uum, and subsequent carrier-mediated transport across the. SE/CC plasma membrane; and (2) direct symplasmic cell- to-cell diffusion via plasmodesmata.
The Plant Cell, Vol. 11, 707–726, April 1999, www.plantcell.org © 1999 American Society of Plant Physiologists
The Dual Function of Sugar Carriers: Transport and Sugar Sensing Sylvie Lalonde,a Eckhard Boles,b Hanjo Hellmann,a Laurence Barker,a John W. Patrick,c Wolf B. Frommer,a,1 and John M. Warda aCenter
for Plant Molecular Biology (ZMBP), University of Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, Geb. 26.12.01, D-40225 Düsseldorf, Germany cDepartment of Biological Sciences, University of Newcastle, New South Wales 2308, Australia bInstitut
INTRODUCTION
SUGAR TRANSPORT SYSTEMS IN HIGHER PLANTS
Sucrose and its derivatives represent the major transport forms of photosynthetically assimilated carbon in plants. Sucrose synthesized in green leaves is exported via the phloem, the long-distance distribution network for assimilates, to supply nonphotosynthetic organs with energy and carbon resources. Sucrose not only functions as a transport metabolite but also contributes to the osmotic driving force for phloem translocation (mass flow) and serves as a signal to activate or repress specific genes in a variety of different tissues. The long-distance transport of sucrose depends on a family of proteins that act as sucrose carriers. The analysis of transgenic plants impaired in sucrose transporter expression has demonstrated that sucrose transporter1 (SUT1) is essential for sucrose translocation in potato and tobacco. These results, together with the localization of SUT1 to sieve elements (SEs), indicate that phloem loading occurs in SEs by transmembrane uptake of sucrose directly from the apoplasm. The sucrose transporters identified so far arise from a single gene family. Some of the newly identified members of the family are involved in specific functions, such as nutrition of developing seeds or pollen. Physiological and molecular studies show that sucrose transport is highly regulated at multiple levels of biological organization and in response to changing sucrose concentrations. Thus, one of the most exciting topics in the regulation of sucrose transport is signal perception. By analogy to yeast, in which members of the sugar transport family serve as sugar sensors, we propose that members of the plant sugar transporter family play a direct role in the signal transduction responsible for regulation of sugar transport and, thus, metabolism in general.
Plant Anatomy: Vascular Tissues
1To
whom correspondence should be addressed. E-mail frommer@ uni-tuebingen.de; fax 49-7071-29-3287.
Nonphotosynthetic tissues and organs, including the entire below-ground portion of the plant, need to be supplied with energy and fixed carbon. Sugars, synthesized in the mesophyll cells, serve as the major exported photosynthetic product. To accommodate long-distance transport of sugars from source (net exporting) to sink (net importing) organs, a vascular network—the phloem—has evolved in land plants. The most abundant compound in the phloem sap of most plant species is the disaccharide sucrose (Zimmermann and Ziegler, 1975). For a minority of plant species, the principle translocated sugars fall into two main groups: the sugar alcohols (mannitol and sorbitol) and the raffinose series (raffinose, stachyose, and verbascose) (Zamski and Schnaffer, 1996). In most cases in which such sugars predominate, however, sucrose is also present. With the exception of a few well-studied species, our knowledge of phloem sap composition is limited to crude analyses (Zimmermann and Ziegler, 1975) derived from stem incision experiments in which sugars present as storage compounds in stems may contaminate phloem sap samples. Therefore, accurate and less invasive techniques, such as in vivo NMR (Köckenberger et al., 1997) or positronemitting tracer imaging system (Hayashi et al., 1997), will be required for a more accurate understanding of phloem sap composition. The phloem of angiosperms consists of several types of cells that are closely associated with the xylem within the vascular bundle. The structure and development of the phloem has been reviewed recently (Sjölund, 1997; Ward et al., 1998; Oparka and Turgeon, 1999, in this issue). The actual conduits in phloem consist of two ontogenetically related cell types: companion cells (CCs) and SEs. These two cells are highly modified and well interconnected by plasmodesmata. SEs, for instance, lose their nuclei, vacuoles, and many other organelles during maturation and form
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tubes of living cells connected by sieve pores. CCs, which are characterized by dense protoplasm, retain a nucleus and numerous mitochondria and are thought to provide functions essential for the survival of SEs via plasmodesmatal links (Lucas et al., 1993). The complete physical pathway of sucrose transport from source to sink is not completely elucidated for any plant, and important differences may exist among species. From the point of sucrose synthesis in the mesophyll, the route to the SE involves several cell types: neighboring mesophyll cells, bundle-sheath cells, phloem parenchyma, and CCs. Cell-to-cell movement of sucrose is considered to occur via plasmodesmata from the point of synthesis up to the SE/CC complex which, in many species, is not well connected to the surrounding cells (Gamalei, 1989). Two principal routes for the delivery of sucrose into the SE/CC complex have been proposed: (1) transporter-mediated export from mesophyll cells, apoplasmic diffusion through the cell-wall continuum, and subsequent carrier-mediated transport across the SE/CC plasma membrane; and (2) direct symplasmic cellto-cell diffusion via plasmodesmata. The extent to which plants utilize either of these pathways and whether plants can switch between these pathways is a question currently under active investigation.
Symplasmic Transport Figure 1 also schematizes the possibility that each transport process outlined above could alternatively take place symplasmically. Based on the systemic movement of plant virus in phloem, plasmodesmal connections between the SE/CC complex and surrounding cells are present even in Solanaceous species which are classified as type 2a (van Bel and
Apoplasmic Transport Plants that primarily utilize an apoplasmic phloem loading mechanism require the transmembrane transport of sucrose and other solutes into the phloem. For such plants, as schematized in Figure 1, we can predict a minimum of five sucrose transport activities along the translocation path. In leaves, the first transport step must be release of sucrose into the cell wall directly from the mesophyll cell (Figure 1, transporter 1). Mechanistically, this first transporter could be a facilitator or an antiporter. Such sucrose efflux systems have been described biochemically (see Delrot, 1989; Laloi et al., 1993). Subsequently, at least one transporter is required for uptake into the phloem (Figure 1, transporter 2). These loading processes are required at various stages of development, such as during germination for sugar export from leaves or for mobilization of stored carbon, events that might require distinct transporters. Reuptake of sucrose along the translocation pathway is necessary to allow solute exchange with the phloem, for example, in stems (Figure 1, transporter 3; Minchin and Thorpe, 1987). In sink tissue, unloading can occur either by transmembrane export of sucrose (Figure 1, transporter 4) or through plasmodesmata. Sucrose efflux transporters involved in phloem unloading have been postulated to function as facilitators or as proton antiporters (Walker et al., 1995). Sucrose in the apoplast of sink tissue can be taken up directly (Figure 1, transporter 5) or through the hydrolysis of sucrose into glucose and fructose by invertase followed by hexose uptake (Figure 1, transporter 6).
Figure 1. Long-Distance Sugar Transport by the Phloem. From its point of synthesis in the mesophyll, sucrose may be loaded into the SE/CC complex either through plasmodesmata or via the apoplasm. The apoplasmic loading mechanism requires sucrose export (1) from the mesophyll or the vascular parenchyma and reuptake (2) into the SE/CC complex. Hydrostatic pressure drives phloem sap movement toward sink tissue. Passive leakage can take place along the path (indicated by wavy arrows). Reuptake (3) also occurs along the path of the phloem. Apoplasmic phloem or postphloem unloading necessitates a sucrose exporter at the sink tissue (4). Import of sucrose and other solutes into sink tissue may occur through plasmodesmata or sucrose transporters (5). In addition to plasmodesmal and transporter-mediated uptake, cells in the sink may take up sucrose, subsequent to its hydrolysis by an apoplasmic invertase, as hexoses (6). The vacuolar transport system could consist of a H 1/sucrose antiporter for uptake and a uniporter for release.
