The Role of Phloem Loading Reconsidered

It is generally assumed that the primary role of phloem loading is to drive long-distance transport by elevating hydrostatic pressure in sieve elements. This concept is consistent with the fact that, in many plants, energy is used to increase the concentrations of photoassimilates in the leaf phloem to levels well above those in mesophyll cells. However, on the basis of recent data, these fundamental assumptions need reevaluation. The data indicate that a large number of woody species—plants with the longest transport distances—load solute passively by maintaining high concentrations of Suc, and in some cases sugar alcohol, in mesophyll cells. Here I suggest that the adaptive advantage of active phloem loading, in the evolutionary sense, is to permit plants to maintain low foliar concentrations of nonstructural carbohydrates (NSCs), i.e. sugars, sugar alcohols, and starch. Economic considerations of inventory costs indicate that maintaining low NSC levels in leaves improves return on investment in carbohydrate synthesis, leading to a substantial increase in growth potential. Münch (1930) did not invoke active phloem loading in his model of long-distance transport. He assumed that the solute produced by photosynthesis generates sufficient hydrostatic pressure in mesophyll cells to drive transport from leaves to sink organs through the symplast. However, subsequent research indicated that the sugar concentration in the phloem is much higher than in mesophyll cells (for an early review, see Crafts, 1961). As the Münch hypothesis gained acceptance, so too did the notion that the role of loading is to energetically concentrate sugars in the phloem, increasing turgor and thereby providing the motivating force for pressure flow. This interpretation of the role of phloem loading has persisted. However, when one considers the primary finding—that there is a disparity in photoassimilate concentrations between the phloem and photosynthetic cells—it is equally reasonable to postulate that the adaptive advantage is derived from keeping the concentrations of these compounds low in the mesophyll. Viewed this way, active phloem loading allows plants to maintain low photoassimilate concentrations in leaves and at the same time elevate pressure in the phloem sufficiently to enable long-distance transport. To understand the difference between these two views, it is important to make the distinction between active and passive loading. In active loading, metabolic energy is used to pump photoassimilate into the phloem, against a concentration gradient. Two species-specific, active mechanisms are known (Fig. 1). In apoplastic loading, the proton motive force is used to carry Suc, and in some cases sugar alcohols, into the phloem via transporters (Lalonde et al., 2004; Sauer, 2007; Braun and Slewinski, 2009). The second mechanism, polymer trapping (Schulz, 2005; Turgeon and Wolf, 2009), is also active although the initial step is not. In the initial step, Suc diffuses from the mesophyll into the minor vein companion cells through plasmodesmata. The Suc in the companion cells is then used to synthesize raffinose and stachyose, a process that increases the concentrations of these sugars in the sieve element-companion cell complex. Although polymer trapping does not involve active transport in the formal sense of moving ions or molecules across a membrane, it is thermodynamically active overall; energy is used to create a concentration difference between the mesophyll and the phloem. There is now convincing evidence, by four independent protocols—histochemistry (Pristupa, 1983), microdissection of minor vein phloem (Haritatos et al., 1996), plasmolysis (Turgeon and Hepler, 1989), and analysis of aphid stylet sap (Voitsekhovskaja et al., 2006)—that the solute concentration in the phloem of polymer trap species is close to, or as high as, that found in plants that load via the apoplast. The polymer trap hypothesis is supported by recent evidence that loading is insensitive to down-regulation of the Suc transporter (Zhang and Turgeon, 2009), but highly sensitive to down-regulation of raffinose and stachyose synthesis (McCaskill and Turgeon, 2007). Schematic diagrams of putative phloem-loading strategies at the minor vein (A–C) and whole leaf (D–F) levels. In A and D, Suc diffuses through plasmodesmata (gaps in walls) into the minor vein companion cells (CC) and sieve elements (SE), a passive process. The Suc concentration in mesophyll cells (M), and therefore the entire leaf, is high; the Suc concentration in the veins is slightly lower. In B and E, Suc diffuses through plasmodesmata into the minor vein CCs (intermediary cells) and is converted to raffinose and stachyose, thus actively elevating the transport sugar concentration in the phloem by polymer trapping; this allows the mesophyll cells to maintain low Suc levels. In C and F, Suc is pumped from the apoplast into the minor vein phloem by transporters (yellow circle), which also enables the leaf to minimize the overall Suc concentration in the leaf blade. Phloem parenchyma cells, which may constitute part of the transport pathway in A and C, are not shown. For a diagram of sugar alcohol loading strategies see Rennie and Turgeon (2009). In contrast to these active mechanisms, passive loading is energetically downhill (Fig. 1). In the leaves of these plants, sugar levels are higher in the mesophyll than in the phloem. Ions and molecules diffuse through plasmodesmata at each interface, without a concentrating step (Turgeon