Os01g0262600

From RiceWiki
Jump to: navigation, search

OsGT1 is a Golgi-localized glycosyltransferase essential for intine construction and pollen maturation, providing new insight into male reproductive development.

Annotated Information

Function

T-DNA Insertions in OsGT1 Cause a Defect in Male Gametophyte Development

We previously generated transfer DNA (T-DNA)-tagged lines of japonica rice (Jeon et al., 2000; Jeong et al., 2002) and determined flanking sequences for the insertion sites (An et al., 2003; Jeong et al., 2006). To identify genes essential for gametophyte development, we genotyped the T-DNA insertion lines; those with a segregation ratio close to 1:1:0 (wild type:heterozygote: homozygote) were selected. From 541 independentlines, we obtained eight with the segregation distortion phenotype. Here, we report detailed analyses of line 3C-00590, which carries a T-DNA insertion in the fifth intron of LOC_Os01g15780 (Fig. 1A). Because the gene encodes a protein in the glycosyltransferase family, we named it OsGT1. We found that another allele (osgt1-2) also harbors T-DNA in the fourth exon of OsGT1 (Fig.1A). This line also exhibited a 1:1:0 segregation (Table I;Supplemental Fig. S1), confirming that this phenotype is due to the T-DNA insertion to OsGT1. These segregation phenotypes suggested that the defect results from a failure in gene transfer through either the male or female gamete. To clarify the function of OsGT1 in the gametophyte, we performed reciprocal crosses between heterozygotes (OsGT1/osgt1-1) and the wild type. When OsGT1/osgt1-1 was used as a pollen receiver (female), wild-type and heterozygous progeny were obtained at a similar frequency (11:13; Table II). However, when OsGT1/osgt1-1 served as a pollen donor (male), only wild-type progeny were obtained (32:0; Table II). Reciprocal crosses between OsGT1/osgt1-2 and wild-type plants produced similar results (Table II). These indicated that mutations in OsGT1 cause defects in the male gamete.

osgt1 Grains Are Defective at the Mature Pollen Stage

To examine further the morphological defects in male gametes of osgt1-1, we collected pollen grains from fully developed anthers and observed them with a bright field microscope. Pollen from wild-type anthers was normal: only a small fraction (nine out of 1,393) was shrunken at stage 12 (Fig. 1B; Supplemental Fig. S2). However, anthers from the heterozygote produced shrunken grains at a 49.5% frequency (915 of 1,847; Fig. 1C; Supplemental Fig. S2), indicating that OsGT1 is required for male gamete development. To determine whether those shrunken grains were caused by the T-DNA insertion, we used a Suc density gradient to separate them from normal grains (Bedinger and Edgerton, 1990; Fig. 1D). The gradient containedfour discrete bands, two lower bands that contained normal pollen with a globular shape (Fig. 1, E and F) plus two upper bands with defective grains that were shrunken or underdeveloped (Fig. 1, G and H). To see whether this defect was due to the mutation, we prepared DNA from each band and performed PCR with gene-specific primers. This resulted in amplification of the OsGT1 fragment from the two lower bands but not from the upper bands (Fig. 1I). Amplification with a gene-specific primer (R2) and a T-DNA primer (L1) did not generate bands from any of the four samples, indicating that normal pollen in the lower bands did not carry T-DNA and that abnormal pollen in the upper bands lacked genomic DNA because they were dead (Fig. 1J). To investigate the exact time when this mutant pollen began to show the phenotypic defect, we compared wild-type and osgt1 pollen at five stages of development (Fig. 2). At the young microspore stage (stage 9), cells from the wild type were released as free microspores from the tetrad (Fig. 2A). These gradually swelled and underwent vacuolation, causing the cytoplasm and nuclei to be distributed in the periphery (Fig. 2B). Afterward, the pollen started to divide mitotically (stage 11a; Fig. 2C; Zhang et al., 2011). Until this stage, all gametophytes in the OsGT1/osgt1-1 anthers appeared normal and were identical to those of the wild type (Fig. 2, F–H). However, at stage 11b, two different types of pollen were observed from the heterozygous anthers: normal grains showing even cytosolic staining and empty pollen with a lumped staining pattern (Fig. 2I). At the mature stage (stage 12), those differences were even more distinct. Whereas all of the pollen from wild-type anthers was normal (Fig. 2E),one-half of the grains from anthers of heterozygotes were shrunken and exhibited exiguous cytosolic contents (Fig. 2J). We tested pollen viability with fluorescein diacetate, which stains only live cells (Heslop-Harrison and Heslop- Harrison, 1970).Whereas globular pollen emitted a bright green signal, no signal was observed from the collapsed grains from mutant plants (Supplemental Fig. S3). These findings indicated that osgt1 pollen cannot form normal mature grains.

