Sucrose accumulation in the sugarcane stem： pathways and control

Sucrose accumulation in the sugarcane stem:pathways and control points for transport and compartmentationAnne L.Rae *,Christopher P.L.Grof,Rosanne E.Casu,Graham D.BonnettCSIRO Plant Industry,Queensland Bioscience Precinct,306Carmody Road,St Lucia,Qld 4068,AustraliaAbstractThe accumulation of high concentrations of sucrose in the stem of sugarcane has been the subject of many studies.Although models have been constructed from the available information,many steps in the transport and accumulation pathway remain unknown.Recent advances in molecular approaches may elucidate some of these processes.Genes encoding proteins associated with sugar synthesis and storage will provide valuable tools.In particular,the use of techniques to localize the sites of expression of sugar transporters and metabolic enzymes will assist in defining possible routes of sugar movement.When combined with an analysis of metabolite concentrations and enzyme activities in cellular and subcellular compartments,these novel approaches will contribute to an integrated picture of stem function.Control points identified will provide useful tools for selection of efficient genotypes and targets for molecular manipulations.#2005Elsevier B.V .All rights reserved.Keywords:Phloem;Sucrose metabolism;Sugarcane;Sugar transporters;Vacuole1.Control points for sucrose storageMature sugarcane stems are capable of accumulat-ing high concentrations of sucrose,approaching 650mM in the storage tissues of some varieties (Welbaum and Meinzer,1990).Although sucrose is commonly found in plant storage organs,it is generally at a low concentration,and starch is the predominant storage carbohydrate (Komor,2000).Storage of assimilate as a small osmotically active molecule might be expected to create metabolicstresses in the cells of the storage tissue.Indeed,a study of the gene transcripts associated with the process of maturation in sugarcane internodes showed increased transcription of many genes involved in stress responses (Casu et al.,2004).The partitioning of sugars into tissues,cells and subcellular compartments is determined by control points in transport pathways.Thus,knowledge of the location of these control elements in the tissue is crucial to an understanding of the sucrose storage process.In addition,these control points will be potential targets for manipulation in strategies to increase sucrose accumulation (Grof and Campbell,2001).Recently,a number of genes which encode enzymes of sugar metabolism or sugar transport in/locate/fcr*Corresponding author.Tel.:+61732142379;fax:+61732142950.E-mail address:anne.rae@csiro.au (A.L.Rae).0378-4290/$–see front matter #2005Elsevier B.V .All rights reserved.doi:10.1016/j.fcr.2005.01.027sugarcane have been identified(Zhu et al.,2000; Carson and Botha,2002;Carson et al.,2002;Grivet and Arruda,2002;Casu et al.,2003).Together with a functional analysis of these enzymes,information on the localization of their expression and activity will enable us to build a model of the pathways and control points for sugar movement into the storage sink.This model will highlight where further investigation is required and will be used to develop strategies for increasing sucrose concentration.2.Source to sink pathwaySucrose is synthesized in the photosynthetic tissue of the leaf mesophyll.In some species,sucrose is able to move from the mesophyll cells to the conducting cells of the phloem via direct cell-to-cell connections known as plasmodesmata.Species with numerous plasmodesmata connecting the phloem cells to surrounding cells have been classified as symplastic phloem loaders(van Bel and Gamalei, 1992).However,there is good evidence that loading of sucrose into the phloem occurs from the apoplast in many species.Sucrose transporter proteins,which would permit the import of sucrose from the extracellular solution,have frequently been localized to the phloem cell membranes(Lalonde et al.,2003). Mutant plants with insertions in a gene encoding a sucrose transporter were unable to export sucrose normally(Gottwald et al.,2000).Further evidence that sucrose passes through the apoplast before entering the phloem was provided by transgenic tomato plants that were engineered to express invertase in the leaf apoplast(Dickinson et al., 1991).In the transgenic plants,starch accumulated abnormally in the leaf cells and growth was severely restricted,indicating that sucrose export from the leaf was impaired.The complete pathway of sucroseflow in apoplastic phloem loaders is not known but the vascular parenchyma is considered to be the most likely site for release of sucrose into the apoplast(Lalonde et al.,2003).In sugarcane,the conducting cells of the phloem are not connected to other cells of the leaf by plasmodesmata(Robinson-Beers and Evert,1991).This suggests