storage in cells

In mature cells, imported sugars enter physical (e.g. vacuoles) and chemical (e.g. starch) storage pools with lesser amounts diverted to respiration (15–20%) and structural components. In contrast to growth sinks, stored carbohydrates are ultimately retrieved from storage pools and used by other storage sinks (e.g. germinating seeds) or translocated to support growth and storage processes elsewhere in the plant. Carbohydrate storage can be brief (hours, days) or extend over considerable periods (months to years). Short-term storage of carbohydrates in stems and roots buffers phloem sap sugar concentrations against changes in photoassimilate export from photosynthetic leaves.

Sugars can also be stored in soluble forms by compartmentation into vacuoles. In this case, the tonoplast provides a physical barrier to protect stored sugars from molecular interconversion by cytoplasmic sugar-metabolising enzymes. Vacuolar sugars are accumulated as sucrose, hexoses or fructans (short-chain polymers of fructose). Sucrose and hexoses can accumulate to molar concentrations (0.1–1.5M) in storage parenchyma cells of roots, stems and fruits. For instance, tap roots of sugar beet and stems of sugar cane accumulate 1M sucrose thereby providing 90% of the world’s sucrose. Hexoses are a common form of sugar storage in fruit, contributing to sweetness of edible fruits such as tomato, grape, orange and cucumber. The wine industry depends upon hexoses accumulating to high concentrations (1.5M) in grape berries to fuel fermentation of ‘must’ in wine making. Fructans are stored in significant quantities in leaf sheaths and stems of temperate grasses and cereals. In pasture species, they contribute to forage quality, and in cereals constitute an assimilate pool that is mobilised to support grain filling.

Alternatively, imported sugars may be stored as starch along the axial transport pathway (available for remobilisation to buffer phloem sap sugar concentrations) or in more long term storage pools of terminal sink organs such as tubers, fruits and seeds. The proportion of photoassimilates diverted into starch differs widely between species and organs. Starch accounts for some 90% of dry weight of potato tubers and cereal grains.

The chemistry of storage products can change during organ development. For instance, starch is the principal storage carbohydrate in young tomato fruit. Later in fruit development, stored starch is hydrolysed and contributes to hexose accumulation in vacuoles of fruit storage parenchyma cells. In other fruits, significant switches between hexose and sucrose accumulation occur during development. All these changes are brought about by ontogenetic shifts in activities of sugar-metabolising enzymes.

5.4.4 - Key transfer events in sugar metabolism and compartmentation

Phloem-imported sucrose can reach the cytoplasm of recipient sink cells chemically unaltered or be hydrolysed en route by extracellular invertase into its hexose moieties. These sugars may then enter a number of metabolic pathways or be compartmented to vacuolar storage (Figure 5.25).


Figure 5.25. Pathways of sugar metabolism and compartmentation within sink cells. Sugars can be delivered to sink cells through either apoplasmic or symplasmic pathways. Within the sink apoplasm, sucrose can be hydrolysed to hexoses by an extracellular invertase. Apoplasmic sugars are transported across plasma membranes of sink cells by proton/ sugar symporters. Alternatively, sucrose enters the sink cytoplasm through a symplasmic path. Within the sink cytoplasm, sucrose can be hydrolysed or compartmented into vacuolar storage. Sucrose hydrolysis provides substrates for energy metabolism or for synthesis of macro-molecules. Invertase activity is important to sustain hexose supply for glycolysis. Sucrose synthesis from the hexose pool is catalysed by sucrose phosphate synthase (SPS). Degradation of sucrose by sucrose synthase generates fructose and uridine diphosphate glucose (UDP-glucose) which enters various biosynthetic pathways including cellulose and starch synthesis. In the case of starch biosynthesis, UDP-glucose and fructose generate glucose-1-phosphate (G-1-P) which is transported across the amyloplast membrane. Accumulated G-1-P is interconverted into adenine diphosphate glucose (ADP-glucose) by the enzyme adenine diphosphate glucose pyrophosphorylase (ADP-glucose PPase). ADP-glucose is the substrate for starch polymer formation. Sucrose compartmentation into the vacuole is mediated by a sucrose/ proton antiporter located on the tonoplast. Within the vacuole, sucrose can be exposed to invertase hydrolysis with the hexose products accumulating or leaking back to the cytoplasm

(a) Sucrose metabolism

Sucrose is metabolically inert and, in order to be metabolised, must be hydrolysed to glucose and fructose. Only two enzymes are capable of metabolising sucrose in green plants. These are invertase and sucrose synthase (Figure 5.25) and they are paramount in sugar metabolism after phloem unloading.

