2.3.1 Introduction
A firm grasp of the control mechanisms of photosynthesis (see section 2.1) and sucrose- related plant growth (see section 2.2) may clarify certain facets of plant metabolism;
however, without knowledge of the interrelationship between the leaf and growing regions of the plant, a proper understanding of how plant development is regulated cannot be fully realised. Carbon assimilation is a function of the balance between the supply by leaf photosynthesis and the demand from growth processes. As such, to properly conceptualise the manner in which plants assimilate and partition carbon, an understanding of the paradigm of ‘sink’ and ‘source’ tissues is required.
The hypothesis that photoassimilate accumulation plays a role in regulating photosynthesis rates is not new. Such a connection was initially highlighted by Boussingault (1868), who suggested the existence of a putative link between leaf (sources) and storage tissues (sinks), instead of a one-way relationship. Close co- ordination of source photosynthetic activity with carbon demand of sink organs has now been clearly demonstrated in several species, where a decrease in assimilation rate is observed when sink demand for carbohydrate is limited (Gucci et al., 1994; Iglesias et al., 2002; De Groot et al., 2003; Quilot et al., 2004; Franck et al., 2006). As plants are sessile, autotrophic organisms, the need to integrate metabolism and growth with external environmental signals is crucial. To do so efficiently, plants must rigorously coordinate both source activity and sink demand, or else risk a fatal ‘economic crisis’
from over-investing in either one or the other. This involves both fine (substrate and allosteric) and coarse (gene expression) regulation, as well as specific sugar-signaling mechanisms (Rolland et al., 2006).
The elucidation of a regulatory relationship between the demand for carbon from growing areas of the plant and the supply from leaves has become a novel and exciting field of molecular and physiological research (Wardlaw, 1990; Pego et al., 2000; Paul &
Foyer, 2001; Rolland et al., 2002; Paul et al., 2001; Paul & Pellny, 2003). Evidence increasingly supports a sink-dependent relationship (Paul & Foyer, 2001), whereby the demand for carbon from sink tissues, or sink-strength, influences the net photosynthetic activity and carbon status of source organs (Paul et al., 2001). However, the dominant
mechanisms through which the sink regulates the source appear not to be simple linear pathways, but rather a set of complex networks with many points of reciprocal feedback control, which together determine the limits within which photosynthesis can be productive and underpin the source-sink interaction (Paul & Foyer, 2001; Minchin &
Lacointe, 2004; Rolland et al., 2006).
The precise metabolic mechanisms that give rise to feedback control are still not fully understood (Paul & Pellny, 2003). However, recognition of the role of sugar transporters and associated regulatory metabolic enzymes in the loading and unloading of sucrose in the phloem has led to the proposition of several models that serve to illustrate the carbon transport pathways between source and sink tissue. Using the source-sink paradigm, this chapter will discuss existing understanding of the physiological and metabolic means by which plants regulate carbon partitioning and accumulation.
Furthermore, it will highlight current mechanistic modeling efforts to predict the observed complexity of the source-sink relationship, with particular reference to sugarcane models.
2.3.2 Phloem transport
Long-distance transport of carbohydrates between sources and sinks occurs in specific cells of the vascular system called the phloem sieve elements. During development, most organelles (including vacuoles and nuclei) of sieve elements are degraded, resulting in an intimate connection via numerous branched plasmodesmata with neighboring phloem companion cells, which in turn supply energy and proteins to the sieve elements (Williams et al., 2000, Stadler et al., 2005). Phloem transport functions via bulk flow, where accumulation of sugars inside the sieve element-companion cell complex (SE-CCC) results in the osmotic uptake of water which then drives sap along the sieve tube. The unloading of sugars at the sink results in a loss of water, and thus a gradient of pressure is maintained (Williams et al., 2000). Despite the occurrence and importance of other phloem solutes, such as amino acids, raffinose and stachyose, hexitols, inorganic ions and the most recently identified component, fructans (Wang &
Nobel, 1998), sucrose is the osmotically dominant compound in the sieve tube sap of most species, including sugarcane (Hartt et al., 1963; Huber et al., 1993; Komor, 2000).