Sugar Transport and Sugar Sensing
Gamalei, 1992). To achieve the pressure difference required for mass flow, the plasmodesmata must normally be closed. The systemic spread of silencing signals via the phloem (Voinnet et al., 1998) indicates that plasmodesmata can be gated open by endogenous plant factors. Symplasmic routes have been identified in the root by determination of dye coupling by using small cytoplasmic fluorescent dyes (Wright and Oparka, 1997). Interestingly, despite the presence of plasmodesmata along the entire length of the transport path, efflux occurs only in restricted regions, such as the region behind the root tip. It is possible that plants may utilize different mechanisms of phloem unloading in different tissues or may even be able to switch between apoplasmic and symplasmic mechanisms depending on growth conditions. Besides these plasma membrane transporters, uptake and release systems are required for subcellular compartments, especially the vacuole, which serves as a transient buffer for sugars and other metabolites (Figure 1; Marty, 1999, in this issue).
The Physics of Mass Flow Uptake of sucrose into the SEs is believed to increase the hydrostatic pressure difference between the ends of the phloem conduits so as to drive the mass flow movement of the phloem sap. The velocity of translocation in the phloem is relatively high, ranging from 0.5 to 3 m hr21 (Köckenberger et al., 1997). The phloem, then, contains an osmotic pump whose efficacy rests primarily on the cooperative functions of three types of plasma membrane proteins: (1) H1/sucrose symporters that accumulate osmotically active sucrose to molar concentrations in the phloem; (2) H 1-ATPases that provide the energy necessary for active transport; and (3) water transporters, potentially aquaporins, that take up water derived from the xylem (for a discussion of aquaporins, see Chrispeels et al., 1999, in this issue; for a discussion of H1-ATPases, see Sze et al., 1999, in this issue). Osmolytes other than sucrose, such as potassium and amino acids, also contribute to the driving force of the sap flow, and the simultaneous withdrawal of osmolytes and water at sink tissues further increases hydrostatic pressure differences. An essential prerequisite for the resulting osmotic pump is that the SE/CC complex be osmotically isolated from neighboring cells. Specifically, if phloem cells are connected to neighboring mesophyll cells, such intercellular connections must be tightly regulated. Differences in the connectivity among different cell types in leaves are in fact discernable and result in the classification of plants as either apoplasmic or symplasmic loaders (van Bel, 1993). Furthermore, evidence for the regulated opening of plasmodesmata has been provided (Oparka and Prior, 1992; Oparka et al., 1994). Of the numerous components required for long-distance photoassimilate transport (Figure 1), only proton-coupled
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sucrose and monosaccharide uptake transporters have been identified at the molecular level. It is unknown whether sucrose release is also proton coupled or if the release carriers are related in sequence to the uptake transporters (Ward et al., 1998). The sucrose binding protein, which in many cases colocalizes with the sucrose transporter, might be responsible for facilitated diffusion of sucrose (Overvoorde et al., 1996; Harrington et al., 1997) and may represent a second sucrose transporter class.
SUGAR TRANSPORTERS
Monosaccharide Transporters Monosaccharide transport activities have also been identified in a variety of plant species (Maynard and Lucas, 1982; Getz et al., 1987; Gogarten and Bentrup, 1989; Tubbe and Buckhout, 1992). Substrates that are efficiently transported include b-D-glucose, 3-O-methyl b-D-glucose, 2-deoxy b-D-glucose, a-D-mannose, and b-D-fructose, whereas b-L-glucose and b-D-ribose are poor substrates for transport (Gogarten and Bentrup, 1989). Plant monosaccharide transporters were first cloned from Chlorella kessleri by exploiting their rapid induction following the addition of hexoses to the growth medium. Specifically, differential screening of cDNAs from autotrophic versus heterotrophic cells enabled the cloning of the first plant hexose transporter gene (HUP1; Sauer and Tanner, 1989). In contrast to the yeast hexose transporters (HXTs), which function as uniporters, the C. kessleri hexose transporter is a symporter (Sauer et al., 1990a; Aoshima et al., 1993). Despite this difference in the transport mechanism, yeast and C. kessleri transporter genes are homologous, encoding proteins composed of 12 putative membrane-spanning domains. At least three hexose transporters are known to exist in C. kessleri (Table 1). The functional proof that these genes encode hexose transporters derives from heterologous expression of the plant genes in yeast (Sauer et al., 1990a). The cloning of higher plant monosaccharide transporters was accomplished by heterologous hybridization (Sauer et al., 1990b). The monosaccharide transporter family in Arabidopsis, as shown in Table 1, contains in excess of 26 genes, and multiple genes have been isolated from other species. STP1 was characterized by heterologous expression in Xenopus laevis oocytes and shown to function as a proton symporter (Aoshima et al., 1993; Boorer et al., 1994). The expression patterns of various plant monosaccharide transporters suggest that these membrane proteins function in hexose uptake in sink tissues (Sauer and Stadler, 1993). Analysis of expression also shows that plant monosaccharide transporters are highly regulated, such as in response to pathogen infection or after wounding (Truernit et al.,
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Table 1. Monosaccharide Transporter Gene Family in Plants
Species Arabidopsis thaliana
Gene Name
AtSTP1 AtSTP3 AtSTP4 ERD6 AtSUGTRPR ATAP22.19 Fi8E5.100a T10M13.6a T01O24.7a F3O9 T20F21.7 AtFCA6 F1N21.10 T22H22.15 T4B21.9 T4B21.10 T7A14.10 T30D6.1 MKD15.12a TACK11J9.5a T14N5.7a F7G19.20a F7G19.23a F9D12.9a F9D12.17a F23E12.140a Beta vulgaris BvMST1 BvMST2 BvMST3 Chlorella kessleri CkHUP1 CkHUP2 CkHUP3 Lycopersicon esculentum LeMST1 Medicago truncatula MtST1 Nicotiana tabacum NtMST1 Picea abies PaMST1 Petunia 3 hybrida PMT1 Prunus armeniaca APR MST1 Saccharum subsp ScfGLUTRAB Ricinus communis RcSTC RcHEX6 RcSTA Vicia faba VfHext Vitis vinifera VvHEXTRAN VvHEXOSET a Amino
Accession Length Number (Amino Acids) X55350 AJ002399 X66857 D89051 Z50752 Z99708 AL022603 AF001308 AC002335 AC006341 AC006068 Z97341 AC002130 AC005388 AF118223 AF118223 AC005322 AC006439 AB007648 AB012239 AB012239 AC000106 AC000106 AF077407 AF077407 AL022604 U64902 U64902 U43629 X55349 X66855 X75440 AJ010942 U38651 X66856 Z83829 AF061106 AF000952 L21753 L08196 L08188 L08197 Z93775 Y09590 AJ001061
522 514 514 496 734 493 508 513 521 518 522 582 453 483 434 442 623 508 514 515 525 454 490 507 526 729 549 545 490 534 540 534 523 518 523 513 510 475 518 523 510 522 516 519 519
acid sequences are deduced from genomic sequence.
1996), thus allowing flexible reallocation of fixed carbon. The expression of monosaccharide transporters in sink tissue also supports an apoplasmic mechanism for phloem or postphloem unloading (Sauer and Stadler, 1993). In this way, plant sink tissues may acquire hexose in a manner similar to that employed by yeast that use an extracellular
invertase to hydrolyze external sucrose. One requirement for this mechanism in plants is the coexpression of plant invertases and monosaccharide transporters (Ehness and Roitsch, 1997). The sugar permease family in yeast contains 34 genes (André, 1995; Nelissen et al., 1997; reviewed in Boles and Hollenberg, 1997), with 20 members belonging to the HXT subfamily (HXT1-17, GAL2, SNF3, and RGT2). The individual HXTs differ considerably with respect to their kinetic properties, with Km values for glucose uptake ranging from 1 mM for HXT7 to 100 mM for HXT1 (Reifenberger et al., 1997). The expression of HXT genes is regulated by glucose concentration, and the transporter homologs SNF3 and RGT2 function as sensors (see below).