that phloem loading occurs from the apoplast in sugarcane leaves.In the phloem,sucrose moves out of the leaf and towards sink tissues,including developing shoot and root apices and storage organs.The movement of sucrose through transport phloem is thought to be driven by concentration gradients(van Bel,2003).In source tissues,loading of sucrose causes influx of water and the resulting high turgor promotes move-ment away from the source.Conversely,removal of the sucrose from phloem causes a reduction in osmotic pressure in sink phloem tissues(van Bel,2003). Sucrose transporters continue to be expressed in transport phloem and may act in retrieval of sucrose lost to the apoplast by leakage(Lalonde et al.,2003). Although no direct measurements have been made in sugarcane,the14CO2studies of Hartt et al.(1963) indicate that sucrose is the major carbohydrate transported in the phloem.In the sink tissues,sucrose is unloaded from the phloem.Several pathways of unloading have been described(Patrick,1997).At junctions between maternal andfilial tissue and at interfaces between host and symbiont,the pathway of unloading always includes an apoplastic step.However,in vegetative tissues,movement may be either apoplastic or symplastic.In potato,the tubers are storage sinks that develop from shoot meristems.The movement of the carboxyfluorescein tracer dye,which is restricted to the symplast,showed that phloem unloading is predominantly apoplastic in stolons.However,the onset of tuber development and starch accumulation is accompanied by a switch to symplastic movement of solutes from phloem to storage parenchyma cells (Viola et al.,2001).In storage tissues where soluble sugars are accumulated,the pathway of unloading includes an apoplastic step at the periphery of the phloem or in subsequent cell layers(Lalonde et al.,2003).For sugarcane,which accumulates soluble sugars into a tissue which is relatively mature,this type of pathway may be more relevant.Accumulation of soluble sugars in tomato fruits is an example of an unloading pathway that involves an apoplastic step.In developing fruits, the switch to soluble sugar accumulation is accom-panied by a reduction in symplastic connectivity, shown by the movement of tracer dyes(Patrick,1997). At this stage,invertase activity in the apoplast and hexose transport activity in the storage cells also develop.These observations suggest that sucrose isA.L.Rae et al./Field Crops Research92(2005)159–168 160released into the apoplast and cleaved by invertase, and that hexoses are mobilized into storage cells where sucrose may be re-synthesized(Patrick,1997).Celery petioles provide a further example of an unloading pathway that includes an apoplastic step.In celery,mannitol is exported from mature leaves in addition to sucrose.Mannitol and sucrose transporters have been identified in the leaves and both sugars are found in the transport phloem(Noiraud et al.,2000, 2001).The major storage sink in celery is formed by the parenchyma cells of the enlarged petiole which have been shown to accumulate mannitol and hexoses. Although the pathway of unloading from the phloem in the storage tissue has not been fully characterized, some steps are known.The expression pattern of sucrose transporters in the petiole suggests that unloading involves an apoplastic step(Salmon et al.,1995).Furthermore,protoplasts isolated from petiole parenchyma were able to take up mannitol and hexoses but not sucrose(Salmon et al.,1995).3.Phloem unloading in sugarcaneThe movement of sucrose from transport phloem to the storage parenchyma in sugarcane is influenced by the structure of the storage tissue.Sugarcane stem vascular bundles are surrounded by a layer of thick-walled cells which become progressively lignified and suberized with development.These layers effectively form a barrier to apoplastic movement of solutes during the period of sucrose accumulation(Figs.1and 2;Jacobsen et al.,1992).Thus sucrose probably cannot reach the parenchyma cells from the phloem by a purely apoplastic path.The presence of numerous plasmodesmata suggests that all cells within the vascular bundle are connected symplastically(Walsh et al.,1996).We have shown that a symplastic tracer dye can move from the phloem into vascular parenchyma cells and then through the thick-walled cells into thefirst layer of parenchyma cells surrounding the vascular bundle(Rae et al.,2005). While movement of a tracer dye may not directly parallel movement of sucrose,the results suggest that sucrose may be able to exit the vascular bundle through symplastic connections.It is possible that sucrose is able to move symplastically throughout the parenchyma tissue.However,in order to maintain a gradient for sucroseflow,it is likely that sucrose is excluded from the symplastic continuum by export to the apoplast or into the vacuole.High concentrations of