Sucrose Transport In The Sugar Beet Plant

Sucrose transport is critical for development and growth in most plant species. Biological membranes like the plasma membrane and the tonoplast are almost impermeable to uncharged and charged molecules under normal conditions unless there is a principle realised within these membranes rendering them selectively permeable to certain molecule species. Some specific sort of pores and/or transport proteins have to be present to ensure selective permeability of these membranes for sucrose as well. Once sucrose has been formed within the cytosol of "source" (net exporting leaf mesophyll) cells, sucrose should be transported through membranes at different stages (fig. 1):

1) Sucrose may be transiently stored within the vacuoles of source leaf cells. Some tonoplast bound transport system for sucrose should be present.

2) Sucrose must cross the plasma membrane of source cells to enter the apoplasm via some transmembrane transport or exocytosis like process if it is not symplastically transported to the sieve elements for long distance transport to sucrose utilising (net importing sink) cells. In those plant species, where phloem loading as the process of transporting sucrose against a concentration gradient into the phloem is essential in long-distance transport of sucrose and carbon partitioning, sucrose transport across the plasma membrane of source cells into the apoplasm is an important transport event.

3) At the plasma membrane of the sieve element companion cell complex (or sieve cells of gymnospermous plant species), a presumably energy dependent uphill transport of sucrose has to occur in many plant species against a concentration gradient.

4) Along the long distance transport pathway, sucrose may leave and re-enter the conductive elements. This may be achieved through plasmodesmata or through trans-plasma membrane transport.

5) In sink regions, sucrose may leave the long distance transport elements into the apoplasm. In this case not only plasma membrane transport of sucrose at the conductive elements has to occur, but also plasma membrane transport at the receiving sink cells unless exocytosis/endocytosis/ pinocytosis like processes provide alternative ways. If sucrose is cleaved by cell wall bound acid invertase into glucose and fructose (41) then hexose transporters must be active. Alternatively or in addition, sucrose transporters may import uncleaved sucrose into the cytosol of recipient cells.

6) In sucrose storing plant species like sugar cane or sugar beet, sucrose will enter the vacuole through tonoplast bound sucrose transport mediating principles like pores, facilitators or carriers unless pinocytosis-/endocytosis-/exocytosis-like processes provide "bulk delivery" of solutes.

Pinocytosis/endocytosis-/exocytosis-like processes have been described tentatively in the past for sugar beet (31, 42, 43, 44) as judged from electron microscopic images showing invaginations of the plasmalemma. Although fixation of the highly vacuolated storage tissue of sugar beet taproots may have caused artefacts (45), this alternative way for membrane passage of solutes cannot be totally excluded (40, 46).

4.1.Sugar transport within the leaves a) Tonoplast transport

In sugar beet leaves, sucrose is not accumulated like, for example, in the relatively closely related spinach. On the contrary, the sucrose concentration is maintained at a fairly low and steady concentration throughout the day (see 47, and literature cited therein). Starch synthesis and synthesis of sucrose supplied from starch degradation mainly contribute to a quite steady rate of sucrose synthesis throughout day and night (47). Based on indirect evidence, sucrose uptake into vacuoles of sugar beet mesophyll cells was qualified as passive but facilitated (47). According to this evidence, the leaf mesophyll vacuoles of sugar beet plants can be considered as short time buffer for sucrose that take up sucrose when there is a more or less steep concentration gradient between the cytosol and the vacuole. Sucrose could be released from the vacuole when its cytosolic concentration decreases below that in the vacuole. By that way, vacuolar sucrose could contribute to export when photosynthesis intensity decreases.

b) Plasma membrane transport

Unloading of sucrose into the source leaf apoplast

Plasmodesmatal frequency, as demonstrated by Evert and Mierzwa (48; but see 49, 50, 51 for discussion), theoretically could allow for symplastic loading of sucrose into the conducting elements of the sugar beet leaf phloem. There is low abundancy of plasmodesmata between phloem parenchyma cells and sieve elements, but plasmodesmatal frequency might be sufficient between mesophyll cells and phloem parenchyma cells at the unloading site as well as between phloem parenchyma, companion cells and sieve elements at the loading site of source leaves. If active phloem loading of sucrose from the apoplast is considered (30, 52, 53), then sucrose has to be unloaded into the apoplast of the sieve element-companion cell complex first.

