characterization of rice functional monosaccharide

† To whom correspondence should be addressed. TelWFax: +81–11–706–2737; E-mail: jjyama＠sci.hokudai.ac.jpAbbreviations : CCCP, carbonyl-cyanide-m-chlorophenyl-hydrazone; 3-OMG, 3-O-methyl glucose

Biosci. Biotechnol. Biochem., 67 (3), 556–562, 2003

Characterization of Rice Functional Monosaccharide Transporter, OsMST5

Budsaraporn NGAMPANYA,1,2 Anna SOBOLEWSKA,3 Taito TAKEDA,3 Kyoko TOYOFUKU,3

Jarunya NARANGAJAVANA,2 Akira IKEDA,1,4 and Junji YAMAGUCHI1,†

1Graduate School of Science, Hokkaido University, Kita-ku N10-W8, Sapporo 060-0810, Japan2Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road,Bangkok 10400, Thailand3Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-0860, Japan4CREST, JST (Japan Science and Technology Corporation)

Received August 29, 2002; Accepted October 15, 2002

cDNA of a monosaccharide transporter in rice,OsMST5 (Oryza sativa monosaccharide transporter 5)was cloned and its sugar transport activity was charac-terized by heterologous expression analysis. The aminoacid sequence and topology were similar to the se-quences and topology of other plant monosaccharidetransporters. Yeast cells co-expressed with OsMST5cDNA transported some monosaccharide substrates.The transport rate increased when ethanol as an electrondonor was added, so the transporter was an energy-dependent active one. Most of the OsMST5 was ex-pressed in panicles before pollination, indicating that itis associated with pollen development in rice.

Key words: energy-dependent active transport; heter-ologous yeast expression; Oryza sativa L.;pollen development; sink cell

As much as 80z of the carbon assimilated duringphotosynthesis is exported from the leaf for themetabolic needs of nonphotosynthetic cells. A cen-tral feature of this resource-partitioning step isphloem loading, and in many plants this process de-pends on an active sugar transporter. Two families ofplant sugar transporters have been identiˆed: sucrose(or disaccharide) transporters and monosaccharide(also either glucose or hexose) transporters. A num-ber of sucrose and monosaccharide transportersrelated to several biological processes during plantgrowth and development have been analyzed.1) Sever-al sink cells can derive sucrose and other photoas-similates by direct access through symplastic con-nections to the phloem. However, other sinks aresymplastically isolated; therefore, sucrose is eitherimported directly from apoplasts via a sucrose trans-porter or taken up by a monosaccharide transporterafter the sucrose is hydrolyzed to glucose and fruc-tose by cell-wall-bound invertases.2)

Various plant monosaccharide transporters havebeen cloned and characterized by heterologous ex-pression in yeast cells.3–5) The expression pattern ofgenes encoding monosaccharide transporters suggest-ed that the transporters are involved in hexose uptakein sink tissue4) and are highly regulated after patho-gen infection or wounding.6) In that is pollen graindeveloping, sinks need carbohydrates for matura-tion, germination, and growth1) and monosaccharidetransporters seem to be in this physiological task. In-deed, genes for Pmt1 and AtSTP2, monosaccharidetransporters in petunia and Arabidopsis thaliana, areup-regulated after pollen mitosis and are involved inthe growth of pollen tubes.7–8) Male gametophytesand pollen grains have no intercellular connections(plasmodesmata) to sporophytic tissue, so the uptakeof nutrients into the cells is exclusively dependent onan external supply of nutrients. Import of carbonand nitrogen is necessary for development of pollengrains in anthers. Pollen thus seems to be an idealsubject in studies of the role and regulation of nutri-tion transport.9)

In rice, sucrose transporters OsSUT110–13) andOsSUT214) as well as monosaccharide transportersOsMST1, 2, and 315) have been cloned. Of monosac-charide transporters in rice, only OsMST3 has beenfound to have a physiological role involving the ac-cumulation of monosaccharides for cell-wall synthe-sis during cell thickening in vegetative organs.15)

Other than OsSUT2, there have been no reportssince 2000 about such aspects of rice; that is a wideˆeld. Here, we investigated sugar transport duringrice ‰owering and grain developing. The monosac-charide transporter OsMST5 and its correspondingcDNA were cloned.