Sucrose Transporters and SUT Genes Sucrose transport activities have been described in a wide variety of systems (Maynard and Lucas, 1982; Giaquinta, 1983; reviewed in Bush, 1993; Ward et al., 1998). To clone plant sucrose transporters, a yeast strain was generated that was unable to hydrolyze extracellular sucrose but was capable of metabolizing internal sucrose due to the presence of a plant-derived sucrose synthase activity (Figure 2). The strain was, furthermore, deficient in maltose utilization, a safeguard against side activities of the yeastendogenous maltose transport systems. The resulting yeast cells grow inefficiently on media containing 0.5% sucrose as the sole carbon source and were consequently used to clone plant sucrose transporters from spinach and potato by functional complementation (Riesmeier et al., 1992, 1993). Detailed transport studies using radioactive tracers have subsequently allowed determination of kinetic properties, pH optima, inhibitor sensitivity, and substrate specificity of various SUT genes. All plant sucrose transporters identified so far are energy dependent and sensitive to protonophores, indicating that they function as proton symporters. The Km for sucrose, in all cases, was found to be in the range of 1 mM (Riesmeier et al., 1993; Lemoine et al., 1996). Similar results were obtained for the Arabidopsis sucrose transporters SUC1 and SUC2. The SUT genes encode highly hydrophobic proteins. They consist of 12 membrane-spanning domains and are distantly related to the hexose transporter family found in many organisms, such as yeast and plants (reviewed in Ward et al., 1997; Rentsch et al., 1998). As for the monosaccharide transporters, sucrose transporters have been characterized by the two-electrode voltage clamp method in X. laevis oocytes (Boorer et al., 1996; Zhou et al., 1997). The stoichiometry of H1/sucrose cotransport was determined to be 1:1, consistent with stoichiometric estimates obtained earlier in plasma membrane vesicles from sugar beet leaves (Bush, 1990; Slone et al., 1991). The SUT family thus corresponds to the protonophore-sensitive high-affinity component of
Sugar Transport and Sugar Sensing
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Figure 2. A Yeast System for Functional Cloning of Sucrose Transporters. Wild-type yeast (Saccharomyces cerevisiae) utilizes sucrose primarily through activities of an extracellular invertase and hexose transporters in the plasma membrane (left). A yeast strain (SUSY7; Riesmeier et al., 1992) was constructed in which the cytosolic and extracellular invertases were genetically knocked out (middle). A plant sucrose synthase expressed in the cytosol allows growth on sucrose as the sole carbon source only if a sucrose transporter is expressed in the plasma membrane (right). The ability (1) or inability (2) of the yeast strains to grow on sucrose or glucose is shown. fruc, fructose; gluc, glucose; INV, invertase; suc, sucrose; SUC2, invertase gene; SUSY, sucrose synthase; SUT1, sucrose transporter 1 gene.
sucrose-uptake kinetics measured in plants (Maynard and Lucas, 1982).
CELLULAR LOCALIZATION OF SUT1 IN THE PHLOEM: CCs AND SEs Based on the transport mechanism (H1 symport) of SUT1, it was expected that the transporter would be involved in phloem loading and thus should be present at the plasma membrane of SE/CC complexes. In situ hybridization indeed demonstrates that SUT1 transcripts are phloem associated (Riesmeier et al., 1993), and the promoters of tomato SUT1 and Arabidopsis SUC2 genes direct the expression of reporter genes in leaf, stem, and root phloem (Truernit and Sauer, 1995; A. Weise, B. Hirner, J.M. Ward, and W.B. Frommer, unpublished results). Hence, SUT1 (or SUC2 from Arabidopsis) might play a role not only in phloem loading but also in retrieval of sucrose leaking from sieve tubes along the translocation pathway (Figure 1, transporter 3). To determine expression at the cellular level, immunolocalization studies have been applied to five species. In Plantago major and Arabidopsis, immunofluorescence with specific
antibodies detects SUC2 in CCs (Stadler et al., 1995; Stadler and Sauer, 1996). By contrast, immunolocalization using immunofluorescence and silver-enhanced immunogold staining assign SUT1 to the plasma membranes of enucleate SEs of tobacco, potato, and tomato (Kühn et al., 1997). The differences in sucrose transporter localization observed in Arabidopsis and P. major compared with tomato, potato, and tobacco may be due to differences in loading mechanisms. However, the transporters studied in Arabidopsis and P. major do not appear to be orthologs of SUT1, which may remain to be identified in those species. This would be consistent with a stepwise manner of sucrose loading, whereby different carriers operate in CCs (SUC2) as opposed to SEs (SUT1); such a scenario is suggested by physiological analyses (Roeckl, 1949). In situ hybridization experiments at the electron microscope level corroborate the localization of SUT proteins to SEs in a remarkable manner. Specifically, Solanaceous SUT1 mRNA localizes mainly to SEs, primarily at the orifices of plasmodesmata (Kühn et al., 1997). In addition, antisense inhibition of SUT1 expression using a CC-specific promoter (from the RolC gene) produces strong phenotypic effects in transgenic plants due to inhibition of sucrose export from leaves (Kühn et al., 1996). These results indicate that
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transcription of the SUT1 gene occurs in CCs and, in conjunction with high turnover rates of mRNA and protein, provide strong evidence for targeting of plant endogenous mRNA, and potentially SUT1 protein, through the plasmodesmata that interconnect CCs and SEs. These data may seem surprising at first sight; however, trafficking of RNA is known to occur in Drosophila and X. laevis during oogenesis and in neurons (St. Johnston, 1995). In maize, moreover, mRNA and protein produced from the KNOTTED-1 gene are transported through plasmodesmata in apical meristems (Lucas et al., 1995). Similarly, microinjection studies in Cucurbita maxima suggest that an RNA binding protein identified in phloem sap of that species is capable of trafficking a number of mRNAs intercellularly, including the SUT1 mRNA (Xoconostle-Càzares et al., 1999). The transport of macromolecules such as mRNAs and proteins through the microchannels of plasmodesmata requires mechanisms of unfolding (Kragler et al., 1998; see also Lazarowitz and Beachy, 1999, in this issue). The function of plasmodesmata thus seems, in this respect and many others, to be highly similar to protein import into organelles such as plastids (see Keegstra and Cline, 1999, in this issue), mitochondria, and the endoplasmic reticulum lumen (Vitale and Denecke, 1999, in this issue). As schematized in Figure 3, two potential pathways for the targeting of SUT1 can be postulated: either (1) mRNA is guided as part of a nucleoprotein complex along the cytoskeleton through the phloem plasmodesmata for subsequent translation; or (2) translation is effected in the CCs and the protein is deposited within the SE at the plasmodesmatal orifices via the endomembrane system (Overall and Blackman, 1996). Other phloem proteins alter the size exclusion limit for plasmodesmata and have been shown to move from cell to cell (Balachandran et al., 1997; Ishiwatari et al., 1998; see Lazarowitz and Beachy,1999, in this issue). Various proteins have been identified in the phloem sap, some of which were found to be specific for SEs, namely, b-amylase, glutaredoxin, thioredoxin, cyclophilin, protein kinases, ubiquitin, and P-protein PP2a (Schobert et al., 1995; Wang et al., 1995; Sjölund, 1997; Szederkényi et al., 1997). The role of most of these proteins in the phloem is unknown, and their origins and biosynthesis are not fully understood. The stability of proteins in the phloem also is generally unknown. The presence of ubiquitin in phloem sap may indicate that protein turnover is occurring. Further research is needed to determine both protein turnover rates in the phloem sap and protein import rates. Mature SEs are living cells despite the fact that they lack a nucleus and many other organelles (Sjölund, 1997). SEs are thought to be dependent on CCs for many cellular functions, but the extent of this dependence cannot be determined until the actual metabolic capabilities of SEs are better understood. For example, it is not clear whether SEs contain plasma membrane proton pumps. In CCs, on
the other hand, the expression of the H 1-ATPase–encoding AtAHA3 gene has been demonstrated (DeWitt and Sussman, 1995; see Sze et al., 1999, in this issue). If SEs do in fact contain an H 1-ATPase, then the supply of ATP to SEs through plasmodesmata would be presumably important. Consistent with this, phloem sap has been shown to contain concentrations of ATP in the range of 1 mM (Kluge et al., 1970). Additionally, the biogenesis of any ATPase in SEs may be dependent on a supply of mRNA or protein from CCs, by analogy to SUT1 biogenesis (Kühn et al., 1997). On the other hand, if SEs do not contain a proton pump or if the proton motive force to drive phloem loading is generated only in CCs, then plasmodesmatal connections are important for the propagation of this driving force (for both electrical coupling and a pathway for protons) into SEs (van Bel, 1996). In addition, trafficking of proteins, nucleic acids, and other molecules through plasmodesmata connecting CCs and SEs is important for
Figure 3. SUT1 Biosynthesis. In Solanaceous species, SUT1 protein is located in the SE plasma membrane (PM), while transcription occurs in CCs. There are two possible pathways: SUT1 mRNA may be delivered through plasmodesmata (pd) by an RNA transport mechanism. Alternatively, SUT1 translation may take place in CCs and delivery to SEs may occur via the plasma membrane or endosomal membranes that are continuous through plasmodesmata. These pathways are not exclusive and could function in parallel. ER, endoplasmic reticulum; sER, sieve element reticulum.