Physiological/biochemical studies with plasma membrane vesicles from sugar beet leaves have demonstrated the existence of a sugar efflux system. This efflux system is different from uptake systems as shown by differential effects on uptake and efflux by pharmacochemicals and polyclonal anti-42 kDa sera that inhibit sucrose uptake (54). Transporter mediated efflux was biphasic (saturation at 5 and 80 mM respectively) and exclusively due to transport events at the plasma membrane, because the presence of tonoplast was excluded. Efflux seemed to be passive, although proton-driven components, i.e. H+-antiport, could not be excluded experimentally. No clear decision can be found up to day whether the efflux transporter(s) function(s) as channels, facilitators or proton driven antiporters. Sucrose efflux transporters have not yet been identified and characterised on the molecular genetic level (55).

Although the plasma membrane preparations have to represent mixtures originating from all cell types present in the leaf tissue including sieve elements and companion cells, these experiments reasonably point to sucrose efflux into the apoplast as possible step before phloem loading. However, these experiments cannot prove this hypothesis or even exclude symplastic loading.

- Apoplastic phloem loading and long distance transport

Fine structural studies would suggest the possibility of symplastic phloem loading in sugar beet leaves. In a maize mutant (sed 1) with structurally modified plasmodesmata as revealed by electron microscopy that was blocked in plasmodesmal transport (56), the export of sucrose was reduced and growth and development of the mutant plant was heavily impaired. This argues for the importance of symplastic loading of sucrose at least in this plant. For sugar beet, however, such mutants are unknown until today.

Beyond physiological evidence for the existence of a supply system for sucrose to the apoplast (54), in sugar beet leaves evidence for the apoplastic route of phloem loading has been presented in the past (57, and literature cited therein, 30, 51, 53). The uptake system(s)

responsible for phloem loading against a concentration gradient may consist of three kinetic components (or more?), two saturable (low and high affinity) and one linear component. For the saturable components, sucrose proton symporters have been described (see 58 for a review). A secondary active sucrose proton cotransport system with a 1:1 stoichiometry was described (59) exhibiting high substrate specificity for sucrose and sensitivity to thiol group modifying agents and to diethyl pyrocarbonate (DEPC). Further kinetic analyses led to the formulation of an ordered binding mechanism best described by a (Suc)-first-on (Suc)-first-off model (60) accounting for the behaviour of the sucrose transporter with regard to the cytosolic ^-concentration (neutral pH). Delrot and co-workers identified a 42 kDa protein from isolated plasma membranes of sugar beet leaves by differential labelling with (203(-|g)-pCMBS (61) or with 3H- and [14C]-NEM (62, 63). Using these polypeptides for immunisation of rabbits, they obtained polyclonal antibodies that recognised a 42 kDa band in Western blots and, most importantly, inhibited sucrose transport (64) in plasma membrane vesicles (65), but also in tonoplast vesicles (66). It was not possible to isolate the carrier gene(s) by screening cDNA-expression libraries using these antibodies (67). However, a polyclonal serum directed against the Arabidopsis thaliana sucrose transporter (AtSUCl) reacted with a 42-kDa band of the sugar beet PMV, confirming previous biochemical identification of this band as a sucrose transporter (68).

- Molecular cloning of genes coding for leafplasma membrane bound sucrose transporters

In more recent times, a certain number of plant sugar transporters have been identified and cloned (for review, see 58, 67, 69, 70, 71). Considering their sequence properties, structure and function, they can be classified into a) the Major Facilitator Super family (MFS), b) sucrose/H+-porters, c) proton-independent transporters. Only few plant sugar transporters seem to be members of the MFS (70, 72). The sugar transporter gene that was cloned from sugar beet (73) may be part of this subfamily and function as hexose transporter. There is no clear-cut indication about the exact localisation or function of the expression product of the cloned sugar beet gene, as far as I know.

Riesmeier et al. (74,75) were the first to be able to isolate sucrose transporter cDNAs from spinach and potato as members of the sucrose/H+-porter subfamily (b), the best characterised group of sugar transporters in plants. They used a modified Saccharomyces cerevisiae mutant strain as an artificial complementation system that was deficient in sucrose uptake and secreted invertase, but able to metabolise imported sucrose as a consequence of expression of a sucrose-cleaving activity. Heterologous screening was subsequently very successful to isolate "similar" genes from other species (the deduced amino acid sequence of the potato sucrose transporter gene StSUTl for example is 68% identical to the spinach sucrose transporter SoSUTl (pS21)), e.g. tobacco, tomato, Arabidopsis, Plantago, sugar beet, carrot, rice (Buschmann et al. cited in 67, 71, 76, 78, 79, 80, 81) and other tissues than leaves, e.g. germinating seedlings of Ricinus communis (82), sink tissues (81) etc., or even more than one carrier within a single species (83). Plant sucrose/H' symporters were reported to be homologous to the melibiose permease of Escherichia coli (84).

