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Characterization of OsSUT2 Expression and Regulation in Germinating Embryos of Rice Seeds

Characterization of OsSUT2 Expression and Regulation in Germinating Embryos of Rice Seeds Rice (2011) 4:39–49 DOI 10.1007/s12284-011-9063-1 Characterization of OsSUT2 Expression and Regulation in Germinating Embryos of Rice Seeds Wei Siao & Jia-Yi Chen & Hui-Hsin Hsiao & Ping Chung & Shu-Jen Wang Received: 15 July 2011 /Accepted: 9 September 2011 /Published online: 21 September 2011 Springer Science+Business Media, LLC 2011 Abstract OsSUT2 encodes a putative sucrose transporter MU 4-Methylumbelliferone containing 12 transmembrane domains in rice plants. 3-OMG 3-O-Methylglucose Subcellular localization of the OsSUT2::GFP fusion protein Pal Palatinose indicated that OsSUT2 is a cell membrane protein. In QRT-PCR Quantitative real-time reverse transcriptase embryos of germinating seeds, the expression of OsSUT2 polymerase chain reaction gradually increased during the early germinating stage. The Sc Scutellum cells developmental regulations of OsSUT2 in germinating Suc Sucrose embryos could be mediated by sugars transported from SUT Sucrose transporter endosperms. OsSUT2 expression was up-regulated by Ubi Ubiquitin glucose through a hexokinase-independent pathway. Exoge- V Vascular bundle nous sucrose was sensed by a sensor localized on the plasma membrane and functioned as an enhancer to promote OsSUT2 expression. Based on OsSUT2 promoter::GUS expression in germinating seeds of transgenic rice, OsSUT2 was signifi- cantly expressed in the embryos and aleurone layers. In Introduction embryos, strong GUS expression was detected in the scutellum and vascular bundle tissues. Developmental In plants, carbohydrates translocate from the source to stage- and sugar-dependent OsSUT2 expression was sug- sink tissues through symplastic and apoplastic pathways. gested to be controlled by transcriptional regulation of the Sugar transport between cells in symplasts is mediated promoter region. by plasmodesmata. In apoplasts, sugar translocators are responsible for carbohydrate uptake into cells. Because . . . Keywords Embryo Oryza sativa Seed germination sucrose is the major transported form of carbohydrate Sucrose transporter between plant tissues, sucrose transporter (SUT) plays an important role in long-distance disaccharide transport. Abbreviations SUT1 in sugarcane functions to partition sucrose between the DAI Days after imbibition vascular bundle and storage cells (Rae et al. 2005). Maize Glc Glucose SUT, ZmSUT1, is responsible for sugar uptake into phloem GUS β-Glucuronidase in source tissues and sugar unloading from phloem into cells Man Mannitol in sink tissues (Carpaneto et al. 2005). Changes in carbohydrate allocation and the inhibition of photosynthesis have been observed in transgenic plants with reduced SUT : : : : W. Siao J.-Y. Chen H.-H. Hsiao P. Chung S.-J. Wang (*) activity due to antisense SUT genes; thus, SUT has been Department of Agronomy, National Taiwan University, indicated to play an important role in carbohydrate partition- No. 1, Section 4, Roosevelt Rd., ing and physiological processing (Kühn et al. 1996; Bürkle Taipei 106, Taiwan et al. 1998). e-mail: shujen@ntu.edu.tw 40 Rice (2011) 4:39–49 To date, SUT genes have been identified from several In the present study, we identified the subcellular plants, most belonging to gene families (Lemoine 2000; localization of OsSUT2. The expression of OsSUT2 in Lalonde et al. 2004). The SUT gene families of dicot and germinating embryos was detected by real-time quantitative monocot species were classified into five sequence-based RT-PCR. The spatial and temporal transcriptional activities groups by Braun and Slewinski (2009). In rice, the SUT of the OsSUT2 promoter were detected in the germinating gene family consists of five genes, OsSUT1 to OsSUT5 seeds of transgenic rice plants carrying the OsSUT2 (Aoki et al. 2003). OsSUT1 and 3 were classified into promoter::GUS fusion gene. Furthermore, the signal trans- group 1. The group 1 SUT family is composed of several duction of sugars for regulating OsSUT2 expression was Poaceae SUT members; so far, no dicot SUTs have been examined and discussed. found in group 1. OsSUT2 was classified with Arabidopsis SUT4 (AtSUT4) in group 2. AtSUT4 has been demonstrated to be responsible for the low-affinity/high-capacity trans- Materials and methods port system (reviewed by Lalonde et al. 2004). OsSUT4 and OsSUT5 were classified into group 3 and 5, respectively. Plant materials and treatment During plant development from seed germination to seed establishment and seedling growth, starch reserved in the Rice (Oryza sativa L. cv. Tainung 67) seeds were obtained seed is the primary carbon and energy source for sprouting from the Hualien District Agricultural Research and and early seedling establishment, and then the sugar Extension Station in Taiwan. For germination, seeds were sources for later stages of seedling growth come from the sterilized in 2.5% sodium hypochlorite with Tween 20 for photosynthetic shoots. In rice plants, carbohydrate transport 20 min and subsequently washed with distilled H O four from germinating seeds to other developing sink tissues is a times. Seeds were then germinated at 37°C in the dark for fundamental process for plant development and growth. 3 days and then moved to the phytotron for growing at 30/ Sugar derived from starch degradation exported to the 25°C under natural daylight. To analyze the sugar content cytosol from storage organelles is the first step for long- and OsSUT2 expression in embryos, the growing shoots distance carbohydrate translocation. In cereal endosperms, and roots were cut and the embryos isolated after 1- to 5- starch granules are attacked by α-amylase (Murata et al. day seed imbibition. 1968), and the maltose and linear/short-branch-chain To isolate embryos from dry seeds, the grain hulls were oligosaccharides are generated for further degradation by removed by machine and the embryos picked by razor α-glucosidase (Stanley et al. 2011). The hexoses produced blade. The isolated embryos were sterilized in 0.25% by starch degradation can be taken up by the scutellum cells sodium hypochlorite for 10 min and subsequently washed of the embryo. Hexoses have been suggested to be re- with distilled H O four times (Matsukura et al. 2000). For synthesized into sucrose in the scutellum (Edelman et al. isolated embryo culturing, the embryos were placed on MS 1959; Nomura et al. 1969). Sucrose in the scutellum is medium and incubated at 28°C in dark or light. For sugar loaded into the vascular bundle, transported, and then and sugar analog treatments, the chemicals were added in unloaded to growing tissues through apoplastic or sym- MS medium. The concentration of all sugars and sugar plastic pathways (Aoki et al. 2006). In addition, it was also analogs used in this study was 100 mM. suggested that sucrose transported from aleurone layers to endosperm at early germination stage could be further Homology analysis of amino acid sequences uptaked by scutellum (Aoki et al. 2006). Based on our and transmembrane domain prediction previous work (Liu et al. 2010), soluble sugar can also be converted to starch in scutellum or the cells surrounding Multiple sequence alignments of SUT amino acid sequen- vascular bundles at the post-germination stage, and the ces were carried out using SDSC Biology WorkBench 3.2 amount of transitory starch in embryonic tissues was (http://workbench.sdsc.edu/). The transmembrane domains dependent on the demand of growing sink tissues. Phloem on the OsSUT2 amino acid sequence were predicted functions as an important pathway for carbon source according to the Hidden Markov Models using TMMOD transport among various plant tissues. In rice germinating software (Kahsay et al. 2005). seeds, OsSUT1 was the first OsSUT family member identified to have a tissue-specific expression pattern RNA extraction (Hirose et al. 1997; Scofield et al. 2007). Developmental expression and regulation of OsSUT1 has been observed in Embryos isolated from ten seeds or harvested from medium germinating seeds (Chen et al. 2010). However, the were ground in liquid nitrogen, homogenized in 1 mL Trizol regulation of other members of the OsSUT gene family reagent (Invitrogen, Carlsbad, CA, USA), and centrifuged at still needs to be established. 