Sugar transporters involved in flowering and grain development of rice

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1 J. Plant Physiol (2001) Urban & Fischer Verlag Sugar transporters involved in flowering and grain development of rice Taito Takeda, Kyoko Toyofuku, Chiaki Matsukura 1, Junji Yamaguchi 2 * Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya , Japan 1 Present address: Orynova K.K., 700 Higashibara, Toyoda-cho, Shizuoka, Japan 2 Present address: Hokkaido University, Graduate School of Science, Kita-ku N10-W8, Sapporo , Japan Received August 30, 2000 Accepted January 12, 2001 Summary Developing seeds offer a fine experimental system for study on sugar transport mechanism(s) in sink tissue. The sugar transporters identified as being specific to the seed development may play a crucial role for flowering and grain development. The rice sucrose transporter OsSUT1 and the monosaccharide transporters OsMST1 3 have been previously characterized. To investigate sugar transport processes during flowering and in developing grains of rice, we newly isolated two genomic clones OsSUT2 (Oryza sativa sucrose transporter 2) and OsMST5 (Oryza sativa monosaccharide transporter 5) and their corresponding cdnas. OsSUT2 and OsMST5 are encoded by open reading frames of 1485 and 1557bp encoding 495 and 519 amino acids, respectively. The putative amino acid sequence of OsSUT2 showed 63.0 and 62.4 % identity to that of OsSUT1 and barley transporter HvSUT1, respectively. Northern blot analysis revealed that OsSUT2 and OsMST5 mrna accumulates in panicles before pollination. In situ hybridization analysis showed that OsSUT2 transcript is specific to the developing pollen. These data presented suggest that both OsSUT2 and OsMST5 play a role during the development at the early stage of the seed development. On the other hand OsSUT1 was expressed at the early stage of the grain development, suggesting a different physiological role compared to OsSUT2. Key words: cereal monosaccharide transporter Oryza sativa L. seed development sucrose transporter Abbreviations: MST monosaccharide transporter. se-cc sieve element-companion cell complex. SUT sucrose transporter Introduction In higher plants, photosynthetically active source tissues export carbohydrates to photosynthetically less active sink tissues such as roots, tubers or seeds. This long distance transport of carbohydrates mainly depends on the plasma * corresponding author: jjyama@agr.nagoya-u.ac.jp membrane sucrose-h + symporters involved in the loading of sucrose from apoplast into sieve element-companion cell complex. Sucrose and monosaccharide transporters have been identified in a variety of plant species (Williams et al. 2000). These transporters consist of 12 highly hydrophobic transmembrane domains (Marger and Saier 1993) and are energy-dependent H + co-transporters. Some of them are ex /01/158/ $ 15.00/0

2 466 Taito Takeda et al. Figure 1. Phylogenetic tree of 10 sucrose transporters. The amino acid sequence deduced from the OsSUT2 cdna is compared with the deduced amino acid sequences of OsSUT1 from Oryza sativa L. (D87819); HvSUT1, 2 from Hordeum vulgare (AJ and AJ272308); ZmSUT1 from Zea mays (AB00864); AtSUC1, 2 and AtSUT2, 4, 8 from Arabidopsis thaliana (X75365, X75382, AC004138, AC and AC005398). The multiple-sequence alignment was constructed with the Clustal W (Thompson et al. 1994) and the Tree View program (Page 1996) was used to generate the image. pressed in sink tissues such as pollen, ovules, roots and seeds, but rate-limiting functions of these sink-specific transporters have not been proven. The developing embryo and endosperm from cereals as well as from other seed plants are symplastically isolated from maternal tissues and need to be supplied with carbohydrates for their development and for the deposition of storage compounds. Therefore active transport system are present on the pathway from the phloem to the endosperm (Gahrtz et al. 1996, Weber et al. 1997). Carbohydrate efflux transporters involved in phloem unloading have been assumed to function as facilitators or as proton antiporters (Walker et al. 1995). In barley caryopses sucrose transporters, HvSUT1 and 2 are expressed in the cells of the nucellar projection and the endospermal transfer layers, indicating that these transporters play a possible role as a control element for the endospermal sucrose concentration (Weschke et al. 2000). The present paper gives an outline on sugar transporters which have been isolated from cereals and describes characterization of newly isolated sugar transporters in rice. Materials and Methods Plant materials Seeds of rice (Oryza sativa L.; Japonica-type cultivar: Nipponbare) were sown and grown in pot. Pollination date (flowering date) was recorded and days after pollination (DAP) were used to monitor the developmental stage.