The sucrose transporter (SUT) genes encode highly hydrophobic proteins with a calculated molecular mass of around 55 kDa, behaving as 47 kDa proteins in denaturing SDS-polyacrylamide gels (85). The protein was predicted to consist of 12 membrane-spanning domains. 45% of the amino acid positions found to be identical when comparing eight sucrose transporters from different plants were located within the putative membrane spanning domains of a topological model (67). Further sequence comparisons led to the hypothesis that the SUT1 structure arose from an ancestral gene duplication event.

The biochemical properties of the SUT1-mediated sucrose transport was quite similar when comparing the heterologously expressed proteins from spinach and potato up to the findings of 1:1 stoichiometry as found for plasma membrane vesicles from sugar beet leaves (59, 67, 86). Two sucrose transporters, called SUC1 and SUC2, were cloned from Arabidopsis thaliana (71). Expression and characterisation and identification of the histidine-tagged protein in baker's yeast also showed similarities with the transporter "in situ" although some differences between the two transporters could be revealed. For example, they all transport sucrose in an energy-dependent manner across the plasma membranes with a specificity for sucrose as described for the respective transporters in planta. The KM-values for sucrose transport are 0.50 mM and 0.77 mM for SUC1 and SUC2 and between 0.3 - 1 mM for SUT1 from spinach and potato, respectively. Transport by all proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) or SH-group inhibitors. Sensitivity towards diethyl pyrocarbonate (DEPC) could be seen as well arguing for DEPC-sensitive histidine residues at or near the sucrose binding site. For SUC1 and SUC2, the VMax but not the KM-values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids. Their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices as predicted for SUT1. A modified SUCl-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS-polyacrylamide gels resembling to the findings of Delrot and co-workers using a biochemical approach (61).

Site directed mutagenesis experiments promise further insight into structure function relationships. Among the proton-sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUCl from Arabidopsis thaliana is reported to be conserved across species. Lu & Bush (87) could determine His-65 as an essential amino acid for transport activity involved in a rate limiting step in the transport reaction using this approach. Heterologous overexpression, purification and crystallisation of the protein (67, and literature cited therein), in combination with site directed mutagenesis, promise to yield a detailed model of the protein and its functions for the relevant sugar beet transporters as well. This may include the determination of a turnover number in order to assess the carrier nature of the symporters.

The findings of Delrot and co-workers (61) during earlier biochemical approaches using the differential labelling technique (44 kDa-protein) seemed to be in contradiction to those obtained for soybean. In the plasma membrane of soybean a 62 kDa polypeptide was identified by the photolabelling technique using a light sensitive sucrose analogue, 6'-HABS (6'-deoxy-6'-(4-azido-2-hydroxy)-benzamido-sucrose) (88, 89). Grimes et al. (90) reported that a soybean 62-kDa sucrose binding protein was associated with the plasma membrane of several cell types engaged in sucrose transport in various cells of different organs. Furthermore, the temporal expression of the gene and the accumulation pattern of the protein closely paralleled the rate of sucrose uptake in the cotyledon. However, molecular cloning and sequence analysis of a full-length cDNA for this 62-kD sucrose binding protein (SBP) indicated that the protein was not an integral membrane protein. It could be demonstrated that SBP is a peripheral membrane protein, organised in homodimeric and -trimeric forms (91). In a yeast expression system SBP was shown to mediate nonsaturable sucrose transport on its own (92). Corresponding to the biochemical properties, a possible role of SBP with the linear component of sucrose uptake observed in many transport studies is discussed (67) and can therefore be classified to the group of proton-independent sugar transporters (c). A regulatory role in phloem sucrose transport through interaction with SUT1 was presumed, because a co-localisation of SBP and sucrose/H' cotransporters was found in the plasma membrane of Vicia faba transfer cells (93). Therefore, the results obtained by different biochemical approaches were not contradictory but seem to be complementary.