8,000×g. The supernatant was treated with 0.2 mL chloro- Rice (2011) 4:39–49 41 form, shaken for 15 s, and incubated at room temperature for embryos 5 days after imbibition (DAI) and RT-PCR was 3 min. After centrifugation at 12,000×g for 15 min at 4°C, performed using specific primers. The forward primer (5′- the upper layer was transferred to a new tube. RNA was TTCTAGAATGCCGCGGCGGCCTAGGCGG-3 ′) precipitated with 0.5 mL isopropanol and incubated for contained the XbaI cloning site sequence, the reverse 10 min at room temperature. After centrifugation, the pellet primer (5′-AGGATCCATCGGTGACCTCTCCTCCTTG- was dissolved in 0.2 mL H O. Before the gene expression 3′) contained the BamHI cloning site, and the stop codon analysis, the total RNA extracted from the embryos was sequence was not included. OsSUT2 cDNA was cloned in- treated with DNase to remove contaminating genomic DNA. frame with the N-terminus of the green fluorescent protein (GFP) gene, driven by a CaMV35S promoter in a transient Quantitative real-time reverse transcriptase-PCR expression vector. The OsSUT2-GFP fusion construct was transiently expressed in barley aleurone layer cells Total RNA (200 ng) was used as the template for quantitative mediated by a He Biolistic particle delivery system (model real-time RT-PCR analysis using the Brilliant SYBR Green PDS-1000, Bio-Rad). Half-de-embryonated barley seeds were QRT-PCR Master Mix (Stratagene, La Jolla, CA, USA), and sterilized before soaking in a shooting buffer (20 mM Na– PCR reactions were performed using a Multiplex 3000P Real- succinate and 20 mM CaCl ,pH 5.0) for 48 h at 24°C in the Time PCR System (Stratagene). The gene-specific RT-PCR dark (Mena et al. 2002). Next, the pericarp was removed primers are listed on Table 1. RT-PCR was carried out as from the seeds and the aleurone layer exposed for bombard- follows: 50°C for 30 min and 95°C for 10 min, followed by ment. After bombardment, the seeds were incubated with 40 cycles of 95°C for 1 min, 58°C for 1 min, and 72°C for shooting buffer for 24 h at 24°C. Finally, fluorescence 1 min. To quantify relative gene expression levels accurately, localization in the aleurone layer cells was observed using an the C values of OsSUT1 to 5 were normalized to the C AXIO Imager M1 fluorescence microscope (Carl Zeiss, T T value of the ubiquitin (Ubi) gene. For all real-time RT-PCR Germany). analyses, three independent experiments were carried out, and the data are presented as mean±SD. Promoter cloning Sugar analysis Rice genomic DNA was extracted from the leaf tissues with plant DNA reagent (Invitrogen). The OsSUT2 DNA ZOL Ten embryos were ground to powder in liquid nitrogen and fragment upstream of the translation start site, located from extracted with 1 mL of 80% (v/v) ethanol at 80°C for 5 min −830 to −1 bp, was amplified by PCR using specific before centrifugation at 3,000×g. The supernatant was primers (forward primer with SacI site: 5′-GAGCTCT analyzed for glucose and sucrose following the method TAAGGAGCACCAA-3′; reverse primer with SmaI site: described by Spackman and Cobb (2002). 5′-CCCGGGCTTCTTCTCGTGTT-3′). Putative cis-ele- ments on the OsSUT2 promoter sequence were character- Subcellular location of OsSUT2 ized by searching for similar motifs in the Database of Plant Cis-acting Regulatory DNA Elements (PLACE) (Higo et al. The coding region cDNA of OsSUT2 was cloned from rice 1999). To generate the plasmid for the promoter activity embryos using RT-PCR. Total RNA was extracted from assay in transgenic rice plants, the OsSUT2 promoter Table 1 Primer pairs for real- Gene Accession number Primer pair Amplicon size (bp) time RT-PCR (F: forward primer; R: reverse primer) OsSUT1 D87819 F: 5′-CTGTGATTTTCCTGTCCCTG-3′ 136 R: 5′-AACACTGCTAGTGGACCAGT-3′ OsSUT2 AB091672 F: 5′-AGGAGGAGAGGTCACCGATAA-3′ 240 R: 5′-CCAACATCCAATGTACAACAGCA-3′ OsSUT3 AB071809 F: 5′-GCCCAAGGTCTCCGTCC-3′ 137 R: 5′-TGCTATAGTACCCGCTCTAA-3′ OsSUT4 AB091673 F: 5′-TTTGGCTGAGCAGAACACCA-3′ 249 R: 5′-ATGTCATTCGGGCAGAGCTT-3′ OsSUT5 AB091674 F: 5′-CTAGTGCGAAACTCCATCAAA-3′ 249 The nucleotide sequences used R: 5′-AAAATATTTGGGTTTCCTGAGAT-3′ for primer designing were pre- Ubi D12629 F: 5′-CGCAAGTACAACCAGGACAA-3′ 101 sented by their accession numbers R: 5′-TGGTTGCTGTGACCACACTT-3′ in GenBank 42 Rice (2011) 4:39–49 (−830/−1) was inserted into the SacI and SmaI sites of the Results pCHY10 vector (a gift from Dr. Chwan-Yang Hong), which contained the first intron of the Ubi gene fused to the β- Subcellular localization of OsSUT2 protein glucuronidase (GUS) reporter gene. We used restriction enzymes to isolate the DNA cassettes containing the To characterize the protein structure and identify the OsSUT2 promoter fragment with the Ubi intron and GUS subcellular localization of OsSUT2 protein, the OsSUT2 gene from the pCYH10 constructs. These cassettes were coding sequence was amplified by RT-PCR. The OsSUT2 then ligated into the SacIand HindIII sites of the cDNA (accession no. HQ875341) encodes a protein of 501 pCAMBIA1302 plasmid. amino acids in length. According to membrane protein topology prediction using Hidden Markov Models in Generation of transgenic rice plants TMMOD software (Kahsay et al. 2005), the OsSUT2 protein contains 12 transmembrane domains (Fig. 1a, b). To produce OsSUT2 promoter::GUS transgenic plants, the The amino- and carboxyl-terminal sequence tails predicted constructed pCAMBIA1302 plasmid was transformed into a cellular localization (Fig. 1b). Based on a previous Agrobacterium tumefaciens EHA105. The cultured A. phylogenetic analysis of the SUT gene family by Braun tumefaciens harboring the constructed plasmids were used and Slewinski (2009), OsSUT2 was grouped with Arabi- to infect rice embryo-derived calli. The transformed calli dopsis AtSUT4. Furthermore, Kühn and Grof (2010) were selected on medium containing 50 μg/mL hygromycin showed that AtSUT4 is also classified with StSUT4 and and 250 μg/mL cefotaxime. Finally, the transgenic rice LeSUT4 in the same group. The amino acid identities plants were regenerated from the transformed calli (Hiei et between OsSUT2 and StSUT4, LeSUT4, and AtSUT4 are al. 1994; Toki 1997). 66%, 66%, and 64%, respectively. The significant differ- ences within the above SUT genes are at the amino Histochemical GUS assay terminus and central inside loop (Fig. 1a). In addition, the central inside loop of OsSUT2 (35 amino acids in length) is Histochemical GUS activity assays were performed as smaller than that of LeSUT2 (94 amino acid residues) and described previously (Jefferson 1987). Half-cut germinated AtSUT2 (87 amino acid residues), which belong to the seeds from transgenic plants were placed in a solution other SUT group (Fig. 1b). To determine the subcellular containing 10 mM EDTA, 0.5 mM potassium ferricyanide, localization of OsSUT2 protein, the expression of OsSUT2- 0.5 mM potassium ferrocyanide, 1.0 mM 5-bromo-4- GFP fusion protein was observed in the aleurone layer cells chloro-3-indolyl-β-D-glucuronide, 0.1% Triton X-100, and of barley seeds. Fluorescence imaging showed that the 0.1 M sodium phosphate buffer (pH 7.0) and incubated at fusion protein was localized on the plasma membrane 37°C for 4 h. The staining reaction was stopped by adding (Fig. 2). 