557557New Functional Monosaccharide Transporter in Rice

Materials and Methods

OsMST5 clone. OsMST5 cDNA was obtainedfrom the Rice Genome Research Program (GenBankaccession number X55350; full-length clone). ThecDNA sequence was identiˆed by dideoxy-chain ter-mination with an ABI373A DNA sequencer (Perkin-Elmer Co., NJ).

Alignment of sequences. A phylogenetic tree (notshown) of monosaccharide transporters from yeasts,mammalian species, and plant species other than ricewas prepared on the basis of the deduced amino acidsequences, in a comparison of OsMST5 with otherknown transporters.

Functional analysis of OsMST5 in yeast cells. Fortesting of the function of OsMST5, an expressionplasmid was constructed with a GAL expression sys-tem in multicopy plasmid pTV3e.16) LBY416 (MATahxt2::LEU2 snf 3::HIS3 gal2 lys2 ade2 trp1 his3 leu2ura3), a mutant strain of Saccharomyces cerevisiae inwhich high-a‹nity glucose transport activity is lowbecause the monosaccharide transporter-relatedgenes HXT2, GAL2, and SNF3, have been interrupt-ed was used as the heterologous expression system. Aplasmid with the open reading frame of OsMST5 in-troduced to replace that of GAL2 in a pTV3e cassettevector between the EcoRI and ClaI sites was in-troduced into LBY416. OsMST5 is expressed underthe control of the GAL2 promoter in the presence ofgalactose.16) The transport activity of subclonedcDNA was compared with that of another monosac-charide transporter in rice, OsMST3 (see Fig. 3),because this other transporter had more activity thanOsMST1 and OsMST2.

Transport activity assay. The monosaccharides in-cluding D-glucose, 3-O-methyl glucose (3-OMG); thenonmetabolizable substrate analogs for D-glucose,and D-xylose were used as transport substrates toanalyze transport activity and for estimation of theenergy dependence of the transport system ofOsMST5. Uptake of glucose and other monosaccha-rides were assayed by procedures described previous-ly.17) Yeast cells were grown to an OD650 of 0.2–0.4 ina synthetic medium containing 2z galactose. Cellswere collected by centrifugation, washed three timeswith a medium for transport assays: 50 mM 2-(N-morpholino)ethanesulfonic acid in NaOH (pH 6.0)containing 2 mM MgSO4. Uptake assays were startedby the addition of 20 ml of radiolabeled sugars to180 ml of a cell suspension, and stopped by the addi-tion of transport assay medium containing 0.5 mM

HgCl2 in stead of 2 mM MgSO4. After incubation at309C, cells were collected by ˆltration under reducedpressure onto a glass ˆber ˆlter (GFWF, Whatman),

and washed with 20 ml of cold assay solution con-taining HgCl2. The radioactivity retained in the ˆlterwas measured by liquid scintillation counting. Theinitial rate of glucose transport was assessed bythe transport of 0.1 mM D-[U14C]glucose (0.5 mCi,CFB96; Amersham Pharmacia Biotech), or D-[U14C]xylose (0.5 mCi, CFB96; Amersham), or 3-O-methyl D-[U14C]glucose (0.5 mCi, CFB96; Amer-sham), for 5 s to 25 min at 309C. Most assays weredone in three or four independent experiments.

Substrate speciˆcity. The substrate speciˆcity ofOsMST3 and 5 expressed in LBY416 yeast cells wasstudied by the addition of several nonradioactive su-gars, added to activated cells 1 min before the addi-tion of [14C]D-glucose. The starting concentration ofthe unlabeled sugar and labeled D-glucose added inthe medium was 0.1 mM.

