Recent progress in phloem physiology
Recently there have been significant advances in understanding of the processes involved in phloem transport, from the route of loading, realization that the pathway is not just a passive conduit, through plasmodesma involvement, to identification and localization of the protein transporters involved (Lalonde et al., 2003; van Bel, 2003). Techniques of molecular biology have led to an explosion in detail about the membrane transporters involved (sugar, water and ions). Here we briefly discuss only those advances that we see as relevant to modelling plant growth.
Several refinements to the Münch theory have been proposed, but the basic mechanism is still thought to be bulk solution flow driven by an osmotically generated pressure gradient. Phloem loading is now understood to involve either an apoplastic or a symplastic step, or a combination of these, between the site of carbohydrate synthesis and the sieve tubes involved in long-distance transport. Apoplastic loading involves flow of carbohydrate, usually sucrose, from the leaf parenchyma into the apoplastic space in the vicinity of the vascular tissue, from where it is actively taken up across the cell membrane into the sieve-tube companion-cell complex. With symplastic loading, carbohydrate flow from parenchyma tissue to the sieve-tube companion-cell complex is entirely through the plasmodesmata, not involving crossing a membrane, probably driven by diffusion. Many temperate-climate woody plants, including most of the tree species, are symplastic loaders (Turgeon & Medville, 1998). This is a very active research area with much interest in the function of plasmodesmata and what drives carbohydrate flow from parenchyma into the sieve tubes within symplastic phloem-loading species. While the specific details of generation of the sieve-tube pressure gradient is unlikely to be important in modelling plant growth, there is evidence for a correlation between the loading route and subsequent net lateral loss along the transport phloem (van Bel, 1996).
Phloem unloading into most tissues is currently thought to be symplastic, through plasmodesmata linking the cells in the sink region. So the end of the symplastic flow is not the terminal sieve elements, but within the receiver cells, with the sink osmotic pressure being kept low by metabolic utilization of the carbohydrate or conversion into less osmotically active polymorphic forms (starch, fructans). There is a growing body of evidence that the region of highest flow resistance is not within the transport phloem linking sources and sinks, but is within the symplastic pathway of the receiver cells (Gould et al., 2004a). Even within developing seeds, where the daughter tissue is symplastically isolated from the parent, making apoplastic transfers essential, phloem unloading from the terminal sieve tubes is symplastic into the seed coat tissues. But, whatever the route of unloading, the kinetics will be saturable.
Early in the study of phloem physiology, high metabolic rates associated with the vascular tissue were thought to be evidence for the driving force for phloem transport being generated along the entire length of the sieve tubes. This is now thought to be associated with the continuous simultaneous leakage and reloading along the transport pathway (Minchin & Thorpe, 1987; Thorpe & Minchin, 1996; van Bel, 2003). The sieve-tube solute concentration can exceed 1m and, as biological membranes are not perfect, it is not surprising that there is leakage into the surrounding apoplast. Reloading is essential if the conduit is not to lose its entire contents before delivery to the terminal sink, although this apoplastic carbohydrate is essential in maintaining stem tissues, and is also probably an important route for the storage and remobilization of carbohydrate within stems, trunks and roots of all plants. Plant species using symplastic phloem loading within the source leaves have been found to have a relatively higher potential for utilization of leaked photosynthate by the stem than found in species utilizing apoplastic loading. If this interpretation is correct, the former will have a tendency for greater lateral sinkiness than the latter, which will tend to favour terminal sinks more. Such a difference could give rise to substantial differences in relative growth rates and affect architectural traits (van Bel, 1996). A direct consequence of leakage and remobilization along the entire length of the transport phloem is to buffer changes in sieve-tube content brought about by changes in supply or demand. For example, a sudden reduction in supply is counteracted by reduced unloading flux with continuation of reloading from the apoplastic pool. This tends to decouple the source and sink for as long as there is an apoplastic pool able to be reloaded into the sieve tubes. (Minchin et al., 1983). The apoplastic pool may be replenished by remobilization of carbohydrate stored within the nearby ray or parenchyma cells.