Physiological relevance of the cloned genes for apoplastic loading

Leaf plasma membrane bound sucrose transporters have been cloned, heterologously expressed and biochemically and physically characterised to some extent (67, 69), but where are they localised exactly? In brief: A number of transporters, recently cloned, have been shown to be phloem associated. For example, for SUT1 this has been demonstrated by RNA in situ hybridisation experiments (75). Immunolocalisation studies could determine the expression at the cellular level. Immunofluorescence with specific antibodies detected SUC2 for Plantago and Arabidopsis in companion cells (94, 95). Tissue print hybridisation analysis of Ricinus hypocotyl revealed that the sucrose transporter transcripts of RcSUTl were localised to the phloem cells of the vascular bundles identified as transfer cells in more detailed studies (82). In tobacco, potato and tomato, silver enhanced immunogold staining and immunofluorescence studies detected SUT1 in plasma membranes of the enucleate sieve elements. In situ hybridisation at the electron microscopic level revealed SUT1 encoded proteins to be mainly localised to sieve elements up to still developing sieve elements, preferentially associated with the orifices of the plasmodesmata between sieve elements and companion cells (67, 85). Antisense inhibition of SUT1 expression under the control of a companion cell-specific promoter indicated synthesis of SUT1 mRNA in the companion cells as expected because of the lack of nuclei in mature sieve elements. The localisation of SUT1 mRNA and protein in the sieve elements together with high turnover rates were taken as indication that SUT1 mRNA was transported through plasmodesmata (85). What targeting and protein synthesis in sieve elements is concerned, more detailed studies are necessary to obtain the answers to yet unsolved problems like presence and abundance of ribosomes and dictyosomes in mature sieve elements.

Most importantly, antisense repression of SUT1 in transgenic plants inhibited sucrose export from leaves (85, 96). Also, overexpression of a yeast invertase in the cell wall of the leaves in transgenic potato plants strongly impaired sucrose export from the leaves (96, 97, 98). In three investigated Solanaceae, the relevant data were consistent with the hypothesis of direct uptake of sucrose by the sieve elements. As observed for transgenic plants with overexpressed yeast invertase in the leaf cell walls, SUT1 antisense plants showed drastic phenotypic effects. For example, plant growth was retarded, leaves were curled and locally bleached, anthocyanins and, most importantly, leaf starch and sucrose content increased. Upon inhibition of SUT1 expression, similar effects on leaf carbohydrate status were observed in tobacco leaves as well (78). In addition, flowering was impaired and the development of the root system delayed (78). These effects are interpreted as the SUT1 gene expression in companion cells being essential for phloem loading (67). The use of transgenic plants specifically impaired in sucrose transporter expression has thus provided strong evidence that SUT1 transporter function is required for phloem loading. Physiological analyses of these plants demonstrated that sucrose transporters are essential components of the sucrose translocation pathway at least in potato and tobacco.

At first sight, results obtained for the localisation of SUT1 in Solanaceae (sieve elements) seem to contradict those obtained for SUC2 in Arabidopsis and Plantago (companion cells). As discussed by Ward et al. (67), the differences could be explained by different sensitivities of detection methods used with respect to the cellular level. They could also be related to species-specific differences in loading mechanisms.

Regulation of sugar transport activity at the source site

Since already phloem loading of sucrose is of essential importance for plant development, the regulation of sucrose transport activity at the leaf sieve element/companion cell-complex might be one of the keys to understand how plants regulate the allocation of photosynthate between competing sink organs. However, our knowledge about the regulation of sugar transport is quite scarce. In general, sugar transport activity could be regulated a) by varying the number of transporters expressed in the membrane, b) by modifying transport activity of already expressed transporters.

The number and the activity of expressed transporters may be hormonally controlled and seem to be dependent on the developmental stage of the leaves and to follow sink-to-source transition (see 67 for a review). A diurnal regulation could be shown for the expression of the sucrose transporter SUT1 supporting the idea that the number of transporters expressed in the membrane contributes to determine sucrose transport activity (85). A decrease of transcripts occurred in the dark in tobacco, potato, tomato, and carrot (81, 85). Sakr et al (68) performed experiments with fresh, cut or aged tissue discs and plasma membrane vesicles of mature sugar beet leaves that contained a 42 kDa-band in denaturing polyacrylamide gels (sucrose transporter) reacting with a polyclonal serum to the Arabidopsis thaliana sucrose transporter (AtSUCl). They concluded from their results that mechanical treatment subjected the sucrose symporter to transcriptional, post-transcriptional and post-translational regulation. Cutting and ageing increased the levels of sucrose symporter mRNA and, ageing more than cutting, of sucrose transporter present in the plasma membrane. Cutting stimulated proton-motive force driven uptake of sucrose in plasma membrane vesicles strongly, ageing only resulted in a slight stimulation. Inhibition by exogenous application of okadaic acid of proton-motive force-driven sucrose transport activity without affecting their proton permeability and without changing the amount of sucrose transporters detectable by ELISA pointed to a direct regulation of the activity H+-sucrose cotransporter by changes in phosphorylation state (99).