3 Sugar transporters in flowering and grain development 467 Figure 2. Phylogenetic tree of sixteen monosaccharide transporters. The amino acid sequence deduced from the OsMST5 cdna is compared with the deduced amino aced sequences of OsMST1 3 from Oryza sativa L. (D25142, D46606 and D40232); SopGlcT from Spinacia oleracea (AF215851); GLUT1 from Homo sapiens (K03195); RGT2 from Saccharomyces cerevisiae (Z74186); HXT2 from S. cerevisiae (M33270); GAL2 from S. cerevisiae (M68547); RcHEX6 from Ricinus communis (L08188); NtMST1 from Nicotiana tabacum (66856); AtSTP1 from Arabifopsis thaliana (X55350); VfHEXT from Vicia faba (Z93775); MtST1 from Medicago truncatula (U38651); ShGLT from Saccharum hybrid (L21753) and CkHUP1 from Chlorella kessleri (Y07520). The multiple-sequence alignment was constructed with the Clustal W (Thompson et al. 1994) and the Tree View program (Page 1996) was used to generate the image. OsSUT2 and OsMST5 clones Complementary DNAs for OsSUT2 and OsMST5 were obtained from the Rice Genome Research Program. The cdna sequences were determined by the dideoxy-chain termination method using an ABI 373A DNA sequencer (Perkin-Elmer Co., NJ, U.S.A.). vulgare), but the sequence was poorly homologous to that of HvSUT2 (Hordeum vulgare) and to those of Arabidopsis transporters having 27.0 to 46.8 % similarity. These data show that the transporters in monocots except of HvSUT2 form a distinct sub-group from those in dicots. Results and Discussion Sequence alignment of known sucrose transporters compared to rice Sucrose transporter (SUT) cdnas have been identified in the monocots rice (Hirose et al. 1997), maize (Aoki et al. 1999) and barley (Weschke et al. 2000). From Arabidopsis, at least seven SUT genes can be identified from the data libraries. The putative amino acid sequence of the newly isolated rice sucrose transporter OsSUT2 was aligned with those of known transporters from other species (Fig. 1). Sequence comparison revealed 62.4 to 63 % similarity of OsSUT2 with OsSUT1 (Oryza sativa), ZmSUT1 (Zea maize) and HvSUT1 (Hordeum Sequence alignment of known monosaccharide transporters compared to rice The phylogenetic tree founded on the deduced amino acid sequences of human, yeast and plant monosaccharide transporters are shown in Fig. 2. There was no significant diversity among the transporters in plants from monocots to dicots. However, SopGlc, plastidic glucose translocator in spinach (Weber et al. 2000) as well as GLUT1 (human) and RGT2, HXT2, and GAL2 (yeast) show high variation from the transporters in plants. The motifs reported to be characteristic for monosaccharide transporters (Henderson et al. 1992) were highly conserved among rice (OsMST1 5), castor bean (HXT6), Arabidopsis (STP1), and humans (GLUT1), with corresponding

4 468 Taito Takeda et al. Figure 3. The diagrammatic representation of rice and legume seeds (a). Carbohydrates efflux to the seed apoplast and retrieval by the filial tissues (b). Filial tissues take up sucrose, or glucose and fructose, which is formed by the hydrolysis of sucrose, might be catalyzed by cell wall invertase (CWINV) in seed apoplast. The sucrose transporter(s) (SUT) and monosaccharide transporter(s) (MST) which are expressed in the most outer-cell layer (transfer cell/aleurone cell in cereals) in endosperm (or embryo/cotyledon) would be involved in sugar uptake from the seed apoplast (endosperm cavity in cereals). VCINV, vacuolar invertase positions of the twelve hydrophobic transmembrane domains (Toyofuku et al. 2000). whether these differences are due to specific characters of SUTs from cereals, detail demonstration for the kinetics of OsSUT1 and 2 would be needed. Functional expression of cereal sucrose transporters in yeast Heterologous expression of SUTs in yeast mutants that are unable to hydrolyze extracellular sucrose has been reported. These experiments suggest that all plant sucrose transporters identified so far are energy-dependent and sensitive to protonophores, showing that they are sucrose-h + symporters. The Km values for sucrose of SUTs in dicots are in the range of 1mmol/L, but that of HvSUT1 and HvSUT2 were 7.5 and 5 mmol/l, respectively (Weschke et al. 2000). To clarify Morphology of seeds from rice, barley and legumues In rice seeds, carbohydrates are carried through the vascular bundle which extends along the length of the grain (Zee 1972). Cuticle layer forms the pericarp/nucellus boundary except beneath the vascular bundle, pigment strand. Nucellar cells symplastically connected with se-cc complexes surround the seed apoplast and the filial tissues and are thought to carry out the membrane efflux of carbohydrates (Oparka and Gates 1981 a, 1981 b) (Fig. 3 b). The stopping of import

Sugar transporters in flowering and grain development 469 Figure 4. Localization of OsSUT1 mrna in leaf sheath of rice. The 5-d-old shoot was used for experiment.