75% ethanol. Developmental expression of OsSUT2 in embryos Quantitative GUS activity assay during germination Developmental regulation of the OsSUT2 promoter was To examine the expression of five OsSUT family genes in analyzed in germinating embryos of transgenic rice seeds. the embryos of germinating seeds, rice seeds were Seeds were germinated in water containing hygromycin, germinated in the dark for 3 days and then grown in and embryos were isolated from 3 and 5 DAI. To assay the phytotron with natural sunlight. The expression levels of effect of glucose on OsSUT2 promoter activity, the seeds OsSUT3 and 5 were lower than those of the other three were imbibed in hygromycin-containing water for 3 days OsSUT genes at 1 and 5 DAI (Fig. 3a). Quantitative RT- and the germinated embryos were isolated from transgenic PCR showed that the OsSUT2 transcript level was seeds. The isolated embryos were cultured in MS medium significantly higher than that of other OsSUT genes at 5 containing 100 mM glucose solution for 5 days at 28°C in DAI (Fig. 3a). Among OsSUT1, 2, and 4, only the OsSUT2 the dark. The protruding shoots and roots were excised and mRNA of embryos was increased at 5 DAI compared to 1 discarded before the embryo proteins were extracted with DAI (Fig. 3a). Furthermore, the expression of OsSUT2 was the buffer containing 100 mM sodium phosphate (pH 7.0), observed to gradually increase during the early germination 5 mM dithiothreitol, 20 μg/ml leupeptin, and 20% (v/v) stage, and the transcript level at 5 DAI was 4.6-fold that of glycerol. Next, 4-methylumbelliferyl β-D-glucuronide was dry seed embryos (Fig. 3b). In addition, the expression of added as a substrate and GUS activities were measured OsSUT2 in embryonic tissues increased with growth stage, fluorometrically in the embryos as described previously even seedlings continuously grew in the dark after (Jefferson 1987). germination (Fig. 4a). However, the phenomenon of Rice (2011) 4:39–49 43 Fig. 1 Multiple amino acid sequence alignment and trans- membrane domain analysis of SUT proteins. a Alignment of amino acid sequences from potato SUT4 (StSUT4; Genbank accession AF237780), tomato SUT4 (LeSUT4; AF176950), Arabidopsis SUT4 (AtSUT4; AY072092), and rice OsSUT2 (HQ875341). TM transmembrane domain. b Comparison of SUT protein structures. The inside and outside membrane regions and transmembrane domains of OsSUT2, StSUT4, AtSUT4, LeSUT4, AtSUT2 (AK226970), and LeSUT2 (AF166498) were predicted using TMMOD software. 44 Rice (2011) 4:39–49 Fig. 2 Subcellular localization of OsSUT2 by transient expres- sion of OsSUT2-GFP fusion proteins in barley aleurone layer cells. a Bright field image. b GFP fluorescence image. c Merged field and fluorescence image. OsSUT2 up-regulation during germination was not obvious considered whether the sugar transported from endosperm in dark-cultured isolated embryos (endosperm-free) during seed germination plays a role in the regulation of (Fig. 4c). OsSUT2 expression in embryos. To evaluate this possibil- ity, the levels of glucose and sucrose were analyzed in the Expression of OsSUT2 regulated by exogenous sugars embryos of germinating seeds from 0 to 5 DAI. Glucose levels gradually increased with the days after imbibition, OsSUT2 expression in germinating embryos was different but sucrose content fluctuated (Fig. 5). The changes in from those with and without endosperm status. It was glucose content corresponded to the expression pattern of OsSUT2 transcripts in embryos during seed germination (Figs. 3b and 5). Even for seed germination in the dark, changes in glucose content and OsSUT2 transcript levels were consistent (Fig. 4a, b). In order to determine whether sugars are the factors up-regulating OsSUT2 expression in the embryos of germinating seeds, the embryos isolated from dry seeds were cultured on sugar-containing MS medium in the dark for 5 days. Although glucose (100 mM) and sucrose (100 mM) slightly enhanced OsSUT2 mRNA levels in 1-day sugar-treated embryo samples, the effect was not significant (Fig. 6a). In 5-day cultured embryos, the OsSUT2 expression was obviously up-regulated by both glucose and sucrose, but not by the same concentration of mannitol (Fig. 6b). Sugar analogs were applied to study the sugar-sensing pathway for regulating OsSUT2 expression. 3-O-Methylglu- cose (3-OMG), a nonmetabolizable glucose analog, was taken up by cells but was not phosphorylated by hexokinase (Dixon and Webb 1979). The results showed that 3-OMG (100 mM) had the same effect as glucose (100 mM) to enhance OsSUT2 expression (Fig. 7a). Palatinose, a non- metabolizable sucrose analog, cannot be imported into plant cells (Bouteau et al. 1999). The effect of palatinose (100 mM) on OsSUT2 gene expression was similar to that of sucrose (100 mM) (Fig. 7b), suggesting that embryo cells sense sucrose signals to regulate OsSUT2 expression through a membrane sensor. Fig. 3 Developmental expression of rice OsSUT in the embryos of OsSUT2 promoter::GUS expression in embryos germinating seeds. a Comparison of the expression of five OsSUT genes in the embryos of 1-DAI and 5-DAI seeds. b Changes in Transgenic rice plants harboring the OsSUT2 promoter:: OsSUT2 expression in embryos during seed germination. The data are presented as mean±SD. GUS construct was used to investigate the spatial expres- Rice (2011) 4:39–49 45 Fig. 5 Changes in the glucose and sucrose content of embryos during seed germination. The data are presented as mean±SD. Glc glucose, Suc sucrose. vascular tissues and scutellum cells of embryos (Fig. 8). In addition, the quantitative GUS activity assay showed that the GUS activity in embryos from 5-DAI seeds was significantly higher than that of 3-DAI seeds (Fig. 9a). To identify the effect of glucose on OsSUT2 promoter activity, the germinated embryos of transgenic rice seeds were isolated and incubated in 100 mM glucose solution for Fig. 4 OsSUT2 expression and glucose content in embryonic cells from light- and dark-grown seedlings. Rice seeds were germinated in the dark for 3 days. Some germinated seeds were moved to a phytotron with natural daylight for 2 days (labeled 3-day D/2-day L), and some germinated seeds were kept in the dark (labeled 5-day D) for growth. The seedlings were collected at 1 and 5 DAI, and the embryos were isolated for OsSUT2 expression analysis (a) and to measure glucose content (b). c The OsSUT2 expression in germinating isolated embryos was analyzed. The embryos were dissected from dry seeds and dark-grown in MS medium. OsSUT2 expression was detected after 1, 3, and 5 days of culture. The data are presented as mean±SD. sion of OsSUT2 in rice embryos. Rice seeds of three independent transgenic lines were germinated in water, and Fig. 6 Effects of sugars on OsSUT2 expression. OsSUT2 transcript GUS expression in germinating seeds was observed. GUS levels were determined in isolated embryos cultured in medium activities were performed in aleurone layers and embryos. containing mannitol (Man), glucose (Glc), or sucrose (Suc) for 1 day (a) and 5 days (b). The data are presented as mean±SD. In embryos, significant GUS staining was detected in the 46 Rice (2011) 4:39–49 Fig. 7 Sugar-sensing pathways for OsSUT2 gene regulation in germinating embryos. a Effect of glucose analog 3-O-methylglucose (3-OMG)on OsSUT2 expression after 5 days of treatment. b Effect of Fig. 8 Histochemical localization of β-glucuronidase activity in the the sucrose analog palatinose (Pal)on OsSUT2 expression after 5 days embryos of transgenic rice plants harboring the OsSUT2 promoter:: of treatment. The data are presented as mean±SD. GUS constructs. The seeds of transgenic rice lines 13, 22, and 32 were germinated in the dark for 3 days, and then the GUS activity was analyzed in half-cut germinated seeds. Sc scutellum cells, V vascular bundle. Bar=1 mm. 5 days. The OsSUT2 promoter activities were obviously enhanced (Fig. 9b). membrane of sieve elements (Reinders et al. 2002). OsSUT2 contains 12 transmembrane domains and was Discussion localized