Recently, Chiou and Bush (100) reported about sucrose levels regulating sucrose symporter message levels and transport activity substantiating the old idea that sink demand might regulate photosynthesis through down regulated phloem sucrose symporters as a result of increased sucrose concentration within the phloem. Decreased phloem sucrose symporter activity should inhibit photosynthesis by increasing carbohydrate levels in the cytoplasm of photosynthetic cells. They suggest decreased extractability of sucrose symporter activity following chilling experiments with sink leaves to be a result of backed up sucrose in the leaf phloem (69). Unfortunately the author does not state whether the decreased symporter activity was measured in source leaves or in the chilled sink leaf, where chilling itself might be responsible for decreased symporter extractability.

4.2.Long distance transport of sucrose

Long distance transport is an energy dependent process. Even in plants with symplastic phloem loading, energy is required to maintain the semipermeability of the conducting channels that has been shown first by Schumacher (101). Localised inhibition of assimilate transport could be demonstrated by Willenbrink and Schuster (102) using low concentrations of the potassium binding ionophore valinomycin. Since no effect on membrane semipermeability could be observed in contrast to comparable experiments with uncouplers such as CCCP (carbonyl-cyanide-m-chlorophenylhydrazone), this indicated a strict compartmentation of potassium for the transport process. This is not proof for the idea of a proton gradient and an opposing potassium gradient across the plasma membrane of the conducting tissue. However, this is consistent with this idea to be realised both in the loading zones and along the whole translocating system as driving force for sucrose loading followed by water influx promoting long distance transport (103).

4.3. Sugar transport within the storage organ a) Phloem unloading and its regulation /plasma membrane transport of sucrose

After loading into the phloem in the lamina of exporting source leaves photoassimilates seem to be unloaded and reloaded along the entire phloem pathway down to the terminal sinks (104, 105 and references cited therein). According to a recent review (105) phloem unloading comprises all the transport events from the sieve elements to the sites of utilisation within the recipient sink cells. In sugar beet, the plasmodesmatal frequency between sieve elements and companion cells was found to be relatively high (106). The first step of phloem unloading, i.e. from the sieve tubes into the cytoplasm of companion cells, could therefore be envisioned as symplastic. Since only few plasmodesmata seem to interconnect the sieve element companion cell complex and the surrounding parenchyma tissue in sugar beet taproots (106), an apoplastic unloading step should be included here. Considering the presence of sucrose transporters located in the plasma membrane of the sieve element companion cell complex of the leaf tissue and probably the petiole, this may be assumed for the taproot as well (107). In carrot roots, expression of a Sue transporter was found but not restricted to the phloem (81). Subsequent metabolism (cell wall bound acid invertase), compartmentation and partitioning between sinks within the taproot (secondary cambial cells, phloem parenchyma, xylem parenchyma, vacuoles) complete the unloading pathway.

Free space invertase was assigned a possible role in phloem unloading (108) by facilitating phloem unloading by maintaining a downhill concentration gradient for sucrose from the phloem towards the apoplast. This was in contradiction with published results on decreasing activity of this enzyme during the beet root development merely detectable or even totally absent in mature beet roots (31). We found that sugar uptake, particularly glucose and fructose (Getz, unpublished result) uptake, was much higher in protoplasts and tissue discs isolated from the bundle region as compared to those isolated from storage parenchyma cells (109). In vitro uptake rates from as low as 1 mM 14C-labeled glucose solutions were found to be sufficient to account for in situ accumulation rates of sucrose. In accordance with these results, we had postulated a cell wall bound acid invertase (cwinv) to be confined to the bundle region of beetroots (29, 109, 110). Cwinv activities could be measured in aged and untreated beetroot tissue (41). In untreated tissue, highest cwinv activities were found within the peripheral, bundle and cambium rich tissue. This was consistent with the generally accepted assumption of cwinv being associated with rapidly growing tissues (utilisation sinks like meristems, sink leaves etc.) (111). The lowest, but still measurable, cwinv activity was found within the storage parenchyma. The bundle rich, but also meristematic cells containing tissue contained three times higher enzyme activity than the storage parenchyma. Km and Vmax data (about 2 mM, 0,12jiMol h"1 g'FW) corresponded well to estimated apoplastic sucrose concentrations and in situ accumulation rates of sucrose. These results argued for a role of cwinv in reinforcing sink strength as originally proposed by Eschrich (108).

Most recently transgenic carrot plants have been created using the 35S promoter of CaMV for antisense expression of cwinv and SuSy (112). Since the promoter was only active in taproots, enzyme activities in source leaves were not affected. Expression of antisense mRNA for cwinv resulted in transgenic plants that did not develop taproots but only small primary-type roots that contained reduced levels of soluble sugars and starch. Instead, photosynthates were used for the development of additional leaves as compared with control plants. As reviewed by Sturm and Tang (113), the possible role of cwinv in phloem unloading is dependent on the plant species, the developmental stage and the tissue region where it is expressed so that the results found for carrot can not be generalised. However, somehow comparable situations are found in the taproot forming sucrose storing plants carrot and beet. As postulated and found for beetroots by biochemical means (41), in carrot the expression of the cwinv gene is thought to be confined to only a few specialised cells, presumed to be located in the crown of the storage organ or the cells surrounding vascular bundles (113).