sucrose have been found in the apoplasticfluid in the storage parenchyma(Welbaum and Meinzer, 1990).This implies that a mechanism exists for export of sucrose into the apoplast at some point outside the thick-walled cells that surround the vascular bundle.Backflow of apoplastic sucrose into the vascular system would be restricted by the hydrophobic nature of the lignified and suberized cell walls surrounding the bundle,thus forming an isolated apoplastic compartment(Jacobsen et al., 1992;Welbaum and Meinzer,1990).The biophysical properties of the storage tissue may be important in determining storage capacity and mechanisms.At maturity,the concentration of sucrose in the stem would generate very high turgor pressures if retained in the parenchyma cells.Measurements of turgor,wall extensibility and membrane conductivity in sugarcane parenchyma tissue suggested that low turgor is maintained by partitioning of solutes between the symplastic and apoplastic compartments(Moore and Cosgrove,1991).4.Role of transporters in unloadingThe role of transporters in the influx of sucrose during phloem loading has been well documented but their role in unloading and post-phloem pathways is less clear.The gradient of sucrose concentrations suggests that post-phloem efflux from the symplast could occur by facilitated diffusion;movement through a transmembrane pore which has specificity for sucrose but which is driven solely by a gradient of the substrate and not energized by direct or indirect consumption of ATP.The proteins responsible for efflux of sucrose into the apoplast in the post-phloem pathway have not been identified.In many species, transporters with sugar influx activity have been localized to sites where efflux would be expected,for example in the nucellar projection cells in developing wheat grains(Bagnall et al.,2000)and in developing Arabidopsis roots(Truernit and Sauer,1995).These transporters demonstrate the capacity for proton co-transport with sucrose when expressed in yeast mutants.However,in some circumstances,theseA.L.Rae et al./Field Crops Research92(2005)159–168161162A.L.Rae et al./Field Crops Research92(2005)159–168Fig.1.Tissues and cells from sugarcane stem internode8.In a section stained with berberine,yellowfluorescence indicates the presence of suberin in the walls of cells surrounding two vascular bundles(A).Staining of a similar section with phloroglucinol shows that these cell walls also contain lignin(B).The rigidity of these cell walls allows the bundles to be pulled intact from the surrounding parenchyma tissue.Isolated vascular bundles shown in longitudinal view(C)and in cross-section(D)were largely free of parenchyma cells but some damaged fragments adhered to thefibre cells.A segment of parenchyma tissue isolated by micro-dissection(E)shows no contamination by thick-walledfibre cells.A higher magnification(F)shows that the cells are intact.(G)In a protoplast released from the isolated parenchyma tissue by enzymatic digestion,a large central vacuole is visible.When protoplasts were incubated with phenosafranin(H),they failed to accumulate the dye,indicating that the plasma membrane is undamaged.Staining of protoplasts with thefluorescent brightener(I),Calcofluor,showed that the cell wall was completely removed,while cellulose debris in the medium retained strongfluorescence(bars A–D,F:100m m;E:300m m;G–I:50m m).sucrose carriers are also capable of transporting sucrose by facilitated diffusion(Lemoine et al.,1996). These observations have led to the suggestion that sucrose/H+influx transporters may also be responsible for sucrose efflux by facilitated diffusion(Lalonde et al.,2003).In the sugarcane stem we have identified a sucrose transporter,designated ShSUT1,which is localized to tissues surrounding the stem vascular bundles(Rae et al.,2005).While this transporter may have a role in returning apoplastic sucrose to the symplast,it is also possible that it is involved in sucrose efflux into the apoplast of the storage tissue. Further experiments will be needed to investigate this possibility.5.Export of sucrose to the apoplastIn pathways of unloading that include an apoplastic step,sucrose that accumulates in the apoplast may be converted to other sugars.In developing barley seeds, sucrose unloaded from the maternal tissues is cleaved by invertases,and both hexose and sucrose transpor-ters are expressed in the transfer cells at the surface of the endosperm to facilitate uptake.There is evidence in this system that co-ordinated expression of invertase and the transporters regulates the hexose/sucrose ratio which may be an important developmental signal (Weschke et al.,2003).Extracellular invertase activity has been detected in sugarcane stem tissue and may be responsible for converting a proportion of the apoplastic sucrose to hexoses(Glasziou and Gayler,1972).It has been