RNA extraction and northern bloting. Because theOsMST5 clone obtained from the Rice GenomeProject was isolated from a panicle cDNA library,Northern bloting was done to detect OsMST5 mR-NAs in several stages during ‰owering RNA was ex-tracted from panicles at various stages with the aurintricarboxylic acid method of Skadsen,18) with minormodiˆcations.19) Total RNA (15 mg) of a sample waselectrophoresed on formaldehyde gel and blottedonto a nylon membrane (Hybond N+; Amersham).Membranes were hybridized at 659C in PerfectHybhybridization solution (Toyobo, Osaka, Japan). Aradiolabeled probe was prepared from gel-puriˆedcDNA fragments (5-end of OsMST5, 1200 bp) byrandom primer labeling with a-[32P]dCTP. We con-ˆrmed that there was no cross-hybridization with thisradiolabeled probe by Southern bloting (not shown).Hybridization and washing were done by the pro-tocol of the manufacturer. Membranes were exposedwith a Fujix BAS2000 Bio-Imaging Analyzer (FujiPhoto Film Co., Ltd., Tokyo, Japan).

Results

OsMST5, a monosaccharide transporter in riceThe putative amino acid sequence of OsMST5 was

30.5–66.2z identical to the sequences of OsMST1, 2,and 315) (Fig. 1). OsMST5 was 518 amino acids long,with a calculated molecular mass of 55.9. Analysis ofthe deduced amino acid sequence by TMHMM (Ver-sion.2.0)20–21) to predict transmembrane helices in theOsMST5 protein gave two sets of putative transmem-brane domains separated by a central long hydrophil-ic region (Fig. 2A). The ˆrst set was ˆve domains andone putative domain inside the cell and the second setwas of six domains. The amino acid sequence of thethird putative domain in the ˆrst set was almost iden-tical to one reported previously as the third trans-membrane domain in OsMST1, 2, and 3.

558

Fig. 1. Amino Acid Aequences of OsMST1, 2, 3, and 5 Alignedwith Monosaccharide Transporters of Other Organisms.

The alignment of the predicted amino acid sequences ofOsMST1 (D251429), 2 (D46606), 3 (D40232), and OsMST5(X55350) with the sequence of RcHEX6 (L08188) from casterbean, SopGlcT (AF215851) from spinach, and GLUT1(KO3195) from humans is shown. Black boxes indicate identicalamino acid residues. Asterisks below the sequences indicate con-served residues. Putative transmembrane domains are under-lined. Multiple-sequence alignment was constructed by theDNASIS-Mac program, version.3.7 (Hitachi SoftwareEngineering Co., Ltd., Yokohama, Japan).

558 B. NGAMPANYA et al.

Alignment of sequencesA phylogenetic tree (not shown) of monosaccha-

ride transporters from yeasts, mammalian species,and plant species other than rice, in a comparison ofOsMST5 with other known transporters showed a lit-tle diŠerent in transporters of monocots and dicots.However, SopGlcT, the plastidic glucose transloca-tor in spinach,22) and GLUT1, from human were verydiŠerent from the plant transporters (see Fig. 1). Theconserved motifs in sugar transport proteinsproposed by Henderson et al.23) were found inmonosaccharide transporters of rice OsMST1, 2, 3,and 5, caster bean (RcHEX6), spinach (SopGlcT),and human (GLUT1). Many of the amino acidresidues that are motifs of sugar transporters (indi-cated by asterisks in the ˆgure) were conserved inOsMST5.

Functional analysis of OsMST5 in yeast cellsThe glucose transport activity of OsMST5 was

about one third that of OsMST3 and nearly twicethat of the empty vector, pTV3e (Fig. 3). Transportactivity was not found after the addition of the SH-group inhibitor HgCl2. The empty vector pTV3e hadtransport activity even in the absence of an inhibitorbecaused of the activity of a minor sugar transpor-ter(s).

Energy for sugar uptake of OsMST5Transport activity for d-glucose (Fig. 4A) was

approximately 8-fold and 14-fold, that of 3-OMG(Fig. 4B) and D-xylose (Fig. 4C). With the empty vec-tor, pTV3e, the uptake level of D-xylose remainedlow level during the 5-min tracing with or withoutethanol (Fig. 4D). Uptake upon energization, startedby the addition of ethanol, was observed with allthree substrates. LBY416 yeast cells harboring thepTV3e vector could not take up D-xylose, andshowed a background level of transport. This ˆndingwas in contrast to those of cells containing OsMST5,which could take up D-xylose rapidly when ethanolwas added (Fig. 4B and Fig. 5). This energization ofthe plasma membrane of yeast cells decreased whenthe uncoupler CCCP, an electron transport inhibitor,was used (Fig. 5). The energy generated by ethanolallowed a high rate of uptake of D-xylose into thecells, where it was rapidly metabolized. With CCCPact as a plasma membrane de-energization, the trans-port of D-xylose was decreased.