In accordance with Tanner's hypothesis on the possible role of abscisic acid (ABA) in phloem unloading we found inhibition of secondary active glucose uptake into protoplasts dependent on the exogenously applied ABA concentrations (114). In addition, we found protoplasts isolated from the bundle region to contain twice as much ABA than those isolated from the storage parenchyma (114). Since beets stressed without leaves merely showed an increase in ABA as compared to intact plants, the assimilatory tissue was assumed to be the production site of ABA (115). This may indicate transport of ABA through the phloem from the assimilatory tissue down to the sink where it is possibly accumulated in form of an apolar conjugate, which remains to be shown. ABA might contribute to facilitate sucrose efflux from the phloem, whereas cytokinins may promote uptake of hexoses and sucrose by the surrounding parenchyma cells either directly as indicated by the stimulating effect of fusicoccin (110, 114) or by induction of mRNAs for cwinv and glucose transporter, as shown for Chenopodium rubrum (116).

Invertase and ABA-promoted phloem unloading of sucrose and subsequent removal of resulting hexoses by hexose uptake by the surrounding parenchyma cells seems to be an attractive model. The idea is supported to some extent by the finding that around 20% of Relabel from 14C -labelled glucose taken up by isolated beet root parenchyma cells was found in sucrose in short term experiments (109, 110) at rates sufficient to explain in-situ rates of sucrose accumulation in beets. However, these results cannot exclude direct uptake of sucrose unloaded into the apoplast, since energy dependent and fusicoccin stimulated sucrose transport was found as well (110). Indeed, one of the two cloned sucrose transporter cDNAs from carrot (DcSUT2) was mainly found to be expressed in sink organs (81). The expression of the sucrose/H* symporter was much higher in storage parenchyma tissues. It would be highly valuable to investigate the importance of a similar sugar beet gene for sucrose storage in beetroot storage parenchyma by using the antisense repression approach in transgenic plants.

b) Tonoplast transport of sucrose

The large central vacuole of beetroot storage parenchyma cells may comprise about 95% of the total cell volume of beet storage cells (117). The composition of the vacuolar content can be highly variable according to the function of the respective cells within the tissue (117, 118). In beets, high amounts of sucrose can be found in the vacuoles of the storage parenchyma. From comparative evaluation of data for sucrose content of tissue discs, isolated protoplasts and vacuoles from red beet roots we concluded that vacuolar sucrose concentration may become higher than 220 mM (119,120). Sugar beets accumulate sucrose up to 20% (relative to fresh weight), whereas fodder beets contain only 3-5%. Indirect evidence suggested cytosolic sucrose content of storage cells from red beets not to exceed concentrations above 5 to 10 mM (110). This and other results reviewed in this chapter are consistent with the view that in beetroot sucrose must be transported uphill into the vacuoles against its concentration gradient, in contrast to the situation in sugar cane reported elsewhere in this book. Sucrose is accumulated in the vacuoles independent of how sucrose enters the cytoplasm of the cells, either by secondary active uptake of glucose and fructose from the apoplast followed by sucrose resynthesis (110, 121), or by direct sucrose uptake from the apoplast, or by symplastic transfer through plasmodesmata. Therefore, energy dependent sucrose transport across the tonoplast represents a potentially important regulatory site for the sucrose accumulation capacity of beetroots.

MgATP dependent uphill transport of sucrose with Michaelis-Menten like kinetics was first shown by Willenbrink and Doll (122, 123) using vacuoles that had been isolated directly from the tissue using a mechanical isolation procedure. It has been documented that sucrose is transported in vitro against an up to two hundred fold concentration gradient in the presence of MgATP (121, 124, 125).