on plasma membrane. Amino acid alignment showed that the number of amino acids in the central inside SUT proteins are important carriers for transporting sucrose loops are similar among OsSUT2 and other SUTs in group across the plasma membrane or vacuolar membranes 4 (according to the classification by Braun and Slewinski (reviewed by Kühn and Grof 2010). Arabidopsis SUT 2009) (Fig. 1); however, the length of the OsSUT2 central protein has also been found on the chloroplast membrane loop is shorter than that of SUTs belonging to group 2, i.e., (Rolland et al. 2003). Rice OsSUT2 is classified in the AtSUT2 and LeSUT2. LeSUT2 is considered to function as same group with Arabidopsis AtSUT4, tomato LeSUT4, a putative sucrose sensor (Barker et al. 2000). The potato StSUT4, Lotus japonicus LjSUT4, and barley conserved domains in the extended cytoplasmic loop of HvSUT2 (Braun and Slewinski 2009; Kühn and Grof LeSUT2 and AtSUT2 play an important role in signal 2010). Some of the above-mentioned SUTs, including sensing and transduction (Barker et al. 2000). Because the AtSUT4, StSUT4, and LjSUT4, have been identified as lengths and amino acid sequences of the central loops are low-affinity/high-capacity transporters (Weise et al. 2000; significantly different between OsSUT2 and LeSUT2, the Reinders et al. 2008). In addition, AtSUT4, LjSUT4, and regulatory mechanism and function of OsSUT2 might not HvSUT2 are vacuolar transporters according to a previous be completely identical to that of LeSUT2. analysis of transient SUT–GFP fusion protein expression Carbohydrate transport from endosperms and embryos to (Endler et al. 2006; Reinders et al. 2008). On the other coleoptiles, shoots, and roots is an important process for hand, LeSUT4 and StSUT4 are located on the plasma supplying developing tissues with a carbon source during Rice (2011) 4:39–49 47 transport to developing shoot and roots but not play a role to transport sucrose from endosperm to embryos (Scofield et al. 2007). The increased OsSUT2 expression in isolated embryos (without endosperms) during germination was not obvious in dark conditions; however, the significant up-regulation of OsSUT2 was observed in the embryos of germinating seeds (with endosperms) in dark conditions. Thus, it was suggested that the sugar transported from endosperm was the key factor for promoting OsSUT2 expression. Sugars not only act as nutrients and energy for supporting plant growth but also function as signals controlling plant development and the expression of various genes (reviewed by Gibson 2005; Rolland et al. 2006). The data in Fig. 6 showed that the transcript levels of rice OsSUT2 could be slightly enhanced by glucose and sucrose after 1 day of treatment and significantly enhanced after 5 days of treatment. Moreover, since the OsSUT2 expression was not affected by mannitol, the sugar-enhanced OsSUT2 expression was not caused by osmotic effect. Sugar- enhanced expression has also been observed with OsSUT1 (Matsukura et al. 2000; Chen et al. 2010). However, the positive effect of sugar on OsSUT1 expression occurs after Fig. 9 Developmental regulation and effect of glucose on OsSUT2 5 days of treatment, and 1-day sugar treatment down- promoter activity. a GUS activity was quantitatively analyzed in the regulates OsSUT1 expression (Chen et al. 2010). Thus, the embryos from germinating seeds of transgenic rice carrying the mechanisms of sugar-mediated regulation are different for OsSUT2 promoter::GUS construct. b GUS activity was determined in isolated embryos cultured in medium with or without glucose. MU 4- OsSUT1 and OsSUT2. Sucrose-induced signal transduction methylumbelliferone. The data are presented as mean±SD, and for gene regulation could involve sucrose as a direct signal statistical significance is set at p<0.05 (asterisk). that is sensed by a sensor located on the cell membrane or an intracellular sensor. In addition, sucrose metabolites, seed germination and seedling establishment. The levels of such as glucose, could be signals to trigger the downstream OsSUT2 mRNA gradually increased from 1 to 5 DAI transduction pathway (reviewed by Halford et al. 1999). (Fig. 3b). However, our previous report showed that the The nonmetabolizable sucrose analog palatinose had a OsSUT1 transcript significantly increased after 1 to 2 DAI similar effect on OsSUT2 expression in rice embryos as and then quickly decreased at 3 DAI (Chen et al. 2010). sucrose, suggesting that sucrose acts as a direct molecule The expression level of OsSUT1 was the highest among for triggering the up-regulation of OsSUT2 expression. In OsSUT members in embryos at early imbibition stages (i.e., addition, the sensor for sucrose signal transduction is DAI 1), but OsSUT2 was the dominantly expressed OsSUT expected to be located on the cell membrane because of member 5 DAI (Fig. 3a). Thus, the functions and the lack of palatinose transport into plant cells (Sinha et al. regulations of individual OsSUT genes are different in the 2002; Rolland et al. 2006). Moreover, since there was no embryos of germinating seeds. As shown by the data in effect of mannitol on OsSUT2 expressions (Fig. 6), it was Fig. 8, the expression levels of OsSUT2 in scutellum and suggested that the up-regulation of OsSUT2 expressions by vascular bundles were higher than in other embryo ground palatinose was not caused by osmotic effect. Moreover, the cells, and the OsSUT2 promoter activity was also signifi- data showing that 3-OMG can also conduct the same cantly observed in aleurone layers. According to the positive effect on OsSUT2 expression in germinating OsSUT2 expression patterns in germinating seeds, it was embryos as glucose suggests that the glucose-induced suggested that OsSUT2 play a role to release sucrose from OsSUT2 expression was mediated by a hexokinase- aleurone layers, transport sucrose into scutellum, and also independent pathway. In contrast, glucose-regulated OsSUT1 load sucrose into the phloem in germinating seeds. On the expression in germinating embryos was via a hexokinase- other hand, OsSUT1 gene was expressed in the scutellar dependent pathway (Chen et al. 2010). vascular bundle of germinating embryos but not in the Changes in the transcriptional activity of the OsSUT2 scutellar epithelial cell layer. Therefore, it was suggested promoter in embryos during germination correlated with the that OsSUT1 functioned to load sucrose into phloem for mRNA levels. OsSUT2 promoter activity was also up- 48 Rice (2011) 4:39–49 Bürkle L, Hibberd JM, Quick WP, Kühn C, Hirner B, Frommer regulated by glucose in embryos. Thus, promoter regulation WB. The H -sucrose cotransporter NtSUT1 is essential for was a key step for controlling OsSUT2 expression. The sugar export from tobacco leaves. Plant Physiol. 1998;118:59– predicted cis-acting elements on the OsSUT2 promoter were searched in the PLACE database (Higo et al. 1999), Carpaneto A, Geiger D, Bamberg E, Sauer N, Fromm J, Hedrich R. Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates identifying a SUSIBA2 transcription factor-binding site sucrose efflux under the control of the sucrose gradient and the proton (WBOXHVISO1; W-box) 477 bp upstream of the ATG motive force. J Biol Chem. 2005;280:21437–43. translation start codon. SUSIBA2 is a sugar-inducible Chen JY, Liu SL, Siao W, Wang SJ. 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An improved hidden Markov model for rice embryos. transmembrane protein topology prediction and its applications to complete genomes. Bioinformatics. 2005;21:1853–8. Acknowledgements We thank Dr. Chwan-Yang Hong from National Kühn C, Grof CP. Sucrose transporters of higher plants. Curr Opin Taiwan University for providing the pCHY10 and pCAMBIA1302 Plant Biol. 2010;13:288–98. plasmids and Mr. Dah-Pyng Shung from Hualien District Agricultural Kühn C, Quick WP, Schulz A, Reismeier JW, Sonnewald U, Frommer Research and Extension Station in Taiwan for providing the rice seeds. WB. Companion-cell-specific inhibition of the potato sucrose This research was supported by grant NSC 99-2313-B-002-006-MY3 transporter SUT1. Plant Cell Environ. 1996;19:1115–23. from the National Science Council of the Republic of China. Lalonde S, Wipf D, Frommer WB. Transport mechanisms for organic forms of carbon and nitrogen between source and sink. Annu Rev Plant Biol. 2004;55:341–72. Lemoine R. 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Weise A, Barker L, Kühn C, Lalonde S, Buschmann H, Frommer WB, The role of the sucrose transporter, OsSUT1, in germination and et al. A new subfamily of sucrose transporters, SUT4, with low early seedling growth and development of rice plants. J Exp Bot. affinity/high capacity localized in enucleate sieve elements of 2007;58:483–95. plants. Plant Cell. 2000;12:1345–55. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Rice Springer Journals