suggested that the activity of the invertases maintains a gradient of sucrose to drive continued unloading. However,both invertase activity and hexose content have been shown to decline with increasing maturity of stem internodes(Welbaum and Meinzer,1990; Rose and Botha,2000;Albertson et al.,2001), suggesting that this pathway may only be significant in immature internodes.In young tissues,demand for glucose as a precursor for cellulose synthesis and for respiration is likely to be higher than in mature tissues.The question of whether sugarcane parenchyma cells are able to take up sugars as sucrose,as hexoses or both has not yet been resolved.Suspension-cultured cells have been shown to take up glucose(Komor et al.,1981).However,there is also evidence that slices of stem tissue can take up sucrose(Lingle, 1989).We have recently identified a sugarcane hexose transporter which is expressed in the parenchyma cells (Rae and Grof,unpublished results).Further investi-A.L.Rae et al./Field Crops Research92(2005)159–168163Fig.2.Possible routes for transport of sucrose into the storage parenchyma of the sugarcane stem.In this model,sucrose is unloaded symplastically from the phloem,and could potentially move symplastically throughout the storage tissue by cell-to-cell connections.However, the presence of sucrose in the apoplast together with the need to maintain a gradient for continued unloading suggests that at some point,sucrose exits the symplastic continuum.Sucrose or the products of invertase activity may be taken up by parenchyma cells via membrane transporters and may subsequently cross the tonoplast.Enzyme activity may interconvert sucrose and hexoses in each of these compartments.The localization of the components of this pathway is critical to completion of this model.The location of sugar transporters and of sugar metabolising enzymes such as sucrose synthase and invertase will be of particular importance.gation of the cell expression patterns of hexose and sucrose transporters in sugarcane may help answer this important question.It has been suggested that glucose and fructose taken up by hexose transporters in the membranes of parenchyma cells could be used to re-synthesize sucrose by intracellular enzymes(Wendler et al., 1990;Whittaker and Botha,1997).The reasons behind this apparent futile cycling are not yet clear,however localization of the components of the sucrose transport pathway may provide the evidence to support or disprove this hypothesis(Fig.2).For example,this pathway would require the expression of both hexose transporters and sucrose synthase or sucrose phos-phate synthase in the parenchyma cells.The genes encoding these metabolic enzymes have been identi-fied in sugarcane and these will be useful tools to clarify the pathway of sugar movement.6.Sucrose in the vacuoles of storage parenchyma cellsSince the vacuole occupies a large proportion of the parenchyma cells in the sugarcane stem,it is likely that much of the stored sucrose resides in this partmentation of sucrose into the vacuole would help to maintain low concentrations in the cytoplasm and thus provide a driving force for continued movement of sucrose into parenchyma cells,either by uptake from the apoplast or through symplastic connections.However the relative con-centrations of sucrose in the vacuole and cytoplasm have not been measured in sugarcane tissue.Recent developments in micro-sampling techniques may permit these measurements with minimum disruption to the tissue.It is known in other species that the activity of H+-ATPase and pyrophosphatase at the tonoplast mem-brane maintain a low pH inside the vacuole and provide a source of energy for active transport across the membrane against a concentration gradient (Maeshima,2001).As both enzymes are modulated by stress(Davies,1997),it would be interesting to measure their activity in the stem of sugarcane,where high concentrations of sucrose could induce osmotic stress.A tonoplast-bound ATPase has been identified in vacuoles isolated from sugarcane suspension-cultured cells(Thom and Komor,1985).Transporters using the H+gradient as a driving force would be expected to act as H+/antiporters and a number of cation transporters of this type have been identified (Maeshima,2001).A sucrose antiporter activity has been reported in membranes from sugarbeet roots (Briskin et al.,1985).However,studies of sugar storage in other species suggest that movement into the vacuole commonly occurs by facilitated diffusion and not via an active antiport mechanism.When partitioning of mannitol inside the parenchyma cells of celery petioles was studied,mannitol was found at a similar concentration in both the cytoplasm and in the vacuole.Analysis of the transport properties of the tonoplast membrane demonstrated the existence of a mannitol-specific carrier that operates by facilitated