Invertase catalyses irreversible hydrolysis of sucrose to its hexose moieties, glucose and fructose. Both acid and neutral invertases occur in plants, with pH optima of about 5 and 7.5, respectively. The activity of invertases varies with plant species, organ type and stage of development. Acid invertases, located in cell wall or in vacuole, are usually active in rapidly growing leaves, stems and fruits and seeds (Ruan et al. 2010), making hexoses available for regulating gene expression and for respiration and biosynthesis. Reduced acid invertase activity in vacuoles during development of sugar cane stems, and its absence from sucrose-accumulating tomato fruit, is a major factor in sucrose accumulation in vacuoles of these tissues. Suppression of cell wall invertase activity led to shrunken seed in maize and small fruit in tomato and loss of pollen fertility in tomato, wheat and rice, demonstrating its critical roles in these reproductive organs. Less is known about the physiological role of neutral invertases..

Sucrose synthase is mainly located in the cytoplasm but recent research also shows that the enzyme may also be associated with plasma membrane and even present in cell wall matrix.  It catalyses sucrose cleavage to fructose and UDP-glucose, a high-energy ester of glucose. UDP-glucose is a substrate for biosynthesis of cellulose and may be converted further for starch synthesis.High activities of sucrose synthase are found in both growing and starch storage tissues. In the cytoplasm of starchy tissues, UDP-glucose is converted by UDP-glucose pyro-phosphorylase to glucose-1-phosphate, which is transported across amyloplast membranes. In amyloplasts, glucose-1-phosphate provides glucose moieties for starch synthesis in a pathway comparable to starch formation in chloroplasts of photosynthetic leaves. The critical role of sucrose synthase in starch synthesis is demonstrated with potatoes transformed with an antisense construct of the gene encoding tuber-specific sucrose synthase. Tuber sucrose synthase activity in transformed plants was depressed significantly while the activities of key starch biosynthetic enzymes were unaltered. Low sucrose synthase activity was directly responsible for a proportional decrease in starch accumulation (Zrenner et al. 1995).

Sinks that accumulate soluble sugars have predictably low sucrose synthase activities. Contrastingly high sucrose synthase activities in phloem vessels may be responsible for energy production for phloem loading or unloading and maintaining cellular function of companion cells.

(b) Hexose metabolism

Hexoses transported to the sink cytoplasm are rapidly phosphorylated to hexose-6-phosphates by glucose- and fructose kinases. In these forms, hexoses can be used as substrates for respiration or for synthesis of new cell constituents. Alternatively, sucrose phosphate synthetase can convert them to sucrose, as in leaves (Chapters 1 and 2). Sucrose synthesized by this reaction can be accumulated in vacuoles (e.g. sugar beet tap roots, sugar cane stems) or be rehydrolysed into hexoses by a vacuolar acid invertase (e.g. grape berries).

5.4.5 - Sink control of photoassimilate partitioning

The pressure-flow hypothesis provides a compelling model to explain sink strength in plants. Evidence such as accelerated import of photoassimilate into roots with artificially lowered P lends empirical support to the model.

Knowledge of cellular and molecular events in phloem unloading and photoassimilate use begins to reveal the array of control steps which underlie photoassimilate unloading and relative sink strength. Photoassimilate import into sinks by apoplasmic pathways or by diffusion through symplasmic pathways (Figure 5.19) is controlled by P in se–cc complexes. In contrast, when phloem unloading is by bulk flow through a symplasmic route (e.g. legume seed coats), P in cells responsible for photoassimilate efflux to the apoplasm controls unloading. Unloading into storage tissues is controlled by P in sink (storage) cells.

These processes at the cell and tissue level must now be related to a whole-plant perspective of sink control of photoassimilate partitioning, taking into account influences of plant development and environmental factors. How plants use photoassimilates (e.g. switch from growth to storage) is accompanied by alterations in the cellular pathway of import. Phytohormones also play a role in photoassimilate partitioning through their influence on development and intercellular signalling.

Plant development generates new sinks, for example in meristems where cells undergo division or growth zones where enlarging cells import photoassimilates.

(a) Meristematic sinks

Potential sink size is set largely during the meristematic phase of development through determination of total cell number per organ. Photoassimilate supply has been implicated as a limiting factor in initiation of leaf primordia at the apical dome and subsequent early development by cell division. Substrate supply for developing seeds (endosperm, embryo) and root and floral apices might also be restricted.

Agricultural yields might therefore increase if plants could be modified to enhance the supply of photoassimilates to meristems. Which factors regulate photoassimilate supply to meristematic sinks? Rate equations describing mass flow of phloem sap (Section 5.2.5) predict that photoassimilate supply reaching a sink will be determined by source output (setting photoassimilate concentration in sap and P in sieve elements at the source) and modulated by Lp of the transport pathway. Increased photoassimilate output from source leaves increases growth activity of primary meristems. Even during reduced source output, photoassimilate import and meristematic sink strength can be maintained by remobilisation of storage reserves. Manipulating competition for photo-assimilates by more established sinks also suggests that source output influences sink behaviour.