Sugar Transport and Sugar Sensing
long-distance signaling and to coordinate development at the whole-plant level in response to environmental factors, nutrients, and water availability (reviewed in Mezitt and Lucas, 1996).
IN VIVO EVIDENCE OF SUT1 TRANSPORTER FUNCTION
If sucrose transport mediated by SUT1 is essential for phloem loading, a reduction in transport activity should affect carbon partitioning and photosynthesis. In SUT1 antisense plants (Riesmeier et al., 1994; Kühn et al., 1996), leaf sucrose and starch content is five- to 10-fold higher than in the wild type, and the increase in hexose content is even greater. In addition, SUT1 antisense plants grow at markedly retarded rates, producing crinkled leaves that exhibit chlorosis and accumulation of anthocyanins. Development of the phenotype depends on the length of the photoperiod and light intensity (Kühn et al., 1996). A similar accumulation of soluble carbohydrates occurs when petioles of potato leaves are cold girdled so as to block phloem translocation (Krapp et al., 1993). Enhanced partitioning into insoluble carbohydrates is also found in a number of studies in which heat girdling is used (Grusak et al., 1990, and references therein). A direct comparison of antisense repression and cold girdling at the ultrastructural level demonstrates that both treatments equally evoke the accumulation of assimilates in all leaf tissues up to the SE/CC complex. However, microscopy reveals that antisense inhibition of loading produces a persistently high sugar level throughout the leaf, whereas cold girdling leads to localized patches containing high sugar and lipid levels (Schulz et al., 1998). Efflux measurements of carbohydrates from excised leaves of antisense plants show strong reduction in phloem transport (Riesmeier et al., 1994). With less carbohydrate transport to sinks, the plants have reduced root growth and tuber yield, phenotypic qualities also observed in transgenic potato plants in which apoplasmic loading is prevented by the overexpression of a yeast invertase in cell walls of leaves (Heinecke et al., 1992; Riesmeier et al., 1994). In tobacco, antisense repression of SUT1 also leads to dramatic growth retardation and accumulation of carbohydrates in leaves (Bürkle et al., 1998). The export of recently fixed 14CO2 is blocked to almost nondetectable levels even in plants inhibited to an intermediate degree, and stronger inhibition was found to be lethal. Thus, proton-coupled carriers seem to be indispensable for phloem loading at least in Solanaceous species. Comparable effects were observed in potato plants in which SUT1 was expressed in antisense orientation under control of the CC-specific RolC promoter (Kühn et al., 1996). It was, however, not possible to estimate the control coefficient of SUT1 because the actual amount of SUT1 protein
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and sucrose transport activity in the phloem was masked by low levels of SUT1 expression presumably in mesophyll cells (Lemoine et al., 1996). The effects observed are therefore in agreement with expected results that SUT1 transcription in CCs is essential for phloem loading. It remains unclear whether antisense repression also affects other members of the SUT gene family. A complete analysis of the role of individual members of the gene family will require approaches such as the use of “knockout” mutants in Arabidopsis. The potential of this approach for studying transport has been elegantly demonstrated in the case of potassium channels (Krysan et al., 1996; Gaymard et al., 1998; Hirsch et al., 1998; see also Chrispeels et al., 1999, in this issue). Identification of “knockout” mutants in sugar transport is in progress in several research laboratories.
SUCROSE TRANSPORTERS IN PHLOEM AND POST-PHLOEM UNLOADING
As detailed above, sucrose and hexoses have to be imported into sink tissues in roots, pollen, seeds, and elsewhere. SUT1/SUC2 expression has indeed been found in these tissues (Riesmeier et al., 1993; Truernit and Sauer, 1995). However, the direct function of such phloem-associated H1/sucrose symporters in sink tissues remains unclear. In several plant species, such as tomato, tobacco, potato, Arabidopsis, P. major, Ricinus communis, Vicia faba, carrot, rice, and pea, additional sucrose transporter genes have been identified, as listed in Table 2 (Gahrtz et al., 1994, 1996; Sauer and Stolz, 1994; Harrington et al., 1997; Hirose et al., 1997; Bürkle et al., 1998; Shakya and Sturm, 1998; Tegeder et al., 1999). Several of these genes/ proteins show highly specific expression patterns. For example, in Plantago, SUC1 is expressed in young ovules (Gahrtz et al., 1996), and sucrose transporter transcripts can be detected in the transfer cells of cotyledons from Vicia seeds and pea (Table 2; Harrington et al., 1997; Weber et al., 1997; Tegeder et al., 1999). Interestingly, these carriers are also expressed in source leaves, indicating a dual function in both phloem loading in leaves and in seed import. Other members of the SUT family are required in unloading zones, such as pollen, ovules, and roots. A pollenspecific sucrose transporter has been identified in tobacco (R. Lemoine, L. Barker, L. Bürkle, C. Kühn, M. Regnacq, C. Gaillard, S. Delrot, and W.B. Frommer, unpublished data). Within sink tissues, sucrose transporters could function in direct transport into sink cells or in sucrose retrieval. This latter function could control sink strength. Osmotic regulation, especially that involved in regulation of phloem or postphloem unloading, has been discussed in detail by Patrick (1997). Carriers in source tissue for efflux from mesophyll,
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Table 2. Sucrose Transporter Gene Family in Plants Species
Gene Name
Accession Number
Length (Amino Acids)
Localizationa
Arabidopsis thaliana
AtSUC1 AtSUC2 AtSUT2 AtSUT4 AtSUT5 AtSUT8 BvSUT1 DcSUT1a DcSUT1b DcSUT2 LeSUT1
X75365 X75382 AC004138 AC000132 AB016875 AC005398 X83850 Y16766 Y16767 Y16768 X82275
513 512 594 511 492 493 523 501 501 515 510
SoL, SiL, Ro, Flb, phloemc SoL, SiL, Flb, CCs
NtSUT1 OsSUT1
X82276 D87819
507 537
Pisum sativum
PsSUT1
AF009922
524
Plantago major
PmSUC1
X84379
503
Solanum tuberosum
PmSUC2 StSUT1
X75764 X69165
510 516
Spinacea oleracea Ricinus communis Vicia faba
SoLSUT1 RcRSC1 VfSUT1
X67125 Z31561 Z93774
525 533 523
Beta vulgaris Daucus carota
Lycopersicon esculentum Nicotiana tabacum Oryza sativa
SoL, Stb St, TP, Fl, Stb CCs,b SEb,d, phloemc SoL, Ro, St, SiL, Flb Pa, LS, LBb Sp, E, Rab Fl, SoL, Ro, St, Sb TC, Sp, SCb, TCe, Spe Fl, V, Fl, St, SiL, SoL, Rob Young ovulese, CCd SoL, St, Ro, Fl, SiL, Frb, CCd SoL, Ro, Flb Phloemc, abaxial phloeme SoLb C, Ro, Hb, E, SiL, SoLb C, SC, Po, SoL, Ro, SiLb, TCe
aC,
cotyledon; CC, companion cell; E, endosperm; Fl, flower; Fr, fruit; H, hypocotyl; LB, leaf blade; LS, leaf sheath; Pa, panicle; Po, pod; Ra, rachis; Ro, root; S, seed, SC, seed coat; SE, sieve element; SiL, sink leaf; SoL, source leaf; Sp, spikelet; St, stem; TC, transfer cell; TP, tap root; V, vascular bundle. Expression of mRNA or localization has been demonstrated using several techniques: bRNA gel blot analysis. cPromoter–b-glucuronidase fusion. dImmunolocalization. eIn situ hybridization.