Kinetic propertiesMSTs generally have broad substrate speciˆcity,

transporting a range of hexoses and pentoses with Km

values for the substrates being typically 10–100 mM.The Km values for glucose transport of OsMST3 andOsMST5 were 0.3 and 0.5 mM, respectively. TheVmax values were 450 and 471 pmolW107 min„1 forOsMST3 and OsMST5, respectively.

559

Fig. 2. Hydropathy Proˆle and a Membrane-spanning Model of OsMST5.Calculation was done by an algorithm published elsewhere.20) Bold, lower, and upper line-blocks in the top indicate the transmem-

brane domain, peptides facing the cytoplasm inside, and peptides facing to the outside of the cell, respectively. Arrow indicates the thirdputative transmembrane.

Fig. 3. Glucose Transport in Yeast Cells with OsMST3 and 5.OsMST3 and 5 were expressed in S. cerevisiae with the GAL2

promoter in a multicopy plasmid, pTV3e, in LBY416 cells. Theexperiment done with the vector pTV3e shows value for trans-port. The ˆnal substrate concentration was 0.1 mM in all experi-ments. The transport activity in the presence of 0.5 mM HgCl2 isshown as ``+HgCl2''.

Fig. 4. Transport of D-Glucose, 3-O-Methyl Glucose (3-OMG)and D-Xylose in Yeast Cells with OsMST5 (A, B, and C), andTransport of D-Xylose in Cells with the Only Vector (D).

Transport in yeast cells with additional energization by100 mM ethanol or without ethanol in the presence of 0.5 mM

HgCl2 or without HgCl2 is shown.


Substrate speciˆcityWith the OsMST3 transporter, the order of inhibi-

tion strong inhibition ˆrst was glucose, galactose,sucrose, xylose, fructose, and, interestingly, fruc-tose, although the order for the last three sugars maybe interchangeable. Galactose inhibited glucose byabout 17z and glucose by about 30z (Fig. 6A).With OsMST5, none of the sugar including glucosecaused inhibition (Fig. 6B).

Expression of OsMST5A strong signal was detected only from panicles be-

fore heading; signals were not detected in develop-mental stages of rice seeds (Fig. 7). Signals not being

detectable in the other tissues and organs tested, em-bryo-derived suspension cultured cells, leaf bladesbefore and after heading, leaf sheaths, roots, dryseeds, and seedlings by Northern bloting (not shown)suggested that this monosaccharide transporter wasassociated with ‰ower development.

Discussion

OsMST5 as a functional monosaccharide trans-porter in rice

The hydropathy proˆles for all plant monosaccha-

560

Fig. 5. EŠects of Ethanol and an Uncoupler (CCCP) on D-Xylose Transport in Yeast Cells with OsMST5.

The starting concentration of D-xylose in the medium was0.1 mM, and the ˆnal concentration of CCCP in the medium was50 mM. Arrows indicate the times of energization with the addi-tion of ethanol to100 mM or CCCP.

Fig. 6. Inhibition of Glucose Uptake of Yeast Cells withOsMST3 (A) or OsMST5 (B) by DiŠerent Sugars, Added to Ac-tivated Cells 1 min Before the Addition of D-Glucose [14C].

The starting concentrations of each unlabeled sugar andlabeled D-glucose added were both 0.1 mM.