Cultivated plants demonstrate these principles. For example, flushing CO2 into glasshouses increases flower set and hence yield of floral and fruit crops. Similarly, applying growth regulators to induce abscission of some floral apices lessens the number of sinks competing for photoassimilates at fruit set and leads to larger and more uniform fruit at harvest. Alternatively, breeding programs have reduced sink strength of non-harvestable portions of crops and hence the severity of competition. For instance, breeding dwarf varieties of cereals has reduced photoassimilate demand by stems with a consequent increase in floret numbers set and grain size.

These observations imply that increases in net leaf photosynthesis and phloem loading should set higher yield potentials. Yet meristems import only a small proportion of total plant photoassimilate. It may be that phloem conductance limits photoassimilate delivery to meristems; increases in source output would amplify the driving force for transport and hence bulk flow through a low-conductance pathway.

Given that mature phloem pathways have spare transport capacity (Section 5.2.5), any transport limitation imposed by low path conductance might be expected within immature sinks. Photoassimilate import into meristematic sinks involves transport through partially differentiated provascular strands that might extend up to 400µm. Movement through this partially differentiated path is symplasmic (Section 5.2.2(b)). Hence, plasmodesmal numbers and transport properties of plasmodesmata could play a critical role in photoassimilate supplies to sinks and determination of sink size (Equation 5.7).

(b) Expansion and storage sinks 

As cells expand and approach cell maturity, photoassimilates are increasingly diverted into storage products. Towards maturity, fully differentiated phloem pathways with spare transport capacity link expansion/storage sinks with photosynthetic leaves. Photoassimilate import by these sinks depends on duration of the storage phase. This can be short term for sinks located along the axial transport pathway and long term for sinks sited at the ends of transport pathways (e.g. tubers, fruits and seeds).

Storage along the axial pathway occurs mainly when photoassimilate production exceeds photoassimilate demands by terminal sinks. However, storage is not necessarily a passive response to excess photoassimilate supply. Stems of sugar cane store large quantities of photoassimilates (50% of dry weight is sucrose) even during rapid growth of terminal sinks. Photoassimilates might be stored as simple sugars (e.g. sugar cane stems) or as polymers (fructans in stems of temperate grasses; starch in stems and roots of subtropical cereals, herbaceous annuals and woody perennials). Photoassimilates stored along axial pathways buffer against diurnal and more long term fluctuations in photoassimilate supply to terminal sinks. In woody deciduous species, axially stored photoassimilates also provide a long-term seasonal storage pool that is drawn on to support bud growth following budburst. Remobilised photoassimilates can contribute substantially to biomass gain of terminal sinks. For instance, in some mature trees, over half the photoassimilates for new growth come from remobilised reserves; similar proportions of stem-stored fructans contribute to grain growth in cereals when photoassimilate production is reduced (e.g. by drought). Physiological switching between net storage and remobilisation is an intriguing regulatory question.

Growth and development of meristems is determined by phloem unloading events and metabolic interconversion of photoassimilates within recipient sink cells. These transport and transfer processes vary between sinks and can alter during sink development. Techniques now exist to alter expression of membrane porter proteins and possibly enhance photoassimilate import by sinks such as seeds which have an apoplasmic step in the phloem unloading pathway. Prospects of altering plasmodesmal conductivity will improve once plasmodesmal proteins are identified and their encoding genes known.

5.4.6 - Follow the flow: unloading of water and its destination

Phloem unloading of nutrients follows water release from vascular system.

As discussed before, because of their low transpiration rates, developing sinks typically import water through phloem, not xylem.  Water unloaded from phloem is used for cell growth or recycled back to the parental bodies.  Water is unloaded from se-cc complexes symplasmically in majority of sinks by bulk flow. For growth sinks such as shoot or root apices, continued symplasmic flow of phloem-imported water can drive cells expansion. In post-phloem unloading pathways interrupted with an apoplasmic step, water must exit cells of the unloading path across cell membranes, facilitated by aquaporins (AQPs). AQPs responsible for water flow across cell membranes are plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins (TIPs). Strong PIP expression in expanding post-veraison grape berries has been shown to correlate with water flows into, and from, the berry apoplasm. For sinks that stop expansion but continuously accumulate biomass, water transported to storage sink apoplasms is recycled back to the parent plant body through a xylem route. Important roles played by AQPs in water recycling are indicated by their high expression at this stage in developing seed, particularly in the vascular parenchyma cells.

In conclusion, this chapter has shown how growth and development of meristems and other sinks is determined by phloem unloading events, and metabolism of the assimilates within the recipient sink cells. These transport and transfer processes vary between specific sinks, and can alter during development. Molecular techniques that alter expression of membrane transporters  can be used to study the pathways and limitations of photoassimilate transport into sinks such as seeds that have an apoplasmic step in the phloem unloading pathway, with the possibility of enhancing the rate of grain growth and crop yield in the future.