5 Sugar transporters in flowering and grain development 469 Figure 4. Localization of OsSUT1 mrna in leaf sheath of rice. The 5-d-old shoot was used for experiment. PAS (Periodic Acid Schiff) staining was carried out to visualize starch granules and cell walls (A) and in situ hybridization (B) was performed. OsSUT1 mrna was specifically localized in phloem companion cells (indicated by arrowheads with CC). ad, adaxial side; ab, abaxial side; Ph, phloem; Xy, xylem; CC, companion cell. Bar = 0.02 mm and the crushing of the nucellus occur simultaneously (Oparka and Gates 1982) showing that the nucellus play a key role in carbohydrate efflux. The membrane influx of carbohydrates by the aleurone cells is predicted (Oparka and Gates 1981a) but the component (s) of this process are still unclear. In barley and wheat seeds, carbohydrates are carried through the single vascular bundle, which traverses the length of the grain (Sakri and Shannon 1975). Subsequently, carbohydrates are delivered through the symplastic pathway being between the vascular bundle and the seed apoplast (endosperm cavity) (Fig. 3 b). This symplastic pathway within the maternal tissue is prevented from a cuticle layer surrounding the inner surface of the pericarp at the crease vein area (Zee and O Brien 1970). The phloem unloading is thought to be carried out through this crease vein area (Thorne 1985). Carbohydrates are unloaded into the endosperm cavity and are taken up from endosperm cavity by the filial tissues and distributed throughout the endosperm (Cook and Oparka 1993, Ugalde and Jenner 1990). Although the filial storage cells of grain legumes are cotyledonary instead of endospermic, these pathways are also proposed for grain legumes (Weber et al. 1997) as well as cereal. Indeed, some sugar transporters associated with the membrane influx of carbohydrates in the filial tissues have been reported (Weber at al. 1997, Weschke et al. 2000). Developing seeds of cereals are useful experimental models to study carbohydrates transport mechanism into sink tissue. To take the solutes from the seed apoplast would be the very powerful way to understand this transport process. In contrast with wheat and barley, the seed apoplast of rice is filled up by endosperm at 4 5 days after pollination. This histological difference makes it difficult to deal with solution in rice seed apoplast compared to wheat and barley. To understand the physiological function of the sugar transporters improved manipulation to take endosperm cavity sap in rice would be expected. Localization of OsSUT1, OsSUT2 and OsMST5 mrnas It has been shown that OsSUT1 (Hirose et al. 1997) and ZmSUT1 (Aoki et al. 1999) are expressed in photosynthetically active tissues, especially in leaf blade, leaf sheath and germinating seed (Hirose et al. 1997). Furthermore, in situ hybridization using the 3 non-translated regions as probe shows that OsSUT1 mrna is localized in the phloem companion cells (Fig. 4; Matsukura et al. 2000). These data suggests that OsSUT1 play a role in sucrose loading into leaf phloem as well as SUT1 subfamily in dicots. Although alignment based on their putative amino acid sequences shows that HvSUT1 revealed high similarity to OsSUT1, the expression of HvSUT1 is mainly restricted in the cells of the maternal-filial boundary in seeds that frequently show transfercell morphology. These data suggest that HvSUT1 may play a distinct role from that of OsSUT1. The abandance of OsSUT2 and OsMST5 mrna was investigated by RNA gel blot analysis using total RNA from various rice tissues. Strong signals for OsSUT2 and OsMST5 were detected in the flower before pollination, but no signals were detectable in the other tissues (data not shown). To investigate the detailed localization of OsSUT2 mrna in the developing flower, in situ hybridization revealed that the Os- SUT2 is expressed in the developing pollen (data not shown). These results suggest that the OsSUT2 plays a role for sucrose influx into developing pollen for starch synthesis and the OsMST5 is also related to early flower development. Monosaccharide transporters having similar functions have been also reported in Arabidopsis (Truernit et al. 1999). Sugar uptake and transport processes in embryos of rice during germination have been clarified as follows (Matsukura et al. 2000): (1) starch degradation products (mainly glucose) are taken up by embryonic epithelium cells through an unknown mechanism(s); (2) sugars are transported from the epithelium cells to the cells around the vascular tissues and transiently deposited as starch granules; (3) the starch is broken down and the resulting compounds are transported and/ or finally converted into sucrose in the vascular cells; (4) the sucrose molecules are loaded into the phloem by means of the sucrose transporter whose expression is under positive regulation by the substrates (sugars). It is likely that sugar transport is also involved in pollen grain development, pollen tube growth and grain development during seed development. The results in this communi-