Characterization of OsSUT2 Expression and Regulation in Germinating Embryos of Rice Seeds

Siao, Wei; Chen, Jia-Yi; Hsiao, Hui-Hsin; Chung, Ping; Wang, Shu-Jen
Rice , Volume 4 (2) – Sep 21, 2011
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Life Sciences; Plant Sciences; Plant Genetics & Genomics; Plant Breeding/Biotechnology; Agriculture; Plant Ecology
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1939-8425
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10.1007/s12284-011-9063-1
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Abstract

Rice (2011) 4:39–49 DOI 10.1007/s12284-011-9063-1 Characterization of OsSUT2 Expression and Regulation in Germinating Embryos of Rice Seeds Wei Siao & Jia-Yi Chen & Hui-Hsin Hsiao & Ping Chung & Shu-Jen Wang Received: 15 July 2011 /Accepted: 9 September 2011 /Published online: 21 September 2011 Springer Science+Business Media, LLC 2011 Abstract OsSUT2 encodes a putative sucrose transporter MU 4-Methylumbelliferone containing 12 transmembrane domains in rice plants. 3-OMG 3-O-Methylglucose Subcellular localization of the OsSUT2::GFP fusion protein Pal Palatinose indicated that OsSUT2 is a cell membrane protein. In QRT-PCR Quantitative real-time reverse transcriptase embryos of germinating seeds, the expression of OsSUT2 polymerase chain reaction gradually increased during the early germinating stage. The Sc Scutellum cells developmental regulations of OsSUT2 in germinating Suc Sucrose embryos could be mediated by sugars transported from SUT Sucrose transporter endosperms. OsSUT2 expression was up-regulated by Ubi Ubiquitin glucose through a hexokinase-independent pathway. Exoge- V Vascular bundle nous sucrose was sensed by a sensor localized on the plasma membrane and functioned as an enhancer to promote OsSUT2 expression. Based on OsSUT2 promoter::GUS expression in germinating seeds of transgenic rice, OsSUT2 was signifi- cantly expressed in the embryos and aleurone layers. In Introduction embryos, strong GUS expression was detected in the scutellum and vascular bundle tissues. Developmental In plants, carbohydrates translocate from the source to stage- and sugar-dependent OsSUT2 expression was sug- sink tissues through symplastic and apoplastic pathways. gested to be controlled by transcriptional regulation of the Sugar transport between cells in symplasts is mediated promoter region. by plasmodesmata. In apoplasts, sugar translocators are responsible for carbohydrate uptake into cells. Because . . . Keywords Embryo Oryza sativa Seed germination sucrose is the major transported form of carbohydrate Sucrose transporter between plant tissues, sucrose transporter (SUT) plays an important role in long-distance disaccharide transport. Abbreviations SUT1 in sugarcane functions to partition sucrose between the DAI Days after imbibition vascular bundle and storage cells (Rae et al. 2005). Maize Glc Glucose SUT, ZmSUT1, is responsible for sugar uptake into phloem GUS β-Glucuronidase in source tissues and sugar unloading from phloem into cells Man Mannitol in sink tissues (Carpaneto et al. 2005). Changes in carbohydrate allocation and the inhibition of photosynthesis have been observed in transgenic plants with reduced SUT : : : : W. Siao J.-Y. Chen H.-H. Hsiao P. Chung S.-J. Wang (*) activity due to antisense SUT genes; thus, SUT has been Department of Agronomy, National Taiwan University, indicated to play an important role in carbohydrate partition- No. 1, Section 4, Roosevelt Rd., ing and physiological processing (Kühn et al. 1996; Bürkle Taipei 106, Taiwan et al. 1998). e-mail: shujen@ntu.edu.tw 40 Rice (2011) 4:39–49 To date, SUT genes have been identified from several In the present study, we identified the subcellular plants, most belonging to gene families (Lemoine 2000; localization of OsSUT2. The expression of OsSUT2 in Lalonde et al. 2004). The SUT gene families of dicot and germinating embryos was detected by real-time quantitative monocot species were classified into five sequence-based RT-PCR. The spatial and temporal transcriptional activities groups by Braun and Slewinski (2009). In rice, the SUT of the OsSUT2 promoter were detected in the germinating gene family consists of five genes, OsSUT1 to OsSUT5 seeds of transgenic rice plants carrying the OsSUT2 (Aoki et al. 2003). OsSUT1 and 3 were classified into promoter::GUS fusion gene. Furthermore, the signal trans- group 1. The group 1 SUT family is composed of several duction of sugars for regulating OsSUT2 expression was Poaceae SUT members; so far, no dicot SUTs have been examined and discussed. found in group 1. OsSUT2 was classified with Arabidopsis SUT4 (AtSUT4) in group 2. AtSUT4 has been demonstrated to be responsible for the low-affinity/high-capacity trans- Materials and methods port system (reviewed by Lalonde et al. 2004). OsSUT4 and OsSUT5 were classified into group 3 and 5, respectively. Plant materials and treatment During plant development from seed germination to seed establishment and seedling growth, starch reserved in the Rice (Oryza sativa L. cv. Tainung 67) seeds were obtained seed is the primary carbon and energy source for sprouting from the Hualien District Agricultural Research and and early seedling establishment, and then the sugar Extension Station in Taiwan. For germination, seeds were sources for later stages of seedling growth come from the sterilized in 2.5% sodium hypochlorite with Tween 20 for photosynthetic shoots. In rice plants, carbohydrate transport 20 min and subsequently washed with distilled H O four from germinating seeds to other developing sink tissues is a times. Seeds were then germinated at 37°C in the dark for fundamental process for plant development and growth. 3 days and then moved to the phytotron for growing at 30/ Sugar derived from starch degradation exported to the 25°C under natural daylight. To analyze the sugar content cytosol from storage organelles is the first step for long- and OsSUT2 expression in embryos, the growing shoots distance carbohydrate translocation. In cereal endosperms, and roots were cut and the embryos isolated after 1- to 5- starch granules are attacked by α-amylase (Murata et al. day seed imbibition. 