carriers in sink tissues for efflux from phloem and postphloem, and carriers involved in transient storage in vacuoles have yet to be identified (Figure 1).
SENSING MECHANISMS IN SUGAR TRANSPORT
A common misconception is that transport processes, especially sugar transport in higher plants, are constitutive and that biosynthetic activities in the source and catabolic activities in the sink are the key factors controlling allocation of carbohydrates. Carbohydrate export rate is increased 10-fold in plants overexpressing pyruvate decarboxylase (Tadege et al., 1998), indicating a large potential for upregulation. Transporters, however, are located in strategic positions along metabolic pathways, and thus an effective regulatory mechanism would be to control uptake and efflux directly.
Indeed, there is strong evidence that sugar transport regulates the distribution of assimilates within the plant through various macromolecular signaling events. In the simplest sensing scenario, cells would contain only an intracellular receptor for a sugar metabolite. Such cells, whether located in source or sink tissues, could modulate metabolic processes, such as photosynthesis or carrier gene expression. However, due to intracellular metabolism, the effector cells involved in these processes must be able to differentiate between biosynthesis and transport, and therefore both intra- and extracellular concentrations of sugars need to be sensed. Furthermore, provided that the cell has a spectrum of carriers of varying affinity and capacity for sugar, extracellular sensors, as shown in Figure 4, can adjust sugar uptake to match requirements, for example, by inducing high-affinity uptake systems at low external concentration. In principle, multiple sensors could be utilized, some for
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high-affinity responses and others for adaptation to highflux requirements. Because the plasma membrane has a limited capacity for protein content, this signal may at the same time lead to an increase in the turnover of low-affinity systems. Increased turnover of transporters can also be controlled via an internal sensor, thus decreasing import if intracellular concentrations exceed the requirements. Although such regulatory networks could also be effective in unicellular organisms like yeast or algae, intercellular transport in higher plants is more complicated. This is because at least two cellular activities—export from one cell and import into the adjacent cell—have to be integrated. Little is known about the molecular mechanisms involved in sugar sensing in relation to transport in higher plants. Yeast could thus serve as a model to help uncover the regulatory networks in higher plants.
YEAST AS A MODEL OF SUGAR SIGNALING
The yeast Saccharomyces cerevisiae contains a large spectrum of .200 integral membrane proteins, many of which
Figure 5. Sugar Signal Transduction in Saccharomyces cerevisiae. HXT-type transporters mediate glucose uptake. Hexokinase PII (HXK2) functions in cytosolic sugar signaling, resulting in transcriptional regulation of enzymes and transporters necessary for sugar utilization. The SNF3 (high affinity) and RGT2 (low affinity) glucose receptors sense the external glucose concentration. These are homologous to HXT-type transporters but contain a C-terminal extension that functions in signaling. The SNF3 and RGT2 signaling pathways affect transcriptional regulation of high-affinity and lowaffinity/high-capacity hexose transporter genes, respectively. See text for details.
Figure 4. Model for Metabolite Sensing by a Combination of Internal and Membrane-Bound Receptors. In addition to an internal receptor (see text), membrane-bound receptors are used to sense external sugar concentrations. Such sensors trigger a signaling cascade regulating transporter biogenesis and insertion or degradation of proteins at the plasma membrane. In this scenario, low sugar concentrations activate a sensor to induce sugar transporter genes (preferentially high affinity). This enables more efficient sugar uptake. If, on the other hand, internal sugar concentration becomes too high, the intracellular sensor may either repress transporter transcription or trigger inactivation via endocytosis and degradation of the transporter, thus reducing the influx. Induction of low-affinity/high-capacity transporters could increase uptake at higher external concentrations.
are clearly involved in transmembrane solute transport. For example, yeast contains .20 permeases for amino acid transport (André, 1995; Nelissen et al., 1997) and .20 permeases for sugar transport (André, 1995; Boles and Hollenberg, 1997). The redundancy of transport systems suggests that complex regulatory networks are absolutely necessary to control the uptake of nutrients in response to a rapidly changing external environment. As shown in Figure 5, yeast has developed a two-pronged regulatory system to ensure coordination between the supply of sugars from the environment and the enzymatic machinery of the cells: (1) the extracellular concentration of sugars is sensed and sugar transport activity is regulated accordingly; (2) sugar transport activity determines the flux of sugars into the cell,
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subsequently generating intracellular signals for further regulatory processes. In S. cerevisiae, multiple transport systems for glucose are regulated at the transcriptional level in response to the external concentration of glucose. For example, HXT2 and HXT7 serve as high-affinity glucose transporters and are induced only by low levels of glucose but repressed at high levels, whereas HXT1 functions as a low-affinity transporter and is induced only by high concentrations of glucose (Özcan and Johnston, 1995; Boles and Hollenberg, 1997). Consequently, sensors of extracellular glucose that respond not only to the kind of carbon source in the medium but also to its concentration are required. Once inside the cell, glucose is phosphorylated by three different kinases: HXK1, HXK2, and GLK1. Finally, it is converted through the glycolytic pathway mainly into ethanol. By contrast to the uptake of glucose, the regulation of the intracellular glucose concentration, phosphorylation, and subsequently, the regulation of flux through glycolysis must be controlled by intracellular signals (Boles et al., 1997). Furthermore, the expression of genes for the utilization of alternative carbon sources like sucrose or galactose, and genes involved in gluconeogenesis, must be shut off in the presence of sufficient amounts of the preferred carbon source, glucose. This is achieved through a mechanism known as glucose (or carbon) catabolite repression (Ronne, 1995; Gancedo, 1998). The glucose signal that triggers induction of hexose transporter genes is generated by the hexose sensors SNF3 and RGT2. On the other hand, the signal that triggers glucose repression is somehow connected to the kinase activity of HXK2 (Ma et al., 1989; Rose et al., 1991). In principle, there are two possibilities for sensory proteins to detect signaling molecules. First, sensors might act as receptors, binding the triggering molecule (e.g., glucose) and transducing the signal via other proteins. Second, sensors might behave like enzymes or transporters and undergo structural changes so as to monitor the presence or absence of the triggering compound directly. Flux measurements by such a sensor protein would involve the recognition of the velocity of the enzymatic reaction as the ratio of active to free enzyme.