Fig. 7. Northern Bloting of OsMST5 mRNA.Total RNA (15 mg) of a sample was electrophoresed on a for-

maldehyde gel and blotted onto a nylon membrane, and hybri-dized with a radiolabeled cDNA probe. After the membrane waswashed, it was examined with a bio-imaging analyzer. Stainingin the rRNA panel was with ethidium bromide. DAP, days afterpollination.


ride transporters are similar and contain 12 mem-brane-spanning domains. The transporters arethought to be members of a major facilitator super-family.24) The ˆrst clone of full-length cDNA of themonosaccharide transporter gene from rice wasreported by Toyofuku et al.15) The OsMST5 reportedhere was a new isolated clone of a rice monosaccha-ride transporter. OsMST5 maybe a membrane-span-ning protein with 12 transmembrane domains region,which is consistent with the structures of other sugartransporters of microbes, mammals, and plants.23)

However, OsMST5 might be unusual membrane pro-tein in having 11 transmembrane domains.

With the empty vector, pTV3e, the uptake level ofD-xylose remained low level during the 5-min tracingwith or without ethanol (Fig. 4D); S. cerevisiae cellsare unable to take up and metabolize D-xylose.26) Inyeast cells, when the substrate cannot be metabo-lized, added ethanol seems to serve as the electrondonor for the electrogenic transmembrane transportof monosaccharides.4) As mentioned above, ethanoland CCCP seem to act as an electron donor and elec-tron transport inhibitor, respectively.15) Results givenhere were consistent with an uptake mechanismcoupled with protons. This ˆnding suggested thatOsMST5 was an energy-dependent active transporterdependent monosaccharide transport system, possi-bly as an H+-symporter similar to previously identi-ˆed plant monosaccharide transporters25–28) and tothe rice monosaccharide transporter OsMST3.15)

However, the Km values for glucose transport byOsMST5 was higher than the Km values for thepreferred substrates of typical monosaccharide trans-porters. Perhaps the diŠerent substrates account forthe diŠerent Km values. The degrees of inhibitionwere less with OsMST5 than OsMST3, perhaps be-cause of lower a‹nities for substrates of the OsMST5


transporter.

Location and function of OsMST5Sugar transporters are found in various parts of

plant cells and tissues. The expression pattern of thetransporters re‰ect their physiological tasks.1) In rice,OsMST3 is to be found in leaf blades, leaf sheaths,calli, and sclerenchyma and xylem cells in the youngroots.15) The abundant of mRNA in sclerenchymaand xylem cells indicated that OsMST3 is involved inthe thickening of cell walls. The sequence of OsMST5was similar to that of OsMST3, even when they werein diŠerent organs. OsMST5 mRNA was detected inpanicles before heading. Bate and Twell29) proposedthat the sequences AGAAA and TCCACCATA inthe promoter region of the lat52 gene in tomato areneeded to code proteins expressed in pollen. Wefound the AGAAA sequence in 10 places and a se-quence similar to TCCACCATA in a single place inthe OsMST5 promoter (not shown). In addition,both sequences were detected in OsSUT2, which ricesucrose transporter is expressed only in the develop-ing pollen.14) These results suggested that OsMST5was a pivotal in early ‰ower development, togetherwith OsSUT2. Monosaccharide transporter involvedin pollen development in petunia and Arabidopsishave been examined.7,8) AtSTP2 and PhPMT1 are ex-pressed after meiosis in pollen mother cells. The im-ported glucose (or carbohydrates) may need develop-ment of uninucleate microspores after supply ofenergy for pollen germination and pollen tubegrowth in those plant species. Takeda et al.14) men-tioned that the OsSUT2 isolated from rice paniclecause the in‰uxe of sucrose into developing pollenfor starch synthesis. Hence, OsMST5 might be asso-ciated with these developmental processes togetherwith OsSUT2. However, additional sugar transport-ers may be involved in these physiological tasks.

Acknowledgments

We thank Dr. M. Kasahara of Teikyo Universityfor providing us with a yeast expression system andDr. T. Sasaki of the Japanese Rice Genome Programfor providing us with expressed sequence tag clones.BN acknowledges a UDC scholarship supported bythe Ministry of University AŠairs, Thailand. Thiswork also was supported by a Research for theFuture grant (JSPS-00L01603) from the Japan Soci-ety for the Promotion of Sciences and CREST ofJapan Science and Technology.

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characterization of rice functional monosaccharide

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