1968), and the maltose and linear/short-branch-chain To isolate embryos from dry seeds, the grain hulls were oligosaccharides are generated for further degradation by removed by machine and the embryos picked by razor α-glucosidase (Stanley et al. 2011). The hexoses produced blade. The isolated embryos were sterilized in 0.25% by starch degradation can be taken up by the scutellum cells sodium hypochlorite for 10 min and subsequently washed of the embryo. Hexoses have been suggested to be re- with distilled H O four times (Matsukura et al. 2000). For synthesized into sucrose in the scutellum (Edelman et al. isolated embryo culturing, the embryos were placed on MS 1959; Nomura et al. 1969). Sucrose in the scutellum is medium and incubated at 28°C in dark or light. For sugar loaded into the vascular bundle, transported, and then and sugar analog treatments, the chemicals were added in unloaded to growing tissues through apoplastic or sym- MS medium. The concentration of all sugars and sugar plastic pathways (Aoki et al. 2006). In addition, it was also analogs used in this study was 100 mM. suggested that sucrose transported from aleurone layers to endosperm at early germination stage could be further Homology analysis of amino acid sequences uptaked by scutellum (Aoki et al. 2006). Based on our and transmembrane domain prediction previous work (Liu et al. 2010), soluble sugar can also be converted to starch in scutellum or the cells surrounding Multiple sequence alignments of SUT amino acid sequen- vascular bundles at the post-germination stage, and the ces were carried out using SDSC Biology WorkBench 3.2 amount of transitory starch in embryonic tissues was (http://workbench.sdsc.edu/). The transmembrane domains dependent on the demand of growing sink tissues. Phloem on the OsSUT2 amino acid sequence were predicted functions as an important pathway for carbon source according to the Hidden Markov Models using TMMOD transport among various plant tissues. In rice germinating software (Kahsay et al. 2005). seeds, OsSUT1 was the first OsSUT family member identified to have a tissue-specific expression pattern RNA extraction (Hirose et al. 1997; Scofield et al. 2007). Developmental expression and regulation of OsSUT1 has been observed in Embryos isolated from ten seeds or harvested from medium germinating seeds (Chen et al. 2010). However, the were ground in liquid nitrogen, homogenized in 1 mL Trizol regulation of other members of the OsSUT gene family reagent (Invitrogen, Carlsbad, CA, USA), and centrifuged at still needs to be established. 8,000×g. The supernatant was treated with 0.2 mL chloro- Rice (2011) 4:39–49 41 form, shaken for 15 s, and incubated at room temperature for embryos 5 days after imbibition (DAI) and RT-PCR was 3 min. After centrifugation at 12,000×g for 15 min at 4°C, performed using specific primers. The forward primer (5′- the upper layer was transferred to a new tube. RNA was TTCTAGAATGCCGCGGCGGCCTAGGCGG-3 ′) precipitated with 0.5 mL isopropanol and incubated for contained the XbaI cloning site sequence, the reverse 10 min at room temperature. After centrifugation, the pellet primer (5′-AGGATCCATCGGTGACCTCTCCTCCTTG- was dissolved in 0.2 mL H O. Before the gene expression 3′) contained the BamHI cloning site, and the stop codon analysis, the total RNA extracted from the embryos was sequence was not included. OsSUT2 cDNA was cloned in- treated with DNase to remove contaminating genomic DNA. frame with the N-terminus of the green fluorescent protein (GFP) gene, driven by a CaMV35S promoter in a transient Quantitative real-time reverse transcriptase-PCR expression vector. The OsSUT2-GFP fusion construct was transiently expressed in barley aleurone layer cells Total RNA (200 ng) was used as the template for quantitative mediated by a He Biolistic particle delivery system (model real-time RT-PCR analysis using the Brilliant SYBR Green PDS-1000, Bio-Rad). Half-de-embryonated barley seeds were QRT-PCR Master Mix (Stratagene, La Jolla, CA, USA), and sterilized before soaking in a shooting buffer (20 mM Na– PCR reactions were performed using a Multiplex 3000P Real- succinate and 20 mM CaCl ,pH 5.0) for 48 h at 24°C in the Time PCR System (Stratagene). The gene-specific RT-PCR dark (Mena et al. 2002). Next, the pericarp was removed primers are listed on Table 1. RT-PCR was carried out as from the seeds and the aleurone layer exposed for bombard- follows: 50°C for 30 min and 95°C for 10 min, followed by ment. After bombardment, the seeds were incubated with 40 cycles of 95°C for 1 min, 58°C for 1 min, and 72°C for shooting buffer for 24 h at 24°C. Finally, fluorescence 1 min. To quantify relative gene expression levels accurately, localization in the aleurone layer cells was observed using an the C values of OsSUT1 to 5 were normalized to the C AXIO Imager M1 fluorescence microscope (Carl Zeiss, T T value of the ubiquitin (Ubi) gene. For all real-time RT-PCR Germany). analyses, three independent experiments were carried out, and the data are presented as mean±SD. Promoter cloning Sugar analysis Rice genomic DNA was extracted from the leaf tissues with plant DNA reagent (Invitrogen). The OsSUT2 DNA ZOL Ten embryos were ground to powder in liquid nitrogen and fragment upstream of the translation start site, located from extracted with 1 mL of 80% (v/v) ethanol at 80°C for 5 min −830 to −1 bp, was amplified by PCR using specific before centrifugation at 3,000×g. The supernatant was primers (forward primer with SacI site: 5′-GAGCTCT analyzed for glucose and sucrose following the method TAAGGAGCACCAA-3′; reverse primer with SmaI site: described by Spackman and Cobb (2002). 5′-CCCGGGCTTCTTCTCGTGTT-3′). Putative cis-ele- ments on the OsSUT2 promoter sequence were character- Subcellular location of OsSUT2 ized by searching for similar motifs in the Database of Plant Cis-acting Regulatory DNA Elements (PLACE) (Higo et al. The coding region cDNA of OsSUT2 was cloned from rice 1999). To generate the plasmid for the promoter activity embryos using RT-PCR. Total RNA was extracted from assay in transgenic rice plants, the OsSUT2 promoter Table 1 Primer pairs for real- Gene Accession number Primer pair Amplicon size (bp) time RT-PCR (F: forward primer; R: reverse primer) OsSUT1 D87819 F: 5′-CTGTGATTTTCCTGTCCCTG-3′ 136 R: 5′-AACACTGCTAGTGGACCAGT-3′ OsSUT2 AB091672 F: 5′-AGGAGGAGAGGTCACCGATAA-3′ 240 R: 5′-CCAACATCCAATGTACAACAGCA-3′ OsSUT3 AB071809 F: 5′-GCCCAAGGTCTCCGTCC-3′ 137 R: 5′-TGCTATAGTACCCGCTCTAA-3′ OsSUT4 AB091673 F: 5′-TTTGGCTGAGCAGAACACCA-3′ 249 R: 5′-ATGTCATTCGGGCAGAGCTT-3′ OsSUT5 AB091674 F: 5′-CTAGTGCGAAACTCCATCAAA-3′ 249 The nucleotide sequences used R: 5′-AAAATATTTGGGTTTCCTGAGAT-3′ for primer designing were pre- Ubi D12629 F: 5′-CGCAAGTACAACCAGGACAA-3′ 101 sented by their accession numbers R: 5′-TGGTTGCTGTGACCACACTT-3′ in GenBank 42 Rice (2011) 4:39–49 (−830/−1) was inserted into the SacI and SmaI sites of the Results pCHY10 vector (a gift from Dr. Chwan-Yang Hong), which contained the first intron of the Ubi gene fused to the β- Subcellular localization of OsSUT2 protein glucuronidase (GUS) reporter gene. We used restriction enzymes to isolate the DNA cassettes containing the To characterize the protein structure and identify the OsSUT2 promoter fragment with the Ubi intron and GUS subcellular localization of OsSUT2 protein, the OsSUT2 gene from the pCYH10 constructs. These cassettes were coding sequence was amplified by RT-PCR. The OsSUT2 then ligated into the SacIand HindIII sites of the cDNA (accession no. HQ875341) encodes a protein of 501 pCAMBIA1302 plasmid. amino acids in length. According to membrane protein topology prediction using Hidden Markov Models in Generation of transgenic rice plants TMMOD software (Kahsay et al. 2005), the OsSUT2 protein contains 12 transmembrane domains (Fig. 1a, b). To produce OsSUT2 promoter::GUS transgenic plants, the The amino- and carboxyl-terminal sequence tails predicted constructed pCAMBIA1302 plasmid was transformed into a cellular localization (Fig. 1b). Based on a previous Agrobacterium tumefaciens EHA105. The cultured A. phylogenetic analysis of the SUT gene family by Braun tumefaciens harboring the constructed plasmids were used and Slewinski (2009), OsSUT2 was grouped with Arabi- to infect rice embryo-derived calli. The transformed calli dopsis AtSUT4. Furthermore, Kühn and Grof (2010) were selected on medium containing 50 μg/mL hygromycin showed that AtSUT4 is also classified with StSUT4 and and 250 μg/mL cefotaxime. Finally, the transgenic rice LeSUT4 in the same group. The amino acid identities plants were regenerated from the transformed calli (Hiei et between OsSUT2 and StSUT4, LeSUT4, and AtSUT4 are al. 1994; Toki 1997). 