The preparation of highly purified antigen (total tonoplasts, prepared from isolated vacuoles) in the case of red beet roots (66), of partially purified antigen (directly prepared from tissue homogenates) in the case of sugar cane (126, 127) were prerequisites for the production of sucrose transport-specific monoclonal antibodies (mAb's) in mice. The use of these mAb's allowed for the identification of a 55-65 kDa-polypeptide fraction (gel permeation chromatography) of solubilised tonoplast proteins from red beet as well as from sugarcane storage tissue. This correlated with the molecular mass of a plasma membrane-bound sucrose/TT-cotransporter from spinach leaf mesophyll cells, of which the gene had been cloned soon afterwards (see above). In SDS-PAGE, this mAb-binding fraction behaved like a protein with a molecular mass of about 40-42 kDa. This corresponded to data published for a polypeptide from plasma membranes of sugar beet leaves which specifically interacted with a polyclonal antibody (61, 62, 63) that also inhibited sucrose transport (64). Reconstituted into artificial proteoliposomes, both fractions - the 42 kDa polypeptide from SDS-gels and the 55-65 kDa fraction from gel filtration columns - exhibited inhibitorsensitive sucrose transport properties, comparable to those found for the 55-65 kDa fraction from red beet root tonoplasts (119, 120). This suggested the localisation of a sucrose transporting protein within the tonoplast of both plants, sugar cane and beets. Only reconstituted proteoliposomes, containing polypeptides from the 55-65 kD-band, took up [14C]-sucrose with linear rates suggesting that this fraction contains the tonoplast sucrose carrier.

Briskin et al. (128) demonstrated sucrose transport to be electrogenic, substrate specific, and accompanied by HT-antiport using light density membranes from sugar beet taproots -presumably tonoplast membranes. Using red beetroot tonoplast vesicles, pH-jump experiments (vesicle interior acidic) could mimic the stimulating effect of MgATP on sucrose uptake (125). This supported the earlier hypothesis of a sucrose/H+-antiport (124, 125, 128, 129) in accordance with the acidic pH within the vacuoles (pH 5.85 to 6.07 according to 3lP/NMR-measurements; 130). Voltage dependency and substrate specificity of presumed sucrose/H+-antiport, demonstrated first by Briskin et al. (128) was confirmed (119). Sucrose transport as well as concomitant vacuole alkalisation could be inhibited in a comparable manner by monoclonal antibodies to the sucrose transporter (119, 120). In addition, similar saturation kinetics and a whole-number stoichiometric relationship (unity) between sucrose transport and antiported protons as well as similar sensitivity towards inhibitors as essential criteria for an electrogenic carrier-mediated sugar transport, coupled to FT as a "driver ion" (86) were demonstrated by Getz & Klein (119). Sucrose/ff-antiport as shown in figure 2 seems to be part of the mechanism that is responsible for sucrose accumulation in the vacuoles of beet taproots.

A sucrose carrier that was inhibited by monoclonal antibodies C50-5-3 and TNP12/8 was immunolocalised on undenatured tonoplast membranes prepared from isolated vacuoles of beetroots harvested during full sucrose storage period using the same sucrose/H+-antiport specific antibodies (120) detected by subtype specific gold labelled antibodies. Taking into account the presumed molecular mass of 55-65 kDa for the sucrose carrier, the immunogold labelling density and the average membrane surface of beetroot vacuoles, a proportion of 0.13 to 0.2 % of total membrane protein content of red beet root tonoplasts could be ascribed to sucrose carriers recognised by the antibodies. This relatively low proportion of the sucrose carriers in total tonoplast integral proteins (TIP) partly explained the instability of the sucrose/H+-antiport mechanism documented by Bush (58). Using Avogadro's number, known data about labelling density, the proportion of tonoplast protein in total protein content and maximal transport rates, turnover rates between 5 and 30 sucrose molecules per carrier and second were calculated characterising the sucrose/H+-antiporter as a carrier (120). The turnover rate may well be lower than estimated for a sucrose/IT-antiport carrier since it was not clear whether the antibodies recognised different epitopes of the same carrier protein or different sucrose carrier proteins. The existence of several different sucrose transporting proteins, perhaps even the development of sucrose porins (131) within the tonoplast at a certain stage of beet root development can not be excluded (fig. 1). One of the cloned genes is reported to code for a presumably tonoplast bound sucrose transport facilitator (73), a protein with 490 amino acids and an estimated molecular mass of 54 kD that is expressed in all vegetative tissues according to RNA gel blot analysis. It cannot be excluded that this facilitator is part of the sucrose transporters active in mobilisation of sucrose (see below). Regarding sugar transport, Greutert and Keller (132) and Niland and Schmitz (133) found comparable results for sucrose and stachyose transport in Japanese artichoke (Stachys sieboldii) demonstrating that sucrose/ H '-antiport is not a unique feature of beetroots.