Rice1.png

Figure 1. Schematic diagrams of OsGT1 and aberrant pollen phenotype in osgt1 mutant. A, T-DNA insertion positions. Start and stop codons are represented as ATG and TGA, respectively; positions of insertion are shown with triangles. Shaded boxes indicate exons; lines connecting exons are introns. Primers for genotyping and expression analysis are marked with arrows. Scale bar = 100 nucleotides. B and C, Pollen grains from wild-type (B) and OsGT1/osgt1-1 (C) anthers. Bars = 20 mm. D, Defective pollen was separated from normal grains through Suc density gradient centrifugation. Bar = 500 mm. E to H, Representative pollen from each band. Bars = 10 mm. I, PCR products by two OsGT1 primers. J, PCR products by OsGT1 primer and T-DNA primer.



Table2.png

Table1.


Table2.png

Table 2.


Image2.png

Figure 2. Light microscopy observation of anthers at different developmental stages. Cross sections are shown from segregating wild type (A–E) and OsGT1/osgt1-1 (F–J) at early microspore stage (A and F), vacuolated stage (B and G), mitotic division stage 11a (C and H), mitotic division stage 11b (D and I), and mature pollen stage (E and J). BP, Binuclear pollen; DP, defective pollen; En, endodermis; Ep, epidermis; M, microspore; MP, mature pollen; T, tapetum; VP, vacuolated pollen. Bars = 25 mm.


osgt1 Pollen Is Defective in Intine Formation

To investigate the defects in osgt1 pollen further, we stained the mature grains with auramine O, which binds to exine (Dobritsa et al., 2011). This revealed that the exine was not markedly different between mutant and wild-type pollen (Fig. 3, A–D; Supplemental Fig. S4). We also monitored for the presence of intine via calcofluor white staining.Whereas defective pollen from heterozygous plants exhibited a very weak signal, wildtype pollen emitted bright blue fluorescence, demonstrating that the mutant pollen was defective in intine (Fig. 3, E–H; Supplemental Fig. S4). Staining with 496-diamidino-2-phenylindole (DAPI) showed that the defective pollen did not carry any nuclei at stage 12, whereas wild-type grains contained one vegetative nucleus and two generative nuclei (Fig. 3, I–L). Because mitotic division precedes intine construction in rice (Lu et al., 2002; Lin et al., 2009), we performed DAPI staining at stage 11, when the binuclear grains are formed. All pollen from the heterozygous plants contained two nuclei (Fig. 3, N and P), suggesting that osgt1 pollen underwent normal mitosis I. Furthermore, amido black staining showed that osgt1 grains did not accumulate cytosolic contents such as proteins (Supplemental Fig. S5), supporting our belief that the pollen abnormality developed later in the mutant. To understand the subcellular changes in osgt1 pollen, we performed transmission electron microscopy (TEM) analysis at stages 11a, 11b, and 12 (Zhang and Wilson, 2009). As revealed via bright-field microscopy, wild-type and osgt1 pollen did not differ significantly at stage 11a (Fig. 4, A–F). At the end of this stage, exine formation is generally considered complete (Lu et al., 2002; Lin et al., 2009). We noted a complete exine structure for both the wild type (Fig. 4, A–C) and osgt1 (Fig. 4, D–F) at stage 11a. However, differences were observed at stage 11b. Whereas wild-type grains had evenly stained cytoplasm (Fig. 4, G and H), mutant pollen showed severely degraded cytoplasm (Fig. 4, J and K). At higher magnification, intine accumulation was observed in wild-type pollen (Fig. 4I), but the intine was defective in osgt1 grains (Fig. 4L). At stage 12, these differences were more apparent. Whereas wildtype grains had accumulated starch granules as well as a thick intine (Fig. 4, M–O), approximately 50% (72 of 129) of the pollen from OSGT1/osgt1 anthers had an altered cytoplasm density and a severe defect in intine accumulation (Fig. 4, P–R). Because intine development precedes the accumulation of starch grains, proteins, and other inclusions in the cytosol (Lu et al., 2002; Lin et al., 2009), we speculated that OsGT1 plays a key role in the former process.