and Medville, 1998; Reidel et al., 2009; Rennie and Turgeon, 2009). (For simplicity, passive transport through plasmodesmata is referred to here as diffusion, although bulk flow may also be possible in plasmodesmata with sufficiently large radii [Fisher and Cash-Clark, 2000; Voitsekhovskaja et al., 2006]). How can a passive transport process motivate long-distance flow in sieve elements? Recall that this is the hypothesis put forward by Münch (1930). The driving force for transport comes from the creation of high concentrations of solutes in the photosynthetic cells. The solutes diffuse into the phloem through plasmodesmata and in the sieve elements the hydrostatic pressure engendered by the solute molecules motivates mass flow (not diffusion) toward the sinks. According to Münch, mass flow in sieve elements occurs as long as the hydrostatic pressure in the phloem is higher at the source than in the sinks, irrespective of how this pressure is developed. It is interesting in this regard that Arabidopsis (Arabidopsis thaliana), though an apoplastic loader with reduced plasmodesmatal numbers in the phloem, is nonetheless able to complete its life cycle when the Suc transporter gene AtSUC2 has been genetically ablated (Srivastava et al., 2009). We recently analyzed loading strategies in 45 herbaceous and woody species (Rennie and Turgeon, 2009). Many of these plants, especially trees, exhibited the characteristics of passive loading. First, vein images were absent in autoradiographs when leaf discs were incubated in 14C-labeled Suc or 14C-labeled sugar alcohol. Autoradiographic vein images are expected if plants load actively, but if loading is passive radiolabeled compounds will pass freely between all symplastically connected cell types in the discs (Fig. 2). Second, concentrations of foliar transport sugars were high—up to 50-fold higher than those in the leaves of many herbs—as required if transport is due to diffusion. Third, all of the species in the survey with the two characteristics discussed above—lack of autoradiographic vein images and elevated sugar concentrations—have abundant minor vein plasmodesmata, a prerequisite for diffusion of large quantities of photoassimilate into the phloem (Gamalei, 1989). Autoradiographs of leaf discs from Arabidopsis and apple (Malus domestica). Abraded discs were incubated in [14C]Suc, washed, freeze dried, and pressed against x-ray film. Minor veins are apparent in Arabidopsis, but not apple, discs. Discs = 8 mm diameter. The data strongly suggest that many plants transport photoassimilate from source leaves to sinks without the need for active phloem loading, in agreement with Münch's original hypothesis. The fact that these plants are almost all trees makes it difficult to argue that energy expenditure at the loading step is needed for efficient phloem transport. If a willow (Salix spp.) or oak (Quercus spp.) tree can transport sugars over tens of meters without active loading, can it really be necessary in an herbaceous plant that is orders of magnitude smaller? This is not to say that active loading has no function in other parts of these plants. Even if the initial loading step from the mesophyll into the minor veins is passive, it is possible, indeed likely, that transporters introduce Suc and other materials into the phloem in other regions of the vasculature to compensate for leakage or to remobilize stored nutrients. It is also possible that active loading in leaves is initiated in certain developmental contexts, or under stress conditions. Phylogenetic analysis also suggests that active phloem loading is not necessary for long-distance transport. Although more work needs to be done on basal angiosperms, available evidence indicates that extensive plasmodesmatal continuity between the mesophyll and the minor vein phloem, an indicator of passive loading, is ancestral in the angiosperms (Gamalei, 1989; Turgeon et al., 2001). Active loading, either by transporters or polymer trapping, appears to be a derived trait. Again, it is difficult to argue that active loading is necessary if early angiosperms, predominately trees, thrived without it. Early experiments in transport physiology indicated that Glc and Fru are not translocated in the phloem (Arnold, 1968), suggesting that the leaf regulates the composition of export sap, presumably at the loading step. However, in symplastic loaders the opportunities for selecting particular substances for export are limited because flux of molecules through plasmodesmata is essentially nonspecific, except on the basis of size (Roberts and Oparka, 2003). How is regulation reconciled with an open, symplastic pathway into the phloem? First, there is actually little evidence that selectivity must be imposed at the loading step. Glc and Fru need not be specifically excluded from the phloem because they are sequestered almost entirely in the vacuoles of leaf cells (Heineke et al., 1994; Voitsekhovskaja et al., 2006; Nadwodnik and Lohaus, 2008). Therefore, they do not have access to the phloem, no matter the loading strategy. Furthermore, phloem sap is not a simple mixture of sugar and selected metabolites. It is a complex broth of organic and inorganic materials including, but not restricted to, ions, amino acids, amides, ureides, organic acids, nucleotides, phosphorylated metabolites of glycolysis, hormones, macromolecules, and several classes of secondary compounds, in addition to nonreducing sugars and sugar alcohols (Ziegler, 1975; Geigenberger et al., 1993; Turgeon and Wolf, 2009). The complexity of the