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In higher plants, sugars are produced in the mesophyll cells of mature leaves which are known as source organs. Heterotrophic cells, such as roots and seeds, are sink organs and rely on the supply of sugars for their nutrition. Thus, the adequate production, storage and transport of sugars are essential to sustain growth and development in plants.
Various sugar transport systems exist in plants. Sugars are transported into surrounding cells apoplastically by sugar transporters across membranes or symplastically through the plasmodesmata. Sugars in phloem sieve cells are loaded apoplastically by sugar transporters and/or symplastically, depending on the plant species, and are transported over long distances from source to sink organs (ap Rees, 1994; Williams et al., 2000; Lalonde et al., 2004; Lim et al., 2006; Büttner, 2007). In addition to these short- and long-distance transport events, sugar molecules are distributed into different subcellular organelles in cells depending on the nutrient requirements. For example, transitory starch is degraded into maltose in chloroplasts, which is then exported into the cytosol at night via the maltose transporter (Niittyläet al., 2004; Weise et al., 2006).
In many plants, the disaccharide sucrose is the principal form of sugar for long-distance transport in phloem sieve cells, whereas the major monosaccharide sugar forms are glucose and fructose. Sugar transporters constitute a large gene family. In Arabidopsis for example, 53 putative monosaccharide transporter genes belonging to seven different subfamilies (Büttner, 2007) and more than 10 disaccharide transporters (Lalonde et al., 2004) have been identified. In addition, rice harbors more than 20 monosaccharide transporter genes and five disaccharide transporters (Hirose et al., 1997; Furbank et al., 2001; Aoki et al., 2003; Lim et al., 2006).
In mature plant cells, vacuoles occupy a large part of the cell volume and are separated from the cytosol by the tonoplast, a semipermeable vacuolar membrane. Vacuoles serve as a main long-term and also temporary storage pool in the cell (i.e. metabolites produced in excess are transported into vacuoles and released according to metabolic requirements; Cairns et al., 2000; Pollock et al., 2000; Wormit et al., 2006; Martinoia et al., 2007; Neuhaus, 2007). In this process, specific tonoplast sugar transporters are required for the vacuolar transport of these nutrients. However, whereas a large number of plasma membrane-localized sugar transporters have been well characterized in various plants (Lalonde et al., 2004; Lim et al., 2006), the existence of tonoplast sugar transporters has only recently been identified in the tonoplast proteomes of Arabidopsis and barley (Carter et al., 2004; Endler et al., 2006). Moreover, to date, only two types of vacuolar sugar transporters, the Arabidopsis thaliana tonoplast monosaccharide transporter (AtTMT) and vacuolar glucose transporter (AtVGT), have been functionally characterized in any detail (Wormit et al., 2006; Aluri & Büttner, 2007).
The tonoplast localization of AtTMT and AtVGT was previously examined by transient expression analysis of green fluorescent protein (GFP) fusion constructs using protoplasts and an active glucose transport ability of AtVGT1 has been demonstrated in isolated yeast vacuoles. Gene expression and mutant analyses of AtVGT1 have further suggested a role in seed germination and flowering (Aluri & Büttner, 2007). By contrast, AtTMT1 and AtTMT2, which possess a very long hydrophilic central loop, were found to be upregulated in response to stress treatments such as drought, salt and cold. The sugar import activities of AtTMT1 have been demonstrated using vacuoles isolated from cold-treated tmt1 mutants. These data collectively suggest a major role for the AtTMTs during stress responses in Arabidopsis that is distinct from that of AtVGT1 (Wormit et al., 2006). Notably also, unlike the AtTMTs, SsGTR, a tonoplast monosaccharide transporter (TMT) homolog of the cyanobacterium Synechocystis sp. PCC6803 that acts as glucose transporter, resides on the plasma membrane of cyanobacteria. SsGTR catalyses proton-coupled sugar import into bacterial cells (Schmetterer, 1990). In other plants, the role of the TMTs remains unknown and the characterization of these genes remains an important issue.
As an approach to elucidating the possible role of TMT proteins during the growth and development of rice, we have isolated and characterized two rice (Oryza sativa) TMTs, OsTMT1 and OsTMT2. The subcellular localization of OsTMT–GFP fusion protein was then examined in both rice and Arabidopsis. Detailed expression patterns were also analysed by reverse-transcription polymerase chain reaction (RT-PCR) and histochemical analysis of transgenic rice plants expressing OsTMT promoter–GUS fusions, and by additional in situ hybridization experiments. The sugar transport ability across tonoplasts was tested in isolated vacuoles from the transgenic Arabidopsis TMT-deficient mutant line (tmt1-2-3) expressing OsTMT1, and complementation tests of sugar levels were performed also in the transgenic Arabidopsis lines. Finally, based on the data we have obtained, the involvement of OsTMTs in sugar retrieval during assimilate transport is discussed.