SUGAR SENSORS: A NEW PERSPECTIVE OF TRANSPORTERS
Cell Surface Sugar Sensors In Escherichia coli, it has long been known that glucose sensing is mediated through glucose phosphorylation during transport by the phosphotransferase system (Postma et al., 1993). However, no related proteins have yet been detected at the plasma membrane of eukaryotic cells. Glucose sensing in yeast seems to involve plasma membrane proteins that resemble glucose transporters but additionally
possess large cytoplasmic signaling domains (Özcan et al., 1996a). A similar mechanism has been discovered recently for amino acid sensing (Didion et al., 1998; Iraqui et al., 1999) and might be a general phenomenon not restricted to yeast. In yeast, SNF3 appears to be a sensor of low levels of glucose and mainly regulates expression of high-affinity glucose transporters, whereas RGT2 appears to be a sensor of high glucose concentrations that regulates expression of low-affinity glucose transporters. Additionally, SNF3 is required at high levels of glucose for repression of the high-affinity transporters HXT2, HXT6, and HXT7 (Liang and Gaber, 1996; Vagnoli et al., 1998). Dominant mutations in both RGT2 and SNF3 that lead to the generation of signals in the absence of glucose have been identified (Özcan et al., 1996a). Despite the homology of SNF3 and RGT2 to glucose permeases, these transmembrane proteins do not seem to be able to mediate significant glucose transport (Liang and Gaber, 1996; Özcan et al., 1998). The large C-terminal extension of SNF3 (303 amino acids) contains two nearly identical repeats of 25 amino acids. One of these repeats is also present in the 218–amino acid C-terminal extension of RGT2 and in RAG4 (GenBank accession number Y14849) from Kluyveromyces lactis, which contains a 250–amino acid C-terminal extension and controls the expression of the lowaffinity glucose transporter RAG1 (Chen et al., 1992). In Neurospora crassa, the glucose sensor RCO3 contains a 119–amino acid C-terminal extension that is dissimilar to that of SNF3, RGT2, and RAG4 (Madi et al., 1997). From mutational analyses, yeast glucose sensors appear to function as two interacting domains (Özcan et al., 1998; Vagnoli et al., 1998): the C-terminal extensions that are required for the transmission of the glucose signal, and the membrane-spanning domain necessary for glucose recognition. Because the glucose transporters HXT1 and HXT2 can be converted into glucose sensors by fusion to the SNF3 C terminus, it is tempting to speculate that the glucose sensors act as glucose transporters with a very low glucose transport capacity (i.e., too low to support growth of mutants that lack glucose transporters), which transduce the glucose signal by a conformational shift during glucose transport. Another protein (SSY1) that appears to function as an amino acid sensor has recently been identified in yeast. SSY1 shows the features typical of the SNF3/RGT2 pair: a transmembrane domain related to amino acid permeases, a long cytoplasmic extension (in this case located at the N terminus of the protein), a low transcription rate, and a low coding probability (André, 1995; Didion et al., 1998; Iraqui et al., 1999). SSY1, rather than being a transporter, controls the transcription of genes encoding amino acid permeases and seems to respond to changes in the extracellular concentration of various amino acids (Didion et al., 1998; Iraqui et al., 1999). The amino acid sequence of the cytoplasmic extension of SSY1 shows no significant similarities to those of SNF3 and RGT2.
Sugar Transport and Sugar Sensing
The only intermediate components so far known to be involved in glucose-induced signal transduction in yeast are the transcription factor RGT1 (Özcan et al., 1996a) and SCFGrr1, a ubiquitin protein ligase complex including the F-box protein GRR1, SKP1, and CDC53 (Li and Johnston, 1997; Skowyra et al., 1997). RGT1 is a zinc cluster protein that binds directly to promoters of the HXT genes (Özcan et al., 1996b). It acts as a repressor of HXT genes in cells growing without glucose and as a transcriptional activator of HXT1 in cells growing on high levels of glucose. Repression of transcription by RGT1 is mediated by the SSN6-TUP1 complex, whereas RGT1-mediated induction is independent of these proteins (Figure 5). The SCFGrr1 protein complex is required for regulation of RGT1 activity (Özcan and Johnston, 1995) mediating the signal generated by SNF3 to inhibit the RGT1 repressor function in response to low levels of glucose. It is also required for conversion of RGT1 into an activator triggered by RGT2 in the presence of high levels of glucose. Ubiquitination of RGT1 or its regulator may target it for protein degradation by the proteasome or directly affect its function. SCFGrr1 complex may be directly stimulated by glucose (Li and Johnston, 1997) or may depend on glucose-activated kinase because target proteins must be phosphorylated to interact with GRR1 (Skowyra et al., 1997). A third sensor protein, MEP2, was recently found in S. cerevisiae to be required for pseudohyphal differentiation in response to ammonium limitation (Lorenz and Heitman, 1998). Under such conditions, diploid yeast cells differentiate into a filamentous, pseudohyphal growth form. MEP2 belongs to a family of three closely related ammonium permeases (Marini et al., 1997). However, unlike the other two members of this transporter family, MEP2 serves as both an ammonium transporter and a component of an ammonium sensor. mep2 mutant strains have no defects in growth rates or ammonium uptake, but no longer form pseudohyphal filaments on media containing limiting amounts of ammonium. Unlike the glucose sensor proteins, however, MEP2 is able to transport its substrate; and as compared with the other members of the ammonium permease family, MEP2 contains no significant amino acid extensions on either its N or C terminus.
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of glucose and is dependent on protein phosphatase 1 (CID1/GLC7). A protein kinase involved in phosphorylation of HXK2 has not been found, and it is possible that phosphorylation of HXK2 could result from substrate-induced autophosphorylation (Fernández et al., 1988). It has been reported that HXK2 has a weak protein kinase activity (Herrero et al., 1989; see above). Moreover, a hxk2 mutant that is not able to undergo phosphorylation can no longer transduce the glucose repression signal (Randez-Gil et al., 1998). These properties might classify HXK2 as an enzyme that additionally, through its enzymatic function, can act as a sensor protein. However, the actual mechanism representing the on–off switch of the glucose signal is not understood. The glucose repression signal finally inhibits the protein kinase SNF1/CAT1, a central element in the regulatory process (Figure 5) and highly conserved in eukaryotes. Glucose inhibition of SNF1 depends on protein phosphatase 1 (GLC7) and its targeting subunit REG1 (Ludin et al., 1998). In the absence of glucose, SNF1 relieves repression by the MIG-SSN6-TUP1 complexes (De Vit et al., 1997) but is also required for the operation of other transcription factors (Gancedo, 1998). In accordance with HXK2 being a sensor for glucose, the triggering of glucose repression in yeast is dependent on glucose uptake. However, the repression is not dependent on a specific hexose transporter protein; the glucose repression signal rather correlates with the extent of glucose influx into cells (Reifenberger et al., 1997). These observations fit very well with the two-pronged regulatory model described above: first, glucose transport activity is adjusted to the extracellular glucose concentration via SNF3 and RGT2; second, glucose transport activity limits provision of intracellular glucose, which then might act as a substrate for HXK2 as well as a global glucose signal (Figure 5). In addition, cross-talk between the two different processes might ensure feedback coordination. Such a function has recently been assigned to HXK2 which, in addition to carbon catabolite repression, seems also to be involved in glucose induction of HXT gene expression (Randez-Gil et al., 1998).