sap indicates that a broad range of materials finds its way into the phloem. There are undoubtedly differences in composition between the cytosol of mesophyll cells and phloem sap, but these differences do not have to be explained by selective loading. The phloem sap could be, and probably is, modified all along the transport route by metabolism in companion cells, leakage from sieve elements, and retrieval of materials from the transpiration stream. Another concern raised by the concept of symplastic loading, either active (polymer trapping) or passive, is that unrestricted access to the phloem could result in an excessive and debilitating drain of essential metabolites from mesophyll cells toward the sinks. However, this seems unlikely when it is considered that minor vein companion cells in all plants cope successfully with such loss. In the minor vein phloem of leaves, net flux of ions and small molecules between companion cells and sieve elements is in the direction of the latter because the contents of the sieve elements are constantly being flushed away. Since the plasmodesmata-pore complex between these cell types is large enough to accommodate GFP (see Turgeon and Wolf, 2009), the companion cells must be constantly losing essential metabolites, even small proteins, to the river of sieve tube sap in the loading zone. Some small molecules are retrieved and returned to the leaf (Ayre et al., 2003), but many others must be resynthesized. This may be a partial explanation for the high metabolic activity of companion cells, especially in minor veins: Not only do they keep the sieve elements alive, they must persistently expend metabolic energy to restore the metabolites needed for their own survival. If companion cells are able to maintain their integrity under these conditions, mesophyll cells should be able to do the same considering that they are far more numerous than companion cells and will therefore suffer proportionately less metabolite loss. Viewed from this perspective, there is an advantage to symplastic loading: Plasmodesmata provide an uninterrupted and relatively unrestricted pathway for materials to replenish the companion cells and to provide essential nutrients of many types to the sinks. It is also possible that certain types of secondary compounds—for example, defensive molecules—have better access to the phloem through plasmodesmata than through the apoplast because there is no requirement for specialized transporters. If apoplastic loading imposes a burden on the plant by requiring the costly and selective use of transporters to maintain what in symplastic loaders is the free and largely unrestricted supply of photoassimilate and other substances to the phloem and sinks, it must confer some compensatory advantage. It does not seem likely that the advantage accrues simply from elevating hydrostatic pressure to motivate export, as discussed above. Another explanation must be sought. The growth of herbaceous plants roughly follows the dictates of compound interest laws; i.e. growth is proportional to present biomass. New leaves create photosynthetic potential, which fuels the growth of yet more leaves. The result is exponential growth. For the same reason, overall plant productivity is most favored when rapid growth occurs early (Harper, 1989; Dohleman and Long, 2009). Not surprisingly, plants with high relative growth rates (RGRs) allocate a large fraction of plant carbon to leaves, i.e. they have a high leaf weight fraction (Poorter and Remkes, 1990). It follows, therefore, that diversion of carbon away from building new photosynthetic machinery, whether by respiration, construction of other organs, or storage, has a negative impact on RGR (Bloom et al., 1985; Lambers and Poorter, 1992). Foliar NSC that is not needed for metabolism or transport can be considered excess inventory in the sense that it is not part of the photosynthetic machinery and is therefore not productive. Some NSC is essential, of course. Leaves accumulate carbohydrate during the day to accommodate respiration and export at night (Smith and Stitt, 2007). However, they do not need substantial reserves once night draws to a close; in a changeable world, at least dawn is predictable. Therefore, predawn NSC levels provide a reasonable measure of excess inventory. Literature values indicate that predawn NSC concentrations are low in many herbaceous plants. Sugar beet (Beta vulgaris) leaves retain only carbon in NSC at the of the night and Since they export carbon over a the predawn is only enough to respiration and export for a Arabidopsis leaves retain only of carbon during the day at the of the night and gene with carbon when the is by to et al., 2008). do herbaceous plants maintain such leaf requiring mechanisms to and (Smith and Stitt, is that carbon growth potential. We the impact of maintaining predawn inventory in the leaves of herbaceous plants the of and data on growth and carbon characteristics of species (Poorter and Remkes, et al., 1990). The is explained in more in The initial of the plant is in the sense that the leaves use all NSC during the have no predawn and all foliar carbon is assumed to be either or to the photosynthetic The initial weight of plant carbon is leaf weight is the leaf weight fraction for plants is (Poorter and Remkes, 1990). In the each of leaf carbon carbon by net photosynthesis (Poorter et al., and this carbon is to new leaves and the of the plant in the original plant and costs carbon required to and are from the produced carbon to of leaf carbon at and and