Image3-.png Figure 3. Phenotypes of pollen grains. A to D, Auramine O staining of grains from segregating wild-type (A and C) and OsGT1/ osgt1-1 (B and D) plants at stage 12 observed under bright-field (A and B) and fluorescence (C and D) microscopy. E to H, Calcofluor white staining of pollen grains from segregating wild-type (E and G) and OsGT1/osgt1-1 (F and H) plants at stage 12 observed under bright-field (E and F) and fluorescence (G and H) microscopy. I to P, DAPI staining of pollen grains from segregating wild-type (I, K, M, and O) and OsGT1/osgt1-1 (J, L, N, and P) plants at stage 12 (I–L) and stage 11 (M–P) plants observed under bright-field (I, J, M, and N) and fluorescence (K, L, O, and P) microscopy. Arrows indicate nucleus. DP, Defective pollen. Bars = 20 mm.


Image4.png

Figure 4. TEM analyses of developing anthers from wild-type (A–C, G–I, and M–O) and OsGT1/osgt1-1 (D–F, J–L, and P–R) plants at stage 11a (A–F), stage 11b (G–L), and stage 12 (M–R). Ba, Bacula; BP, binuclear pollen; Cy, cytosol; DP, defective microspores; En, endothecium; Ep, epidermis; Ex, exine; In, intine; MP, mature pollen; Ne, nexine; Or, orbicule; SG, starch granule; Te, tectum. Bars = 10 mm (A, D, G, J, M, and P), 2 mm (B, E, H, K, N, and Q), and 0.5 mm (C, F, I, L, O, and R).


OsGT1 Functions at a Late Stage of Pollen Development

Recent genetic and biochemical investigations have uncovered several key genes required for pollen wall development in rice. For example, UDP-glucose pyrophosphorylase1 (UGP1) catalyzes the reversible production of Glc-1-P and UTP to UDP-Glc and pyrophosphate. In Ugp1-silenced plants, callose deposition is disrupted during meiosis. Consequently, the pollen mother cells begin to degenerate at the early meiosis stage, eventually resulting in complete pollen collapse (Chen et al., 2007). This indicates that Ugp1 plays a very early role (stage 6) in anther development (Fig. 8). TAPETUM DEGENERATION RETARDATION (TDR), GAMYB, PERSISTENTTAPETAL CELL1 (PTC1), CYP704B2, DEFECTIVE POLLEN WALL (DPW), POST-MEIOTIC-DEFICIENT ANTHER1 (PDA1), OsC6, and WAX-DEFICIENT ANTHER1 (WDA1) are critical for exine development (Jung et al., 2006; Zhang et al., 2008, 2010; Aya et al., 2009; Hu et al., 2010; Li et al., 2010a, 2011; Shi et al., 2011). In their mutants, microspores display a defective exine layer. TDR and GAMYB are transcription factors that control lipid biosynthesis and metabolism. PTC1 is orthologous to MS1 and regulates timely degradation of tapetal cells as well as pollen exine development. CYP704B2 catalyzes the production of v-hydroxylated fatty acid with 16- and 18-carbon chains. DPW is a fatty acid reductase and produces 1-hexadecanol with higher specificity against the palmiltoyl-acyl carrier protein (Shi et al., 2011). OsC6 is a lipid transfer protein; expression of OsC6 is positively regulated by TDR. WDA1 is involved in the biosynthesis of cuticular waxes. Mutants defective in these genes start to show defects at stages 8 through 10 (Fig. 8). We observed that the defective phenotypes of osgt1 pollen began to appear at stage 11b, which is much later than the stages when the phenotypes became obvious in the exine-defective mutants. Mutationsin RICE IMMATURE POLLEN1 (RIP1), which encodes a WD40 protein, result in defects at late stage 11b (Han et al., 2006). Because the rip1 mutant develops sperm cells normally, and the mutant pollen grains are nearly normal at the mature stage, we conclude that RIP1 functions downstream of OsGT1 (Fig. 8).