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Assimilated sugar in source leaves is either transiently stored or distributed to heterotrophic sink tissues where it can serve either as an energy source for growth and development or as a supply of nutrient storage reserves. Hence, efficient sugar retention during translocation to release sites is necessary for optimal plant growth and productivity. It is postulated that sugar transporters can function to retrieve sugars that have leaked into the apoplasm (Ritchie et al., 2003; Scofield et al., 2007). Analyses of the biochemical properties of sugar transporters using protoplasts from developing seed coats have indicated that in Phaseolus and Vicia seed coats, they enable the retrieval of sugars that have leaked to the coat apoplasm during the symplasmic passage of these molecules along the post-sieve element pathway. This suggests that sugar recovery contributes to the movement of photoassimilates from the importing sieve elements to the cells responsible for sucrose release that are located at the interface between maternal and filial tissues (Ritchie et al., 2003). Similarly, it has been proposed in rice that the primary role of OsSUT1, a plasma membrane sucrose transporter, is the retrieval of sucrose from the apoplasm and its transport to the phloem sieve cells to maintain supply to the filling grain on the panicle (Scofield et al., 2007). These data raise questions about the nature of sugar retention in vacuoles within these transport paths and the involvement of vacuolar sugar transporters (Thom et al., 1982; Chiou & Bush, 1996).
The TMT family is the first vacuolar sugar transporter group to be characterized, and the TMT proteins themselves possess a large central loop between transmembranes 6 and 7. The TMT proteins have been shown to transport monosaccharides, glucose and fructose in a proton/sugar antiport manner, demonstrated by the finding that the protonophore ammonia (NH4Cl) inhibits glucose transport in isolated vacuoles (Wormit et al., 2006). In our present study, we have characterized two rice TMT genes, OsTMT1 and OsTMT2. Localization experiments using GFP fusion proteins clearly show that both of these rice TMTs reside on tonoplasts (Figs 2, S3). Glucose transport experiments using isolated vacuoles from the Arabidopsis tmt1-2-3 lines expressing OsTMT1 further demonstrated that OsTMT1 exhibits glucose transport ability across the tonoplasts (Fig. 6). Taken together with the earlier findings in Arabidopsis (Wormit et al., 2006), our data strongly suggest that the TMT proteins function in vacuolar monosaccharide transport in both monocot and dicot plants. In addition, our phylogenetic analysis further indicates that the previously reported hexose transporters, HvSTP1 and HvSTP2 from barley, PST type 2a from sugarcane and VvHT6 from grape, are likely also to be tonoplast monosaccharide transporters belonging to the TMT subfamily (Casu et al., 2003; Weschke et al., 2003; Agasse et al., 2009) (Fig. 1). It will be of great interest and value in the future to determine if these proteins all localize at the tonoplasts and retain monosaccharide transport abilities.
In Arabidopsis, AtTMT1 expression is prominent in mesophyll cells, in the cells surrounding the vascular tissue, and in the flower organs including petals, filaments and pollens (Wormit et al., 2006). However, developing Arabidopsis seeds did not exhibit AtTMT1 expression. AtTMT2 has been shown to be expressed in the root stele, in the edges of mature leaves, and in petals and filaments of flowers in Arabidopsis (Wormit et al., 2006). By contrast, AtTMT3 expression was found to be low in all Arabidopsis tissues and not detectable by northern blotting. In particular, the AtTMT1 and AtTMT2 genes have been shown to be upregulated upon cold, drought and salt stresses, and glucose transport into vacuoles was found to be stimulated by cold treatment. Thus, the AtTMT proteins have been suggested to contribute to the molecular response to osmotic stress stimuli (Wormit et al., 2006; Neuhaus, 2007).