The sieve elements are intimately associated with the companion cells, both being derived from a common mother cell. During maturation the sieve elements lose almost all their cytoplasm and organelles to become highly dependent on their adjacent companion cell, forming the sieve element–companion cell complex. Phloem loading involves carbohydrate transport into the sieve element–companion cell complex and through specialized plasmodesma between sieve elements and companion cells. The plasmodesmata allow the passage of a wide range of molecular species between these cells. Phloem sap has been shown to contain at least 150 different proteins: mRNA and RNA fragments, as well as carbohydrates and small molecules (van Bel, 2003). The roles, if any, played by this wide range of molecules in the coordination of plant growth and development are still quite uncertain, and these molecules may simply be the products of unselective loss from companion cells (Oparka & Santa Cruz, 2000), although there is growing evidence that short strands of RNA transported within the phloem sap are involved in gene silencing within the sink tissue (Yoo et al., 2004). Ayre et al. (2003) have shown that, while smaller sized metabolites from the companion cells do enter the translocation stream indiscriminately, subsequent membrane leakage and selective reloading has the effect of cleaning out such material from the long-distance pathway. Phloem-transported macromolecules have the potential to alter sink function by modulating the unloading kinetics, but this is still to be demonstrated.
Numerical modelling of Münch's basic hypothesis has demonstrated that, while this mechanism is able to account for both the observed specific mass flow and the transport speed within herbaceous plants, files of sieve tubes longer than several metres are not able to support observed flow rates (Thompson & Holbrook, 2003). Lang's (1979) suggestion of short files of contiguous sieve tubes acting as a relay, with unloading and reloading necessary to move photoassimilate from one short conduit to the next, would overcome this problem. This has not yet been confirmed, and will be difficult to distinguish from continuous unloading and reloading along the pathway.
New research tools
Molecular biology has added a large number of new methods for looking at the detailed processes involved in phloem transport, as well as providing transgenic plants which enable the whole-plant response to the complete knockout, or the downregulation or upregulation of specific genes. These approaches have confirmed the importance of apoplastic phloem loading; of continuous reloading along the pathway; and of the sucrose transporters within sink tissues. A large body of new mechanistic detail supporting Münch's original hypothesis has resulted, which has recently been reviewed by van Bel (2003).
For modelling purposes we need measurements of the dynamic behaviour of the intact phloem system, which can be obtained only by noninvasive methods, as the phloem system is very sensitive to mechanical probing. Hence noninvasive measurement techniques are playing an increasingly important role in phloem physiology. Confocal microscopy allows in vivo observation of phloem function (Wright & Oparka, 1997), while nuclear magnetic resonance has been used to image functional phloem (Peuke et al., 2001). Short-lived isotope techniques, specifically C-11, allow in vivo measurement of phloem transport with a very fine time resolution (seconds), but is limited by its short decay time (t½ 20.4 min; Minchin & Thorpe, 2003). In vivo measurement has been fundamental to the development of the minimalist Münch-based model discussed in the following section.
Aphids feed on phloem sap, so their ability to find and connect their stylets into individual functioning sieve tubes has long been exploited as a means of sampling phloem sap. Wright & Fisher (1980) showed how a manometer can be attached to the stylet of an aphid; more recently Gould et al. (2004b) used the pressure probe continuously to monitor the hydrostatic pressure within functional sieve tubes while applying experimental treatments to the phloem system. This work has produced results consistent with Münch's hypothesis of osmotically driven bulk flow.
Another potentially useful tool in phloem studies is metabolic control theory, developed by Kacser & Burns (1973) and Fell (1997), to provide a sound theoretical framework for studies of controlling flow through complex metabolic systems. This resulted in radically new ways of understanding flux control, and has demonstrated that the concepts of limiting factors and bottlenecks are not appropriate. A top-down form of this theory (Quant, 1993), involving lumping the parts of the overall system, has been developed. Using this approach on a simple plant system consisting of a single source and a single sink, it was found that the source leaf had 80% of the control of photosynthate flux between leaf and sink (Sweetlove et al., 1998; Farrar & Jones, 2000), and this high control by the source leaf was maintained for plants deprived of N, cooled, or at low light levels (Sweetlove & Hill, 2000). This approach has not been used to look at flows with multiple sinks; such work is badly needed, as there is a general belief that it is the sinks that determine C flows into the individual sinks.