Image8.png

Figure 8. Schematic diagram of rice pollen wall development based on stages described by Li et al. (2010). Genes involved inthis process are represented. Arrows indicate times when mutant phenotypes become visible.

Expression

Expression of OsGT1 Is High at Later Pollen Developmental Stages

To understand the role of OsGT1 in pollen development, we performed quantitative reverse transcription (RT)-PCR with gene-specific primers. Transcripts were weakly detected in the roots, shoots, and shoot apical meristems of seedlings and mature leaves as well as in reproductive organs such as inflorescences and developing seeds (Fig. 5A). During anther development, the OsGT1 expression signal increased at stage 9 (young microspore) and was high at stage 12 (mature pollen; Fig. 5A), suggesting that this latter developmental stage had the greatest requirement for OsGT1 activity.

We used anthers from wild-type and heterozygous plants to measure OsGT1 expression, because separation of osgt1 pollen from wild-type grains at stage 11 by Suc density gradient centrifugation was unsuccessful. As expected, the transcript level in heterozygous plants was about one-half that measured from the wild type (Fig. 5B).

To explore the comprehensive expression patterns of OsGT1, we generated transgenic rice plants expressing the GUS reporter gene under the control of the OsGT1 promoter. Strong GUS activity was detected in the anthers and carpels (Fig. 5C). In mature anthers, activity was preferentially detected in the pollen. Only one-half of the mature grains were GUS positive, because we had used primary transgenic plants (Fig. 5D).

Image5.png

Figure 5. Expression pattern of OsGT1. A, Quantitative RT-PCR analyses in various organs. Transcript levels were normalized to UBIQUITIN and calculated by the comparative cycle threshold method. Error bars indicate SD. 7DR, Seven-day-old seedling roots; 7DS, 7-dold seedling shoots; SAM, shoot apica meristem; ML, mature leaves; IF, immature panicles (2 cm long); 3DAPS, seeds at 3 d after pollination; stages 7 to 12, anthers at different developmental stages. B, Quantitative real-time RT-PCR analyses of OsGT1 in wild-type and heterozygous anthers at mitotic stage. C, GUS expression in a spikelet harboring an OsGT1 promoter::GUS construct. An, Anther; Ca, carpel; Pa, palea. Bar = 1 mm. D, GUS staining of pollen grains. Bar = 10 mm.