4.4. Sucrose accumulation and control

Provided that photosynthesis and sucrose synthesis are not rate limiting (overexpression of SPS has to be effectuated in source and sink, see above), in the sink region (a) variation of the transport activity of individual transport system(s) for sucrose and (b) variation of the number of active sucrose transporters per tonoplast area could be the factors regulating sucrose accumulation. The number of sucrose transporters itself may be under hormonal and/or developmental control. If sucrose is confined to the vacuolar compartment as estimated earlier for red beets (109, 134), then metabolic energy has to be spent to achieve uphill transport of sucrose and a specific transport system is required to utilise this energy adequately. The

Cell Wall Plasma- Cytoplasm Tono- Vacuole (Apoplast) membrane (Symplast) plast (Cell Sap)

Sucrose Proton Symporter

Figure 2. Sucrose transport at the plasma membrane and at the tonoplast. At the plasma membrane, an inside directed H+-gradient is established by a P-type ATPase. The energy of this If-gradient can be utilized to drive sucrose transport across the plasma membrane through an I I/Suc-cotransporter following the proton gradient. On the tonoplast, a pyrophosphatase and a V0V]-ATPase pump protons out of the cytoplasm into the vacuole establishing and maintaining an inside the cytosol directed proton gradient. As a result, sucrose and proton gradients are oriented in the same direction, i.e. into the cytosol. Like at the plasma membrane, the downhill gradient of protons drives sucrose transport. In contrast to the situation at plasma membrane, a II'/sucrose (Sue) antiporter transports sucrose uphill against its concentration gradient across the tonoplast into the vacuole. It is not known, whether an additional transporter might be responsible for the linear uptake phase. Information about sucrose efflux on the tonoplast of storage parenchyma cells is still rather limited. (Modified from 120)

Figure 2. Sucrose transport at the plasma membrane and at the tonoplast. At the plasma membrane, an inside directed H+-gradient is established by a P-type ATPase. The energy of this If-gradient can be utilized to drive sucrose transport across the plasma membrane through an I I/Suc-cotransporter following the proton gradient. On the tonoplast, a pyrophosphatase and a V0V]-ATPase pump protons out of the cytoplasm into the vacuole establishing and maintaining an inside the cytosol directed proton gradient. As a result, sucrose and proton gradients are oriented in the same direction, i.e. into the cytosol. Like at the plasma membrane, the downhill gradient of protons drives sucrose transport. In contrast to the situation at plasma membrane, a II'/sucrose (Sue) antiporter transports sucrose uphill against its concentration gradient across the tonoplast into the vacuole. It is not known, whether an additional transporter might be responsible for the linear uptake phase. Information about sucrose efflux on the tonoplast of storage parenchyma cells is still rather limited. (Modified from 120)

activity of given transporters may be regulated by the energy status of the membranes (plasma membrane and tonoplast have to be considered) or directly, e.g. by phosphorylation as shown for sucrose/H+-symporters of the plasma membrane (99).

The density of immunogold decoration on tonoplasts prepared from sprouting beets in the beginning of their second vegetation period was only about one third to one half of that found on membranes from sugar accumulating beets (120) indicating loss of carrier protein recognised by the sucrose/H~ antiport specific mAb between the sucrose accumulation phase and that of sucrose remobilization. The enzymes required for a futile cycle as discussed for sugar cane by Wendler et al. (135) and Komor (136) are present in the cytoplasm of sugar beet roots (29). If such a futile cycle of sucrose synthesis and sucrose degradation takes place in beetroots, a powerful H+/sucrose- antiport system would greatly counteract and prevent its energy wasting effects during the sucrose accumulation phase. Loss of H+/sucrose-antiport carriers in the vacuolar membrane could then favour the futile cycle in the beginning of the second vegetation period and thereby remobilization of sucrose. For this particular stage of beet development, rising activity of vacuolar acid invertase was discussed to be responsible for sucrose remobilization (134). Recently, it was demonstrated (137) that vacuolar acid invertase was not involved with the mobilisation of sucrose in sprouting beetroots under natural conditions, i.e. without injury, washing and/or ageing of the tissue.

As mentioned previously (120) an increase of sucrose exporting transporters (activity or number?) could contribute to sucrose mobilisation during sprouting. At this time, it can only be speculated whether these transporters, tentatively symbolised by uniporting sucrose transporters in fig. 2, belong to the MFS family (the cDNA cloned by Chiou & Bush [73]?), the proton-independent transporter group, to the sucrose/H+-symporter group or some sort of porins. At this time, the beetroot storage cells are to be considered as source cells, whereas the developing reproductive organs represent the strongest sinks. It can only be speculated at this time, how much changing ionic conditions may influence the membranes' permeability (138), and how much the overall switch from sucrose accumulating to sucrose exporting vacuoles might be under hormonal control (ABA, V-ATPase?). This has to be elucidated by relevant experiments.

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