carbon in et al., are also the of predawn on plant growth. strategy. In the of the carbon is in leaves, in the of the to of leaf carbon is excess inventory and the of the leaf carbon is carbon from the of the leaf is to the plant in its original construction costs are from the and from the leaves at are also is weight of plant carbon against excess leaf from to of leaf The initial weight of plant is The photosynthetic of leaf carbon either or carbon carbon Literature values for the of leaf carbon in at predawn are on the for Arabidopsis and and sugar beet and and et al., et al., apple et al., and and In the the weight of carbon in the plant increases to on day (Fig. It must be that plant growth is to numerous developmental and and is far more complex than the model However, the model does provide a for the growth of plants with of stored carbon in conditions. To the of excess inventory in leaves, the of leaf carbon to predawn NSC is in the model from to without the carbon to leaves (Fig. The of carbon in excess inventory is during the growth i.e. predawn NSC The growth for maintaining NSC is day a plant with excess inventory only of that in the plant (Fig. The in agreement with other of the of on growth (Harper, 1989), that maintaining carbon in leaves has a on growth potential. values of predawn NSC in leaves of several species are in values are not to or the growth characteristics of these plants, only to how concentrations of predawn NSC found in species growth potential. Although the are generally found in the leaves of woody there are NSC concentrations are high in an herbaceous species that Suc and from the apoplast (Rennie and Turgeon, 2009). Foliar solute against stress et al., and the model suggests that the of this on RGR is that NSC levels in and developmental For example, growth as plants and it is therefore unlikely that the RGR is over a as in the predawn foliar have an on a of plant et found that, in Arabidopsis growth with low and sugar levels in leaves at the of the low inventory levels as well as If productivity in the model is reduced from carbon to carbon as in the or by growth is reduced (Fig. The advantage by plants without excess inventory in these is not as as when productivity is it is substantial (Fig. Therefore, there is a advantage to foliar inventory in plants that are with one even in a If active phloem loading is so do trees not it more Here it is important to that most trees leaves in in the than by addition of new leaves the as is the with herbaceous plants of the carbon used to growth comes from 1989). Therefore, the advantage by keeping NSC leaf not to most woody species to the same as it does to herbaceous plants. In the RGR of trees is well that of and Poorter, 1992). There may be other for maintaining low NSC in leaves. in the of more carbon from leaves with inventory. are to compounds more than but the of carbon is nonetheless if more carbon is et al., Another for maintaining low Suc concentrations could be to of Although the and are not well there is convincing evidence that carbon and are and et al., active phloem loading plants by leaves to maintain concentrations of We have that and to on Suc, thus diffusion of photoassimilate into the phloem without of photosynthesis et al., 2009). is growth and in source and sink and regulation of and of carbon and all by and We will not understand how phloem loading is into this complex understand its primary There is little that high turgor is needed in source phloem to drive long-distance transport. concentrations of solute in the phloem also the sieve elements to maintain and to to when the plant is under stress and and However, there is to that active loading is needed to and maintain this pressure many plants with the longest transport function without it. In this suggests that active phloem loading for Economic considerations suggest that the adaptive advantage accrues from maintaining low foliar NSC thus carbon available for growth. It could be that the is that active loading to drive phloem transport and that over time plants advantage of this to NSC levels in mesophyll cells, thus increasing growth potential. However, there are two with this First, analysis indicates that active loading, either by transporters or polymer trapping, is a derived in the Second, many trees transport nutrients over long by passively photoassimilate into the phloem. are difficult to with the hypothesis that active loading is essential for efficient pressure flow and for that the other the hypothesis that active loading not to motivate transport but to plants to foliar carbon reserves is with the data and the of phloem-loading mechanisms in and in life According to this the of herbaceous plants, by leaf and rapid possible in part by active loading, in more efficient use of In apoplastic loaders there may have been a to in of restricted access to the phloem for nutrients and specialized compounds, but the costs for species with each other on the basis of rapid growth. available evidence it appears that predawn NSC levels in herbaceous plants, though as a they are than those of woody plants. It may be that some are in this growth by foliar reserves as a If this is and reserves are no needed in conditions, of in leaves could result in increases in the growth of plants. The fact that relatively minor differences in predawn carbohydrate levels in Arabidopsis, a species with low NSC with differences in growth et al., suggests that these could be The materials are available in the of this strategy. I for and also Wolf, and for of the