Thus, in these species, sucrose is not only the main transport metabolite, but also the
primary contributor to the osmotic driving force for phloem translocation (Hellman et al., 2000).
2.3.2.1 Phloem loading
Recently, there have been several advances in understanding of the processes involved in phloem loading at the source (Lalonde et al., 2003; van Bel, 2003; Minchin & Lacointe, 2004). Depending on species, phloem loading is now believed to involve an apoplastic step, a symplastic step, or both, between the site of sucrose synthesis and the sieve tubes involved in transport (Komor, 2000). Apoplastic loading involves flow of sucrose from the leaf mesophyll into the apoplastic space in the vicinity of the vascular tissue, from where it is actively taken up across the cell membrane by sugar transporter proteins into the SE-CCC, or directly into the sieve element (Hellman et al., 2000;
Minchin & Lacointe, 2004). However, with symplastic loading, sucrose flows from cell- to-cell to the SE-CCC entirely through plasmodesmata, probably via diffusion, and thus does not cross the membrane (van Bel & Gamalei, 1992). However, it should be noted that plasmodesmata are not simply intracellular ‘holes’ which facilitate passive transport, but rather dynamic, complex structures through which the transport of macromolecules is highly regulated (Lucas et al., 1993). An association of cytoskeletal elements, such as actin- and myosin-like proteins, with plasmodesmatal structures has been demonstrated (White et al., 1994; Radford & White, 1998). These cytoskeletal elements may play a role in the targeting and transport of macromolecules through the plasmodesmata (Radford and White, 1998).
As most studies of the leaf have revealed that the sucrose concentration in the sieve tube sap is higher than in the mesophyll, active transport is likely to be involved at the loading site (Komor, 2000). Similar to other active transport processes, the rate of active sucrose export would thus depend on the sucrose concentration in the leaves according to Michaelis-Menton-type kinetics (Komor, 2000). In soybean, such a linear relationship between sucrose content in the leaf and net export rate has been shown by manipulating sucrose content with varying light intensities (Fader & Koller, 1983). A similar 14CO2- labelling study was undertaken using leaves of a variety of species, with particular emphasis on the difference between C3 and C4 plants (Grodzinski et al., 1998). C4
plants were clearly shown to have higher leaf solute concentrations and export rates (Grodzinski et al., 1998).
In C4 plants, such as sugarcane, where a two cell-type (exterior mesophyll and interior bundle sheath) configuration exists, sucrose is still produced primarily in the mesophyll (Lunn & Furbank, 1997). Thus, in C4 plants, sucrose must additionally pass through the bundle sheath cells to be loaded into the phloem through either a plasmodesmatal or apoplastic step, or both (Fig. 2.5) (Lunn & Furbank, 1999; Walsh et al., 2005). However, in sugarcane, the conducting cells of the phloem are not connected to other cells of the leaf by plasmodesmata (Robinson-Beers & Evert, 1991), suggesting that phloem loading occurs from the apoplast (Rae et al., 2005a).
2.3.2.2 Phloem unloading
Phloem unloading in most species is believed to favour symplastic movement, through plasmodesmata linking the cells in the sink region (Minchin & Lacointe, 2002). In immature potato tubers, phloem unloading is predominately apoplastic, although the onset of tuber development and starch accumulation is accompanied by a switch to symplastic transfer of solutes to storage parenchyma cells (Viola et al., 2001). The destination of the symplastic flow is not the terminal sieve elements, but rather within the receiver cells, where the sink osmotic pressure is kept low by metabolic utilization of the carbohydrates or conversion into less osmotically active polymorphic forms, such as starch (Minchin & Lacointe, 2002). Growing evidence indicates that the region of highest flow resistance is not within the transport phloem linking sources and sinks, but rather within the symplastic pathway of the receiver cells (Gould et al., 2004; Walsh et al., 2005).