66%, 66%, and 64%, respectively. The significant differ- ences within the above SUT genes are at the amino Histochemical GUS assay terminus and central inside loop (Fig. 1a). In addition, the central inside loop of OsSUT2 (35 amino acids in length) is Histochemical GUS activity assays were performed as smaller than that of LeSUT2 (94 amino acid residues) and described previously (Jefferson 1987). Half-cut germinated AtSUT2 (87 amino acid residues), which belong to the seeds from transgenic plants were placed in a solution other SUT group (Fig. 1b). To determine the subcellular containing 10 mM EDTA, 0.5 mM potassium ferricyanide, localization of OsSUT2 protein, the expression of OsSUT2- 0.5 mM potassium ferrocyanide, 1.0 mM 5-bromo-4- GFP fusion protein was observed in the aleurone layer cells chloro-3-indolyl-β-D-glucuronide, 0.1% Triton X-100, and of barley seeds. Fluorescence imaging showed that the 0.1 M sodium phosphate buffer (pH 7.0) and incubated at fusion protein was localized on the plasma membrane 37°C for 4 h. The staining reaction was stopped by adding (Fig. 2). 75% ethanol. Developmental expression of OsSUT2 in embryos Quantitative GUS activity assay during germination Developmental regulation of the OsSUT2 promoter was To examine the expression of five OsSUT family genes in analyzed in germinating embryos of transgenic rice seeds. the embryos of germinating seeds, rice seeds were Seeds were germinated in water containing hygromycin, germinated in the dark for 3 days and then grown in and embryos were isolated from 3 and 5 DAI. To assay the phytotron with natural sunlight. The expression levels of effect of glucose on OsSUT2 promoter activity, the seeds OsSUT3 and 5 were lower than those of the other three were imbibed in hygromycin-containing water for 3 days OsSUT genes at 1 and 5 DAI (Fig. 3a). Quantitative RT- and the germinated embryos were isolated from transgenic PCR showed that the OsSUT2 transcript level was seeds. The isolated embryos were cultured in MS medium significantly higher than that of other OsSUT genes at 5 containing 100 mM glucose solution for 5 days at 28°C in DAI (Fig. 3a). Among OsSUT1, 2, and 4, only the OsSUT2 the dark. The protruding shoots and roots were excised and mRNA of embryos was increased at 5 DAI compared to 1 discarded before the embryo proteins were extracted with DAI (Fig. 3a). Furthermore, the expression of OsSUT2 was the buffer containing 100 mM sodium phosphate (pH 7.0), observed to gradually increase during the early germination 5 mM dithiothreitol, 20 μg/ml leupeptin, and 20% (v/v) stage, and the transcript level at 5 DAI was 4.6-fold that of glycerol. Next, 4-methylumbelliferyl β-D-glucuronide was dry seed embryos (Fig. 3b). In addition, the expression of added as a substrate and GUS activities were measured OsSUT2 in embryonic tissues increased with growth stage, fluorometrically in the embryos as described previously even seedlings continuously grew in the dark after (Jefferson 1987). germination (Fig. 4a). However, the phenomenon of Rice (2011) 4:39–49 43 Fig. 1 Multiple amino acid sequence alignment and trans- membrane domain analysis of SUT proteins. a Alignment of amino acid sequences from potato SUT4 (StSUT4; Genbank accession AF237780), tomato SUT4 (LeSUT4; AF176950), Arabidopsis SUT4 (AtSUT4; AY072092), and rice OsSUT2 (HQ875341). TM transmembrane domain. b Comparison of SUT protein structures. The inside and outside membrane regions and transmembrane domains of OsSUT2, StSUT4, AtSUT4, LeSUT4, AtSUT2 (AK226970), and LeSUT2 (AF166498) were predicted using TMMOD software. 44 Rice (2011) 4:39–49 Fig. 2 Subcellular localization of OsSUT2 by transient expres- sion of OsSUT2-GFP fusion proteins in barley aleurone layer cells. a Bright field image. b GFP fluorescence image. c Merged field and fluorescence image. OsSUT2 up-regulation during germination was not obvious considered whether the sugar transported from endosperm in dark-cultured isolated embryos (endosperm-free) during seed germination plays a role in the regulation of (Fig. 4c). OsSUT2 expression in embryos. To evaluate this possibil- ity, the levels of glucose and sucrose were analyzed in the Expression of OsSUT2 regulated by exogenous sugars embryos of germinating seeds from 0 to 5 DAI. Glucose levels gradually increased with the days after imbibition, OsSUT2 expression in germinating embryos was different but sucrose content fluctuated (Fig. 5). The changes in from those with and without endosperm status. It was glucose content corresponded to the expression pattern of OsSUT2 transcripts in embryos during seed germination (Figs. 3b and 5). Even for seed germination in the dark, changes in glucose content and OsSUT2 transcript levels were consistent (Fig. 4a, b). In order to determine whether sugars are the factors up-regulating OsSUT2 expression in the embryos of germinating seeds, the embryos isolated from dry seeds were cultured on sugar-containing MS medium in the dark for 5 days. Although glucose (100 mM) and sucrose (100 mM) slightly enhanced OsSUT2 mRNA levels in 1-day sugar-treated embryo samples, the effect was not significant (Fig. 6a). In 5-day cultured embryos, the OsSUT2 expression was obviously up-regulated by both glucose and sucrose, but not by the same concentration of mannitol (Fig. 6b). Sugar analogs were applied to study the sugar-sensing pathway for regulating OsSUT2 expression. 3-O-Methylglu- cose (3-OMG), a nonmetabolizable glucose analog, was taken up by cells but was not phosphorylated by hexokinase (Dixon and Webb 1979). The results showed that 3-OMG (100 mM) had the same effect as glucose (100 mM) to enhance OsSUT2 expression (Fig. 7a). Palatinose, a non- metabolizable sucrose analog, cannot be imported into plant cells (Bouteau et al. 1999). The effect of palatinose (100 mM) on OsSUT2 gene expression was similar to that of sucrose (100 mM) (Fig. 7b), suggesting that embryo cells sense sucrose signals to regulate OsSUT2 expression through a membrane sensor. Fig. 3 Developmental expression of rice OsSUT in the embryos of OsSUT2 promoter::GUS expression in embryos germinating seeds. a Comparison of the expression of five OsSUT genes in the embryos of 1-DAI and 5-DAI seeds. b Changes in Transgenic rice plants harboring the OsSUT2 promoter:: OsSUT2 expression in embryos during seed germination. The data are presented as mean±SD. GUS construct was used to investigate the spatial expres- Rice (2011) 4:39–49 45 Fig. 5 Changes in the glucose and sucrose content of embryos during seed germination. The data are presented as mean±SD. Glc glucose, Suc sucrose. vascular tissues and scutellum cells of embryos (Fig. 8). In addition, the quantitative GUS activity assay showed that the GUS activity in embryos from 5-DAI seeds was significantly higher than that of 3-DAI seeds (Fig. 9a). To identify the effect of glucose on OsSUT2 promoter activity, the germinated embryos of transgenic rice seeds were isolated and incubated in 100 mM glucose solution for Fig. 4 OsSUT2 expression and glucose content in embryonic cells from light- and dark-grown seedlings. Rice seeds were germinated in the dark for 3 days. Some germinated seeds were moved to a phytotron with natural daylight for 2 days (labeled 3-day D/2-day L), and some germinated seeds were kept in the dark (labeled 5-day D) for growth. The seedlings were collected at 1 and 5 DAI, and the embryos were isolated for OsSUT2 expression analysis (a) and to measure glucose content (b). c The OsSUT2 expression in germinating isolated embryos was analyzed. The embryos were dissected from dry seeds and dark-grown in MS medium. OsSUT2 expression was detected after 1, 3, and 5 days of culture. The data are presented as mean±SD. sion of OsSUT2 in rice embryos. Rice seeds of three independent transgenic lines were germinated in water, and Fig. 