SUGAR-MEDIATED REGULATION OF SINK AND SOURCE GENES
Intracellular Sugar Sensing: Enzymes as Sensors In the triggering reaction of glucose repression in yeast, HXK2 seems to play an important and probably more direct role. Mutations in HXK2 abolish glucose repression of invertase and other glucose-repressed genes (Entian, 1980). The catalytic and regulatory functions of HXK2 are inseparable from glucose repression and inversely correlated to its sugar-phosphorylating activity (Ma et al., 1989; Rose et al., 1991). HXK2 exists in a dimeric–monomeric equilibrium that is affected by phosphorylation (Randez-Gil et al., 1998). In vivo, dephosphorylation of HXK2 is promoted upon addition
A large spectrum of genes is regulated by sucrose and monosaccharides (reviewed in Thomas and Rodriguez, 1994; Koch, 1996). The interplay of regulatory processes interconnected with sugar regulation provides the plant with valuable mechanisms to adjust to environmental conditions and also to control developmental and physiological processes (e.g., photosynthesis and flowering) (Bernier et al., 1993; Jang and Sheen, 1994; Corbesier et al., 1998). Carbohydrate-responsive genes can be classified as initiating “feast or famine” responses. Genes for photosynthesis and resource mobilization are induced by carbohydrate depletion
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(the famine response), whereas increasing sugar concentrations stimulate gene expression for utilization and storage (the feast response) independently of location (i.e., in source or sink tissues) (Rocha-Sosa et al., 1989; Müller-Röber et al., 1990; Sheen, 1990; Graham et al., 1994; Jang and Sheen, 1994; Thomas and Rodriguez, 1994; Koch, 1996). Sucrose, as the principal transport form of sugars, can specifically control the expression of a number of genes. Examples of sucrose-regulated genes include an Arabidopsis leucine zipper gene, ATB2, and the RolC promoter from Agrobacterium (Smeekens and Rook, 1997; Yokohama et al., 1997). Externally supplied sucrose exceeding concentrations of 25 mM lead to a repression of ATB2 transcription (Rook et al., 1998). In sugar beet, high sugar concentrations lead to a repression of sucrose transport activity, correlating with a reduction in steady state mRNA levels of BvSUT1 (Chiou and Bush, 1998); glucose and fructose affect transport activity to a lesser extent. Sucrose can repress transcription of photosynthetic genes encoding, for example, chlorophyll a/b binding protein (CAB), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and plastocyanin. Sucrose-uncoupled (sun) mutants have been identified, in which sucrose concentration is uncoupled from the repression of such genes (Dijkwel et al., 1996, 1997). Taken together, these studies indicate the presence of sucrosespecific signal perception and transduction processes in which both internal and external sensors might be involved. Glucose-specific responses have also been described (Thomas and Rodriguez, 1994; Koch, 1996). Moreover, glucose and sucrose seem to control different pathways. Whereas glucose favors cell division, sucrose relates to storage compound accumulation in seeds (Weber et al., 1998). A specific hexose-sensing system in which hexokinase plays a central role, potentially as an intracellular sensor, has been identified in the repression of typical “famine” genes (Figure 6; Sheen, 1990; Graham et al., 1994; Jang and Sheen, 1994). Specifically, nonmetabolizable glucose analogs (e.g., 6-deoxyglucose or 3-O-methylglucose) that are taken up into cells but are not metabolized by hexokinase do not repress gene induction in photosynthetic or glyoxylate cycles. Only substrates of hexokinase that are taken up and phosphorylated mimic glucose-specific repression (Graham et al., 1994; Jang and Sheen, 1994). Additionally, phosphorylated hexoses do not alter gene expression, but inhibition of hexokinase blocks the 2-deoxyglucose– and mannose-dependent repression. Thus, hexokinase activity per se is involved in sensing, a finding that is further strengthened by studies of transgenic Arabidopsis plants in which the expression of the hexokinase-encoding genes AtHXK1 and AtHXK2 had been prevented (Jang et al., 1997). In antisense plants, the glucose-dependent repression of typical famine genes, such as Rubisco and CAB, is impaired, thereby leading to reduced sensitivity to high external glucose concentrations. Interestingly, Arabidopsis plants overexpressing a yeast HXK gene,
thus bypassing sugar flux through the plant’s endogenous hexokinases, behave similarly to the AtHXK antisense plants (Jang et al., 1997). These data support a direct role for hexokinase itself in signaling in addition to its kinase activity. However, the situation seems to be more complex. Transgenic tobacco plants overexpressing a yeast invertase in the cytosol, apoplasm, or vacuole all accumulate elevated levels of hexoses. However, only in plants with apoplasmic or vacuolar invertase expression was gene expression repressed by the elevated sugar content (Herbers et al., 1996). Furthermore, because the introduction of sugar phosphates into cells by electroporation does not modify the expression of carbohydrate-responsive genes (Jang and Sheen, 1994), it seems to be the flux of sugars undergoing phosphorylation that is sensed rather than the mere internal concentration of sugar phosphates. A more recently identified hexokinase-independent glucose-signaling system, schematized in Figure 6, seems to be preferentially responsible for controlling “feast” and pathogen-related gene expression. In photoautotrophic Chenopodium rubrum cell suspensions, the expression of invertase and sucrose synthase genes is induced upon treatment with 6-deoxyglucose (Godt et al., 1995; Roitsch et al., 1995). In transgenic Arabidopsis, 3-O-methylglucose activates a patatin class I promoter fused to b-glucuronidase, which suggests that the sensing mechanism for this nonphosphorylatable analog occurs before hexokinase in the signaling pathway. Martin et al. (1997) have identified several Arabidopsis mutants with reduced sugar response (rsr) that harbor a patatin promoter–b-glucuronidase construct. These mutants, in which the patatin promoter does not respond to sugars, offer a good model for studying hexokinase-independent sugar sensing. Specifically, despite high intracellular concentrations of hexoses caused by specific enzyme inhibitors, patatin expression was downregulated strongly, thereby supporting a model in which extracellular concentrations or fluxes are sensed at the cell exterior (Müller-Röber et al., 1990; Zrenner et al., 1995). As in the case of the hexokinase-dependent sensing mechanism, fluxes are more important than steady state levels of carbohydrate to initiate a response. Studies on sugar-induced pathogen-related genes, moreover, provide strong evidence for the existence of sensing mechanisms, located at the plasma membrane, which might correspond to the RGT2/SNF3-type sensing system present in yeast. Despite the central importance of sugars as key regulators of gene expression, very little is known about the signal transduction mechanisms in which they take part. Several studies have provided evidence that mechanisms similar to yeast phosphorylation events are involved in sugar-specific signal transduction cascades. Several kinase genes have been found in plants with homology to SNF1 of which some complement the snf1 yeast mutant. In vivo function has been demonstrated in transgenic potato plants expressing
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Figure 6. Current Representation of Sugar Sensing in Plants. Monosaccharide transporters allow the uptake of hexose analogs useful for probing the sugar signaling pathway. 6-Deoxyglucose and 3-O-methylglucose can be transported but are not substrates for hexokinase; therefore, signal pathways requiring hexokinase activity are not triggered by these analogs, whereas genes like patatin class I, invertase, or sucrose synthase genes are induced via a hexokinase-independent pathway. Mannose and 2-deoxyglucose serve as substrates for hexokinase and can thus repress photosynthetic genes via the hexokinase-dependent pathway. Sucrose can be metabolized and hexoses used in the hexokinase-dependent pathway.
an SNF1-related protein kinase gene in antisense orientation such that a decrease in sucrose synthase expression is observed in tubers (Halford and Hardie, 1998). Rubisco gene repression and invertase gene induction by sugars involve the action of both kinases and phosphatases (Ehness et al., 1997). Also, in the case of the regulation of sporamin and b-amylase gene expression, the involvement of protein phosphorylation is likely inasmuch as treatment with specific inhibitors of kinases and phosphatases reduces sugar-spe-
cific induction (Ohto and Nakamura, 1995; Takeda et al., 1994).