6 470 Taito Takeda et al. cation indicate that the OsSUT1, 2 and OsMST5 are associated with these developmental processes. However, additional sugar transporters involved in these processes could not be ruled out. Further investigations will be needed for clarifying sugar-transport and -regulation in the flowering and seed development. Acknowledgements. We thank Dr. T. Sasaki of the Japanese Rice Genome Program for EST clones. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (no ) and no from the Ministry of Education, Science, Sports and Culture, Japan. References Aoki N, Hirose T, Takahashi S, Ono K, Ishimaru K, Ohsugi R (1999) Molecular cloning and expression analysis of a gene for a sucrose transporter in maize. Plant Cell Physiol 40: Cook H, Oparka KJ (1983) Movement of fluorescein into isolated caryopses of wheat and barley. Plant Cell Env: Gahrtz M, Schmelzer E, Stolz J, Sauer N (1996) Expression of the PmSUC1 sucrose carrier gene from Plantago major L. is induced during seed development. Plant J 9: Henderson P, Baldwin JSA, Cairns MT, Charalambous BM, Dent HC, Gunn F, Liang WJ, Lucas VA, Martin GE, McDonald TP, McKeown BF, Muiry JAR, Petro KR, Rooberts PE, Shatwell KP, Smith G, Tate CG (1992) Sugar-cation symport systems in bacteria. Int Rev Cytol 137A: Hirose T, Imaizumi N, Scofield GN, Furbank RT, Ohsugi R (1997) cdna cloning and tissue specific expression of a gene for sucrose transporter from rice. Plant Cell Physiol 38: Marger MD, Saier MH (1993) A major super-family of trans-membrane facilitators that catalyses uniport, symport and antiport. Trends Biochem Sci 18: Matsukura C, Saitoh T, Hirose T, Ohsugi R, Perata P, Yamaguchi J (2000) Sugar uptake and transport in rice embryo. Expression of companion cell-specific sucrose transporter (OsSUT1) induced by sugar as well as light. Plant Physiol 124: Oparka KJ, Gates P (1981a) Transport of assimilates in the developing caryopsis of rice (Oryza sativa L.). Ultrastructure of the pericarp vascular bundle and its connections with the aleurone layer. Planta 151: Oparka KJ, Gates P (1981b) Transport of assimilates in the developing caryopsis of rice (Oryza sativa L.). The pathways of water and assimilated carbon. Planta 152: Oparka KJ, Gates P (1982) Ultrastructure of the developing pigment strand of rice (Oryza sativa L.) in relation to its role in solute transport. Protoplasma 113: Sakri FAK, Shannon JC (1975) Movement of 14C-labelled sugars into kernels of wheat (Triticum aestivum). Plant Physiol 55: Thorne JH (1985) Phloem unloading of C and N assimilates in developing seeds. Annu Rev Plant Physiol 36: Toyofuku K, Kasahara M, Yamaguchi J (2000) Characterization and expression of monosaccharide transporters (OsMSTs) in rice. Plant Cell Physiol 41: Truernit E, Stadler R, Baier K, Sauer N (1999) A male gametophytespecific monosaccharide transporter in Arabidopsis. Plant J 17: Walker NA, Patrick JW, Zhang W, Fieuw S (1995) Mechanism of photosynthate efflux from seed coats of Phaseous vulgaris: A chemiosmotic analysis. J Exp Bot 46: Wang HL, Patrick JW, Offler CE, Wardlaw IF (1993) A novel experimental system for studies of photosynthate transfer in the developing wheat grain. J Exp Bot 44: Weber A, Servaites JC, Geiger DR, Kofler H, Hille D, Groner F, Hebbeker U, Flugge U (2000) Identification, purification, and molecular cloning of a putative plastidic glucose translocator. Plant Cell 12: Weber H, Borisjuk L, Heim U, Sauer N, Wobus U (1997) A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds. Plant Cell 9: Weschke W, Panitz R, Sauer N, Wang Q, Neubohn B, Weber H, Wobus U (2000) Sucrose transport into barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation. Plant J 21: Williams LE, Lemoine R, Sauer N (2000) Sugar transporters in higher plants a diversity of roles and complex regulation. Trends Plant Sci 5: Zee SY (1972) Vascular tissue and transfer cell distribution in the rice spikelet. Aust J Biol Sci 25: Zee SY, O Brien TP (1970) Studies on the ontogeny of the pigment strand in the caryopsis of wheat. Aust J Biol Sci 23:

Analysis of Sugar Content and Expression of Sucrose Transporter Genes (OsSUTs) in Rice Tissues in Response to a Chilling Temperature

JARQ 51 (2), 137-146 (217) https://www.jircas.go.jp Analysis of Sugar Content and Expression of Sucrose Transporter Genes (OsSUTs) in Rice Tissues in Response to a Chilling Temperature Shinichiro TAKAHASHI