In monocots, previous in situ localization analysis has shown that PST type 2a, a putative TMT homolog from sugarcane, is expressed mainly in the phloem companion cells and associated parenchyma cells in the mature stem (Casu et al., 2003). The expression of HvSTP1 and HvSTP2, two putative barley TMT homologs, has also been observed in young developing caryopses by in situ hybridization (Weschke et al., 2003). HvSTP2 transcripts were found to be present mainly in the region of the dorsal vascular bundle of the maternal pericarp tissue during early barley seed development, whereas HvSTP1 was shown to be expressed in the filial syncytial endosperm layer. These data likely suggest the functional involvement of these proteins in sugar translocation in sink organs. In our present study, we analysed transgenic rice, plants expressing pOsTMT1-GUS or pOsTMT2-GUS reporter constructs. Analysis of the GUS expression patterns in these transgenic plants revealed that OsTMT1 and OsTMT2 are highly expressed in vascular tissues and its peripheral regions in both source and sink organs (Fig. 5). These results from RT-PCR, promoter GUS assay and in situ hybridization experiments suggest that OsTMT1 and OsTMT2 are likely also to be centrally involved in sugar translocation in both source and sink organs.
We were unable to observe any significantly altered expression of OsTMT1 and OsTMT2 in response to environmental stress stimuli such as drought, cold or salt treatments, suggesting that they may not function in stress responses in rice. It is noteworthy in this regard that OsTMT1 and OsTMT2 are expressed at considerable levels in the photosynthetic mesophyll cells. It has been shown previously that a number of cereals, including rice, store relatively high ratios of soluble sugars to transitory starch in their leaves, whereas Arabidopsis plants primarily store starch (Nakano et al., 1995; Winder et al., 1998; Murchie et al., 2002; Trevanion, 2002; Lee et al., 2008). This likely suggests that rice requires functioning vacuolar sugar transporters to facilitate sugar turnover in these organelles and thereby sustain plant growth under normal conditions. By contrast, this role of the vacuolar sugar transporters may not be essential for plant growth in starch-storing plants including Arabidopsis. Thus, the TMTs in starch-storing plants may have evolved a stress response role.
The results of studies of plasmodesmatal frequencies and transport pathway experiments using 14C-labeled assimilates have suggested that sucrose moves symplastically from phloem sieve elements of the pericarp to the circumferential maternal nucellus, and is then apoplastically taken up through the aleurone and subaleurone cells into the endosperm cells (Oparka & Gates, 1981a,b; Furbank et al., 2001; Lim et al., 2006). In the apoplastic space in seeds, sucrose is cleaved by a cell-wall invertase such as GRAIN INCOMPLETE FILLING1 (GIF1; also called OsCIN2) in rice, Miniature1 (Mn1) in maize, and HvCWIN1 and HvCWIN2 in barley, to produce hexose compounds (Miller & Chourey, 1992; Hirose et al., 2002; Weschke et al., 2003; Roitsch & Gonzalez, 2004; Cho et al., 2005; Wang et al., 2008). GIF1 expression in rice has also been found to be restricted to the main vascular tissues of seed coats (Wang et al., 2008). Interestingly, the barley HvCWIN1 localization profile was found to be strongly associated with expression of the putative barley TMT homolog, HvSTP1. Similarly, HvCWIN2 was found to be preferentially expressed in seed coats, correlating roughly with the expression of another putative barley TMT homolog, HvSTP2 (Weschke et al., 2003). Hence, our current data and previous findings suggest that sucrose transferred into the apoplastic space in early developing seeds is hydrolysed by cell-wall invertases and that the resulting excess hexose compounds are retrieved and temporally stored in the vacuoles of vascular tissues and peripheral cells by TMTs. The generation of OsTMT1 and OsTMT2 loss-of-function rice plants in which these genes have been suppressed will be very valuable for future studies involving a more detailed characterization of the TMTs in rice, an agronomically vital crop species.
An extremely long hydrophilic loop, which is characteristic of TMT proteins, is also observed in the sucrose transporters localized on plasma membranes such as AtSUT2/AtSUC3 (Barker et al., 2000; Aoki et al., 2003). In addition, an extended large cytoplasmic domain is also found in the carboxy-terminal regions of the yeast glucose sensors SNF3 and RGT2. The existence of these extraordinarily long hydrophilic regions has initiated considerable debate about a possible function as a sugar sensor. However, no robust evidence for this has yet been presented and it will thus be interesting to determine the specific function of this large loop domain in TMT proteins in the future.
In conclusion, we have isolated and characterized the TMT genes in rice, OsTMT1 and OsTMT2, and demonstrated their involvement in vacuolar monosaccharide transport. Significantly, while the Arabidopsis TMTs play a major role in osmotic stress responses, our current data indicate that rice TMTs function in sugar retrieval during sugar translocation. Thus, our data suggest that the TMT transporters are evolutionarily conserved in many plants but are likely to have functionally diverged in monocot and dicot plants.