HORMONAL REGULATION OF SUGAR TRANSPORT
Many studies have provided evidence that sugar transport can be adapted to the changing needs of the plant. Indeed,
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comparison of the transport activities in developing versus mature leaves has shown that H1/sucrose cotransport is differentially active and develops during leaf maturation (Lemoine et al., 1992). Hormones such as auxin and cytokinin have long been known to increase the rate of phloem transport. Fusicoccin and auxin can rapidly promote sucrose uptake, whereas abscisic acid acts as an inhibitor (Malek and Baker, 1978; Sturgis and Rubery, 1982; Vreugdenhil, 1983). In broad bean, the direct promotion of assimilate export by the application of gibberellin was reported (Aloni et al., 1986). Phloem loading in isolated bundles of celery seems to be directly affected by gibberellin and auxin (Daie et al., 1986). However, a principle problem of such studies is the difficulty to differentiate between effects operating within the network. Proton-coupled sugar transporters can be regulated in two major ways: (1) indirectly by regulating H 1-ATPase activity, or (2) more specifically by controlling the expression of sugar transporters at the transcriptional and post-transcriptional levels. Several factors are known to regulate ATPase activity and/or transcription: fusicoccin (Oecking et al., 1997; Baunsgaard et al., 1998), salicylic acid (Bourbouloux et al., 1998), and anaerobiosis, which probably acts at the level of ATP supply for the H 1-ATPase (Sowonick et al., 1974; Giaquinta, 1977; Servaites et al., 1979; Thorpe et al., 1979; Maynard and Lucas, 1982; see also Sze et al., 1999, in this issue). Sucrose can also induce accumulation of two plasma membrane ATPases, LHA4 and LHA2 (Mito et al., 1996). This induction is dependent on sugar uptake and metabolism, inasmuch as mannitol and 3-O-methylglucose have no effect. These results suggest that the induction of expression of H1-ATPase genes by metabolizable sugars may be part of a generalized cellular response to promote cell growth in the presence of abundant carbon sources (Mito et al., 1996). Potato SUT1 and Arabidopsis AtSUC2 are expressed in source and sink tissues (Riesmeier et al., 1993; Truernit and Sauer, 1995). The expression of the sucrose transporter SUT1 is diurnally regulated at both the mRNA and protein level (Kühn et al., 1997), in accordance with diurnal regulation of export rates from leaves (Heinecke et al., 1994). SUT1 mRNA and protein levels can, furthermore, be induced by the addition of auxin and cytokinin to detached leaves (C. Kühn and W.B. Frommer, unpublished results), whereas no such effect was observed for either of the two major H1-ATPase genes expressed in potato leaves (Harms et al., 1994). Sucrose itself was shown to be involved in regulating sucrose transporter activity, potentially at the transcriptional level (Chiou and Bush, 1998). Inhibitor experiments indicate that SUT1 activity is also regulated at the post-translational level by phosphorylation (Roblin et al., 1998). Protein kinase activities, which might be responsible for phosphorylation of sucrose transporters in the SEs, have been detected in the phloem sap (Nakamura et al., 1993). As described in yeast, sugars may also regulate the stability of transporters (Jiang et al., 1997). Inhibition
studies using cycloheximide show that the half-life of SUT1 is in the range of a few hours (Kühn et al., 1997). This high turnover rate may indicate specific mechanisms controlling the number of active carriers in the plasma membrane and may suggest involvement of endocytosis, as in the case of mammalian glucose transporters and yeast amino acid permeases (Hein et al., 1995; Thorens, 1996). Concerning monosaccharide transport, no clear function has been demonstrated using antisense or “knockout” strategies. Very little is also known about its regulation. Coordinated regulation of mRNAs for extracellular invertase and a monosaccharide transporter in C. rubrum has been described (Ehness and Roitsch, 1997). Because sucrose synthase and invertase gene expression is regulated by sugars, one may expect a complex network coordinating uptake of both monosaccharides and sucrose with the metabolic events involving both extracellular and intracellular sensors as sketched in Figure 6.
PLANT TRANSPORTER FAMILIES IN SUGAR SENSING
Are there known members of the plant transporter families that could be sensors? Indications, as presented above, suggest that transporters might be involved in sensing hexoses (Figure 6; Martin et al., 1997). As described above, such sensors in yeast often contain additional cytosolic domains that are thought to play a role in transmitting the signal. Analysis of the plant monosaccharide transporter MST family (Table 1) indicates that the Arabidopsis genome contains at least 26 MST genes. Although none of the predicted AtMST proteins share obvious similarities with the C-terminal extensions found in the yeast RGT2 and SNF3, two Arabidopsis genes (AtSUGTRPR and F23E12.140; Table 1) that are closely related to each other contain extended central loops. Additionally, one member of the tomato and Arabidopsis SUT family is characterized by the presence of a long central loop predicted to be cytosolic (L. Barker, C. Kühn, B. Hirner, E. Boles, H. Hellmann, A. Weise, J.M. Ward, and W.B. Frommer, unpublished results). The availability of the full sequence from Arabidopsis will provide access for more detailed analysis. Because the putative tomato sucrose sensor colocalizes with SUT1 and because no sucrose transport activity is detectable, one might speculate that it is involved in controlling the expression and turnover of SUT1 in SEs (Figure 7). Because transporter-like sensors have also been identified for amino acids and ammonium, one may speculate that the transporter families also contain respective sensors. Within the large amino acid transporter families, no homologs containing large cytosolic domains similar to SSY1 have been found to date (Fischer et al., 1998). Because in the putative yeast ammonium sensor MEP2 no extended loops or other features that specify it as a sensor have been identified and because it represents a functional transport
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an essential component responsible for phloem loading, as was shown by antisense inhibition in transgenic potato and tobacco. Physiological studies have shown that plant sugar transport and metabolism are highly regulated by sugars at the transcriptional and post-transcriptional levels. In yeast, sugar regulation of transporters is controlled by sensors at the plasma membrane. The availability of genes for members of the plant MST and SUT family and other phloem sap-specific proteins provides the tools to further explore and understand the mechanism and regulation of long-distance transport and its role in sugar sensing and regulation in plants.
ACKNOWLEDGMENTS Figure 7. A Potential Sucrose-Sensing Mechanism in SEs. Solanaceous SUT1 and a sucrose sensor colocalize in SEs (by analogy with yeast SNF3 and RGT2 proteins). The putative sensor regulates SUT1 either directly or via an indirect signaling mechanism such as phosphorylation. The high turnover rates of SUT1 may indicate that regulation occurs at the level of protein inactivation and degradation; however, because SUT1 is transcribed in companion cells, a regulatory mechanism involving transcriptional control is more complicated but still possible.
We are grateful to Bruno André (Université Libre de Bruxelles, Belgium) for critical reading of the manuscript and helpful discussion. This work was supported by grants from Deutsche Forschungsgemeinschaft (Grant No. SFB446) and the European Union Biotechnology Program (Grant Nos. BIO4 CT96-0583 and BIO4 CT96-0311) and by an Alexander von Humboldt fellowship to S.L.
REFERENCES
protein, sequence comparisons seem inappropriate to identify plant homologs. None of the plant ammonium transporters that are highly related to the yeast MEP genes contain extended cytosolic loops (N. von Wirén and W.B. Frommer, unpublished results). In all cases, experimental proof will be required to identify and characterize putative plant metabolite sensors. One approach could be to determine if, in “knockout” mutants, the transcriptional and post-transcriptional regulation of other transporters of the same family is affected.
CONCLUSIONS
In summary, sensors have probably evolved from transporters due to their suitability to recognize their substrates. This could have occurred by the addition of a signaling domain to an existing transporter. Plants have to adapt to changing environments, and sensing functions are thus essential. As compared with yeast, the identification of sensors in plants will be more difficult, but as in the case of functional expression to identify plant transporters, knowledge from yeast may serve as a tool to identify plant, and potentially animal, sensors as well. Yeast complementation has indeed allowed the identification and characterization of both monosaccharide and sucrose transporters from plants. Both classes of carrier are encoded by large gene families. SUT1, localized in SEs, is
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