diffusion(Greutert et al.,1998).In other examples,the H+efflux characteristic of active transport could not be detected when sugars were added to purified tonoplast vesicles from tomato fruits(Milner et al.,1995)or lime fruits(Echeverria et al.,1997).Purification of vacuoles from sugarcane has been problematical and has produced conflicting results on sugar transport across the tonoplast membrane(Moore,1995).In preparations of vacuoles that accumulated sucrose, there was no evidence for active transport,suggesting that facilitated diffusion may be responsible for accumulation of sugars(Priesser and Komor,1991; Williams et al.,1990).The tonoplast proteins that mediate vacuolar uptake of sugars in sugarcane have not yet been identified.7.Developing tools to elucidate control pointsfor transport and accumulation of sucrose Although many questions still remain,a few steps in the pathway of sucrose transport to the storage parenchyma in sugarcane can now be tentatively defined(Fig.2).For example,the evidence suggests that sucrose is exported from the vascular bundles by a symplastic pathway.Symplasticflow of solutes may be influenced by differences in osmotic pressure between cells.These could be generated by regulating the intracellular and apoplastic concentrations of solutes through the activity of invertases and other sugar metabolising enzymes(Patrick,1997).Trans-porters that control the trans-membrane movement ofA.L.Rae et al./Field Crops Research92(2005)159–168 164salts have also been implicated in osmotic regulation. Interestingly,one of the most strongly up-regulated transcripts in maturing stem of sugarcane is homo-logous to a Na+/H+antiporter from Arabidopsis(Casu et al.,2004).This may indicate an increased need for osmotic adjustment as sucrose content increases in the stem.Alternatively,water movement can be regulated by the activity of aquaporins,or trans-membrane water channels.A number of cDNA clones with sequences homologous to aquaporins have been isolated from maturing sugarcane stem(Casu et al.,2004).Symplastic transport may also be regulated by control of solute movement through plasmodesmata (Roberts and Oparka,2003).Small molecules appear to move through these membrane-lined pores by simple diffusion,thus forming a rate-limiting step in transport pathways.However,transport through plasmodesmata is sensitive to a wide range of physiological changes and regulation of transport capacity is feasible.The molecular components of plasmodesmata have been difficult to identify.Some information was derived by inference from ultra-structure and from the movement of dyes with a known molecular size.The study of viral movement proteins demonstrated that effective pore size could be manipulated(Roberts and Oparka,2003).A small number of mutants with defects in plasmodesmata function have been identified.The sxd1mutant in maize was unable to export sucrose normally from leaves due to blockages in plasmodesmata connecting the bundle sheath and vascular parenchyma cells (Russin et al.,1996).It now appears that sxd1is not a plasmodesmata protein and the defect in plasmodes-mata function is a pleiotropic effect of a mutation that affects differentiation of bundle sheath cells(Mezitt Provencher et al.,2001).Recently,a novel approach to high-throughput screening of genes by localization of protein-GFP fusions has identified11cDNAs that encode plasmodesmata proteins(Medina Escobar et al.,2003).This approach should permit significant advances in our understanding of plasmodesmata function in the near future.Transfer of solutes between the apoplastic and symplastic compartments is controlled by carrier proteins embedded in the plasma membrane.These proteins are considered to be rate limiting steps in many pathways of nutrient uptake(Kunze et al.,2002) and are a common target for genetic manipulation.A number of putative transport proteins were identified in a collection of cDNA clones from maturing sugarcane stem(Casu et al.,2003).We are currently investigating the sugar transport capacity and cellular localization of these proteins in the sugarcane stem. Two approaches have been useful for localization. Antibodies raised to peptides that match the sequence of the transporter protein have been used in immunolabelling experiments to detect the proteins in tissue sections.Transcripts corresponding to the genes encoding the transporters have also been used to pinpoint sites of gene expression by in situ hybridiza-tion.The information from these localization studies has been used to suggest roles for the transporters in the sugarcane stem.We have shown that a hexose transporter is probably involved in retrieval of sugars into the phloem(Casu et al.,2003)and that a sucrose transporter may play a role at the interface between transport and storage tissue(Rae et al.,2005). Experiments to localize the other sugar