Importantly, symplastic unloading rarely operates independently, as the pathway of unloading in storage tissues, in which soluble sugars are accumulated, often includes an apoplastic step at the periphery of the phloem or in subsequent cell layers (Lalonde et al., 2003). A preference for apoplastic unloading has been demonstrated in tomato and developing fruits by the movement of tracer dyes (Patrick, 1997). Thus, in sugarcane, where sugars accumulate in tissues that are relatively mature, the apoplastic pathway may be pre-eminent (Rae et al., 2005a; Rae et al., 2005b). The sugarcane culm contains high levels of apoplastic sucrose (Hawker & Hatch, 1965; Welbaum & Meinzer, 1990), which suggests that sucrose is unloaded from the phloem complex directly into the apoplast. Sucrose is then cleaved in the apoplast by CWI and taken up by
1997). At culm maturity, the concentration of sucrose would generate very high turgor pressures, if retained in the parenchyma cells. Measurements of turgor, wall extensibility and membrane conductivity in sugarcane parenchyma tissue have suggested that low turgor is maintained by partitioning of solutes between the symplastic and apoplastic compartments (Moore & Cosgrove, 1991).
An added complexity occurs within the sugarcane culm due to the structure of the vascular bundles, which are surrounded by a sheath of lignified fibre cells that serves to isolate the xylem water from the apoplast of culm storage tissues (Fig. 2.5) (Jacobsen et al., 1992; Welbaum & Meinzer, 1990). As the culm matures, the sheath cell layers progressively lignify to form a barrier to apoplastic movement of solutes during the period of sucrose accumulation (Jacobsen et al., 1992). Thus, sucrose probably cannot reach the parenchyma cells through a purely apoplastic path (Rae et al., 2005a). The symplastic passage of sucrose through the fiber sheath has been examined by Walsh et al. (2005) and is suggested to be a likely point of rate limitation in sucrose transport from phloem to storage parenchyma. Upon export from the vascular bundle, it is possible that sucrose may then move symplastically throughout the parenchyma, as has been partially demonstrated by tracer dyes (Rae et al., 2005b). However, in order to maintain a gradient for sucrose flow, sucrose may be excluded from the symplastic continuum by export to the apoplast or into the vacuole (Rae et al., 2005a). Backflow of apoplastic sucrose into the vascular system would then be restricted by the hydrophobic nature of the lignified and suberized cell walls surrounding the vascular bundle, thus forming an isolated apoplastic compartment (Jacobsen et al., 1992). Consequently, a comprehensive model for sucrose unloading in sugarcane should incorporate both symplastic and apoplastic transfer.
Fig. 2.5. Possible pathways for sucrose flux from source to sink in sugarcane. Sucrose is produced in the source mesophyll and either loaded symplastically via the bundle sheath or exported to the apoplasm and imported into the sieve element companion cell complex (SE-CCC). Sucrose leakage and re-uptake may occur along the SE-CCC.
Sucrose may then be unloaded in the culm sink symplastically through the fibre sheath, whereupon it flows from cell-to-cell through the plasmodesmata. Alternatively, sucrose may be exported from the symplastic continuum and then actively removed from the apoplast by surrounding parenchyma tissue via sugar transporter proteins ( ), or hydrolyzed to hexoses, taken up via hexose transporters ( ), and then resynthesized within the cell. Interconversion of sucrose and hexoses (‘futile cycling’) may co-exist within the vacuole and/or cytoplasm, and even indirectly extend into the apoplastic space due to sugar transporter activity (modified from Hellman et al., 2000 and Rae et al., 2005b).
sucrose
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hexoses CWI
hexoses VACUOLE
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sucrose sucrose
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sucrose CYTOPLASM
Apoplastic loading Symplastic
loading sucrolytic
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