6 Effects of sugars on OsSUT2 expression. OsSUT2 transcript GUS expression in germinating seeds was observed. GUS levels were determined in isolated embryos cultured in medium activities were performed in aleurone layers and embryos. containing mannitol (Man), glucose (Glc), or sucrose (Suc) for 1 day (a) and 5 days (b). The data are presented as mean±SD. In embryos, significant GUS staining was detected in the 46 Rice (2011) 4:39–49 Fig. 7 Sugar-sensing pathways for OsSUT2 gene regulation in germinating embryos. a Effect of glucose analog 3-O-methylglucose (3-OMG)on OsSUT2 expression after 5 days of treatment. b Effect of Fig. 8 Histochemical localization of β-glucuronidase activity in the the sucrose analog palatinose (Pal)on OsSUT2 expression after 5 days embryos of transgenic rice plants harboring the OsSUT2 promoter:: of treatment. The data are presented as mean±SD. GUS constructs. The seeds of transgenic rice lines 13, 22, and 32 were germinated in the dark for 3 days, and then the GUS activity was analyzed in half-cut germinated seeds. Sc scutellum cells, V vascular bundle. Bar=1 mm. 5 days. The OsSUT2 promoter activities were obviously enhanced (Fig. 9b). membrane of sieve elements (Reinders et al. 2002). OsSUT2 contains 12 transmembrane domains and was Discussion localized on plasma membrane. Amino acid alignment showed that the number of amino acids in the central inside SUT proteins are important carriers for transporting sucrose loops are similar among OsSUT2 and other SUTs in group across the plasma membrane or vacuolar membranes 4 (according to the classification by Braun and Slewinski (reviewed by Kühn and Grof 2010). Arabidopsis SUT 2009) (Fig. 1); however, the length of the OsSUT2 central protein has also been found on the chloroplast membrane loop is shorter than that of SUTs belonging to group 2, i.e., (Rolland et al. 2003). Rice OsSUT2 is classified in the AtSUT2 and LeSUT2. LeSUT2 is considered to function as same group with Arabidopsis AtSUT4, tomato LeSUT4, a putative sucrose sensor (Barker et al. 2000). The potato StSUT4, Lotus japonicus LjSUT4, and barley conserved domains in the extended cytoplasmic loop of HvSUT2 (Braun and Slewinski 2009; Kühn and Grof LeSUT2 and AtSUT2 play an important role in signal 2010). Some of the above-mentioned SUTs, including sensing and transduction (Barker et al. 2000). Because the AtSUT4, StSUT4, and LjSUT4, have been identified as lengths and amino acid sequences of the central loops are low-affinity/high-capacity transporters (Weise et al. 2000; significantly different between OsSUT2 and LeSUT2, the Reinders et al. 2008). In addition, AtSUT4, LjSUT4, and regulatory mechanism and function of OsSUT2 might not HvSUT2 are vacuolar transporters according to a previous be completely identical to that of LeSUT2. analysis of transient SUT–GFP fusion protein expression Carbohydrate transport from endosperms and embryos to (Endler et al. 2006; Reinders et al. 2008). On the other coleoptiles, shoots, and roots is an important process for hand, LeSUT4 and StSUT4 are located on the plasma supplying developing tissues with a carbon source during Rice (2011) 4:39–49 47 transport to developing shoot and roots but not play a role to transport sucrose from endosperm to embryos (Scofield et al. 2007). The increased OsSUT2 expression in isolated embryos (without endosperms) during germination was not obvious in dark conditions; however, the significant up-regulation of OsSUT2 was observed in the embryos of germinating seeds (with endosperms) in dark conditions. Thus, it was suggested that the sugar transported from endosperm was the key factor for promoting OsSUT2 expression. Sugars not only act as nutrients and energy for supporting plant growth but also function as signals controlling plant development and the expression of various genes (reviewed by Gibson 2005; Rolland et al. 2006). The data in Fig. 6 showed that the transcript levels of rice OsSUT2 could be slightly enhanced by glucose and sucrose after 1 day of treatment and significantly enhanced after 5 days of treatment. Moreover, since the OsSUT2 expression was not affected by mannitol, the sugar-enhanced OsSUT2 expression was not caused by osmotic effect. Sugar- enhanced expression has also been observed with OsSUT1 (Matsukura et al. 2000; Chen et al. 2010). However, the positive effect of sugar on OsSUT1 expression occurs after Fig. 9 Developmental regulation and effect of glucose on OsSUT2 5 days of treatment, and 1-day sugar treatment down- promoter activity. a GUS activity was quantitatively analyzed in the regulates OsSUT1 expression (Chen et al. 2010). Thus, the embryos from germinating seeds of transgenic rice carrying the mechanisms of sugar-mediated regulation are different for OsSUT2 promoter::GUS construct. b GUS activity was determined in isolated embryos cultured in medium with or without glucose. MU 4- OsSUT1 and OsSUT2. Sucrose-induced signal transduction methylumbelliferone. The data are presented as mean±SD, and for gene regulation could involve sucrose as a direct signal statistical significance is set at p<0.05 (asterisk). that is sensed by a sensor located on the cell membrane or an intracellular sensor. In addition, sucrose metabolites, seed germination and seedling establishment. The levels of such as glucose, could be signals to trigger the downstream OsSUT2 mRNA gradually increased from 1 to 5 DAI transduction pathway (reviewed by Halford et al. 1999). (Fig. 3b). However, our previous report showed that the The nonmetabolizable sucrose analog palatinose had a OsSUT1 transcript significantly increased after 1 to 2 DAI similar effect on OsSUT2 expression in rice embryos as and then quickly decreased at 3 DAI (Chen et al. 2010). sucrose, suggesting that sucrose acts as a direct molecule The expression level of OsSUT1 was the highest among for triggering the up-regulation of OsSUT2 expression. In OsSUT members in embryos at early imbibition stages (i.e., addition, the sensor for sucrose signal transduction is DAI 1), but OsSUT2 was the dominantly expressed OsSUT expected to be located on the cell membrane because of member 5 DAI (Fig. 3a). Thus, the functions and the lack of palatinose transport into plant cells (Sinha et al. regulations of individual OsSUT genes are different in the 2002; Rolland et al. 2006). Moreover, since there was no embryos of germinating seeds. As shown by the data in effect of mannitol on OsSUT2 expressions (Fig. 6), it was Fig. 8, the expression levels of OsSUT2 in scutellum and suggested that the up-regulation of OsSUT2 expressions by vascular bundles were higher than in other embryo ground palatinose was not caused by osmotic effect. Moreover, the cells, and the OsSUT2 promoter activity was also signifi- data showing that 3-OMG can also conduct the same cantly observed in aleurone layers. According to the positive effect on OsSUT2 expression in germinating OsSUT2 expression patterns in germinating seeds, it was embryos as glucose suggests that the glucose-induced suggested that OsSUT2 play a role to release sucrose from OsSUT2 expression was mediated by a hexokinase- aleurone layers, transport sucrose into scutellum, and also independent pathway. In contrast, glucose-regulated OsSUT1 load sucrose into the phloem in germinating seeds. On the expression in germinating embryos was via a hexokinase- other hand, OsSUT1 gene was expressed in the scutellar dependent pathway (Chen et al. 2010). vascular bundle of germinating embryos but not in the Changes in the transcriptional activity of the OsSUT2 scutellar epithelial cell layer. Therefore, it was suggested promoter in embryos during germination correlated with the that OsSUT1 functioned to load sucrose into phloem for mRNA levels. 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Rice – Springer Journals

Published: Sep 21, 2011

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