transporters within the stem tissue in a similar way will further define possible routes of sugar transport.Fig.2highlights the paucity of information regarding the cellular and subcellular location of enzymes that interconvert sugars in the sugarcane stem.Recently,genes encoding a number of enzymes have been identified from sugarcane gene libraries (Zhu et al.,2000;Carson and Botha,2002;Carson et al.,2002;Grivet and Arruda,2002;Casu et al., 2003).As described for the transporters,these genes and the encoded protein sequences will be valuable tools for the localization of enzymes in sugarcane tissue.Together with an analysis of metabolite concentrations in the cellular and subcellular compart-ments,this information can be used to establish pathways and control points for metaboliteflux.The metabolic activities of storage parenchyma cells are not well understood.Some of the activities suggested in the model(Fig.2)are derived from studies of suspension-cultured cells,which may differ markedly from cells in intact tissue.In mature internodes of the sugarcane stem,the parenchyma cells are large and highly vacuolated.Both micro-scopic examination and analysis of gene expression suggest that these cells are no longer actively dividing. Transcripts were also reduced in maturing internodes for genes that encode enzymes associated with transcription and translation mechanisms(CasuA.L.Rae et al./Field Crops Research92(2005)159–168165et al.,2004).However,the metabolic activities of the parenchyma cells are presumably important to maintaining and regulating the high concentrations of sucrose in the tissue.We are using several new approaches to investigate gene expression in parench-yma cells.Vascular and parenchyma tissue can be physically separated by micro-dissection of the sugarcane internode(Fig.1C–F).The vascular bundles and their surrounding sheath of thick-walledfibre cells can be pulled away from the parenchyma cells relatively easily,allowing the extraction of RNA from separate tissue types.We have used this RNA to test the tissue-specific expression of genes that were identified by microarray experiments as being strongly up-regu-lated in mature stems.Few of these genes show significant activity in the parenchyma tissue(Rae et al.,unpublished results).This suggests that parenchyma genes may be poorly represented in cDNA libraries made from whole stems,consistent with low transcriptional activity in this tissue.It may be necessary to develop new approaches to target parenchyma genes specifically.RNA isolated from the parenchyma cells will be used to construct a single-tissue cDNA library,so that the genes expressed in these cells can be characterized.This approach may allow the identification of proteins that are involved in sugar accumulation but which have previously been difficult to isolate,such as plasma membrane and vacuolar transporters.As a means of testing the functional properties of parenchyma cells,we have also developed a procedure for releasing protoplasts from the stem parenchyma cells using enzymic digestion(Fig.1G–I).Intact protoplasts will be an important tool to further dissect parenchyma cell function.8.ConclusionsIn summary,information on the cellular and sub-cellular location of the proteins that facilitate sugar transport will be immensely valuable in building a complete picture of the pathways and control points.It is important to be aware that the metabolic activities in the storage tissue are not uniform from one internode to another but will change with development.As the internodes mature,the balance between synthesis,growth and storage of carbohydrates changes.This is reflected by changes in sucrose/hexose ratios,inver-tase activity and expression of transporters in older internodes.A comprehensive model of sugar transport and accumulation pathways will need to take these developmental changes into account.This review has covered some of the recent developments in thefield;for more information the reader is referred to reviews by Moore(1995),Komor (2000)and Grof and Campbell(2001).An under-standing of sugar transport pathways will be the basis for strategies to increase theflux of sugars and ultimately the amount of sucrose in the storage tissue (Grof and Campbell,2001).A broad genetic resource of varieties and species of sugarcane with a range of sucrose contents is available to help define which steps of the pathway are critical.Control points identified during this process will be useful as markers for enhanced selection in breeding programs and may also be targets for genetic manipulation to increase sucrose accumulation.AcknowledgementThe authors’research is supported by the Sugar Research and Development Corporation,Australia and the Cooperative Research Centre for Sugar Industry Innovation through Biotechnology. 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