Evolution

OsGT1 Encodes a Glycosyltransferase

To examine whether OsGT1 has glycosyltransferase activity, we purified a glutathione S-transferase (GST)- OsGT1 fusion protein after expression in Escherichia coli (Supplemental Fig. S6). Enzymatic activity was tested by measuring the conversion of quercetin to quercetin monoglucosides. HPLC analysis of the reaction product showed that quercetin was converted to quercetin-3-Oglucoside and quercetin-7-O-glucoside (Fig. 6F). Using AtGT-2 protein as a positive control, we found a similar HPLC pattern in which quercetin monoglucosides were produced from quercetin (Fig. 6E; Kim et al., 2006). By contrast, the negative-control GST protein did not produce monoglucosides (Fig. 6D). These results confirmed that OsGT1 is a newly discovered member of the glycosyltransferase family. Our attempt to identify a specific target molecule for OsGT1 was unsuccessful. Identifying a specific target will be difficult because the GT4 family has both functional and sequence diversity (Kawai et al., 2011). Rice has 609 glycosyltransferases that are classified into 41 GT families (Cao et al., 2008). OsGT1 belongs to the GT4 family, members of which utilize diverse substrates such as proteins, lipids, and sugars (Dörmann et al., 1999; Silverstone et al., 2007). Moreover, OsGT1 is structurally distinct from other members in the family (http://ricephylogenomics.ucdavis.edu/cellwalls/gt/), suggesting that it has a unique role.

Image6.png

Figure 6. Glycosyltransferase assay. A to C, Quercetin (A) and its glucosides, quercetin-3-O-glucoside (B) and quercetin-7-Oglucoside (C), were separated by HPLC. D and E, Chromatograms of assay products using GST protein as a negative control (D) and AtGT-2 protein as a positive control (E). F, Chromatogram of OsGT1 reaction products. Peak Q, Quercetin; peak 3, quercetin-3-O-glucoside; peak 7, quercetin-7-O-glucoside.


OsGT1 Is Localized in the Golgi Apparatus

Most glycosyltransferases act along a secretory pathway that includes the endoplasmic reticulum and the Golgi, where they transfer monosaccharide units to various receptor molecules (Priest et al., 2006; Yin et al., 2010). To investigate the subcellular localization of OsGT1, we generated a fusion construct of OsGT1 and GFP. This fusion molecule was placed under the control of the maize (Zea mays) UBIQUITIN (Ubi) promoter, and the chimeric molecule was transformed into mesophyll protoplasts. As a control, we cotransferred the MANNOSIDASE I (ManI)-RED FLUORESCENT PROTEIN (RFP) gene, which localizes at the Golgi (Kim et al., 2010). After incubating the transformed cells for 12 h, we monitored transient expression of the introduced molecules using a confocal microscope (Fig. 7, A and B). The GFP and RFP signals overlapped, as indicated by orange coloring, suggesting that OsGT1 is colocalized with ManI in the Golgi (Fig. 7C). To confirm this, we generated another OsGT1-GFP fusion constructunder the control of the 35S promoter and introduced the molecule together with ManI-RFP into onion (Allium cepa) epidermal cells via bombardment. From the mesophyll cells, we observed an overlap of the green signal generated by OsGT-GFP and the red signal by ManI-RFP (Fig. 7, E–G). In contrast, we found green signals produced by GFP alone in the nuclei and cytoplasm of mesophyll protoplasts (Fig. 7I) and onion epidermal cells (Fig. 7J). These observations demonstrated that OsGT1 is localized to the Golgi apparatus.

Image7.png

Figure 7. Subcellular localization. A to D, Mesophyll protoplasts prepared from young seedlings were cotransformed with fusion constructs Pubi::OsGT1-GFP and P35S::ManI-RFP. Images observed under GFP channel (A) and RFP channel (B) were merged (C); D is a bright-field image. E to H, Onion epidermal cells were cobombarded with fusion constructs. The GFP channel image (E) and RFP channel image (F) were merged (G); H is a bright-field image. I, GFP image of mesophyll cells expressing control construct Pubi::GFP. J, GFP image of onion epidermal cell expressing control construct P35S::GFP. Bars = 5 mm for mesophyll protoplasts and 50 mm for onion cells.




You can also add sub-section(s) at will.

Labs working on this gene

Crop Biotech Institute and Department of Plant Molecular Systems Biotechnology, Kyung Hee University, Yongin 446–701, Korea

State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

References

Moon S, Kim S R, Zhao G, et al. Rice glycosyltransferase1 encodes a glycosyltransferase essential for pollen wall formation[J]. Plant physiology, 2013, 161(2): 663-675.

Structured Information