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Journal of Integrative Plant Biology 2013, 55 (8): 710–720

Research Article

The ATP‐binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice Bai‐Xiao Niu1††, Fu‐Rong He1†, Ming He1†, Ding Ren1, Le‐Tian Chen1,2* and Yao‐Guang Liu1* 1

State Key Laboratory for Conservation and Utilization of Subtropical Agro‐bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China 2 Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China † These authors contributed equally to this work. †† Present address: State Key Laboratory of Genetic Engineering, Institute of Plant Biology, Center for Evolutionary Biology, School of Life Sciences, Fudan University, Shanghai 200433, China Corresponding authors Tel: þ86 20 8528 1908; Fax: þ86 20 8528 0200; E‐mail: [email protected], [email protected] Articles can be viewed online without a subscrption. Available online on 9 April 2013 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipb doi: 10.1111/jipb.12053

Abstract Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map‐ based cloning and sequence analysis, we identified a 1,459‐bp deletion in an adenosine triphosphate (ATP)‐binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very‐long‐chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development. Keywords:

ABC‐transporter; male sterility; pollen exine; rice (Oryza sativa L.).

Niu BX, He FR, He M, Ren D, Chen LT, Liu YG (2013) The ATP‐binding cassette transporter OsABCG15 is required for anther development and pollen fertility in rice. J. Integr. Plant Biol. 55(8), 710–720.

Introduction Rice (Oryza sativa L.) is not only an important food crop but also an excellent model monocot plant for genetic and functional genomic studies (Jung et al. 2008). The fertility of rice pollen grains is a critical agronomic trait affecting rice yield, and pollen sterility eliminates the difficulty of emasculation, thus it is a

© 2013 Institute of Botany, Chinese Academy of Sciences

simple but effective way for hybrid rice production (Cheng et al. 2007; Li et al. 2007). Production of pollen is a critical process of sexual plants during anther development in the lifecycle (Itoh et al. 2005). Briefly, anther primordial cells generate meiocytes and four lobed cell layers, including the epidermis, endothecium, middle layer, and tapetum. After meiosis, microspores are released from tetrads, and the tapetum

Rice OsABCG15 Functions in Pollen Fertility

functions as a nutrient source for developing microspores (Ma 2005; Wilson and Zhang 2009). These processes involve a large number of genes expressed in various tissues of the anther (Guo and Liu 2012). Adenosine triphosphate (ATP)‐binding cassette (ABC) transporters belong to a super proteins family in bacteria, yeasts, plants, and animals (Guidotti 1996). There are 120 and 121 ABC transporter members in the genome of Arabidopsis and rice, respectively; and these proteins can be subgrouped into seven major subfamilies of ABC ATPases (A–G) (Garcia et al. 2004). Most of these proteins transport various substrates via transmembrane domains using energy released by ATP hydrolysis at nucleotide‐binding domains (Schwiebert et al. 1998). The substrates of ABC transporters include organic anions, heavy metals, hormones, fatty acids, ions and secondary metabolites, thus these gene family members contribute to almost all aspects of plant development. They are also involved in responses to biotic (pathogens) and abiotic (e.g., heavy metals, drought, cold) stresses in plants (Verrier et al. 2008). The subfamily G of the ABC family is markedly expanded in plants, formally called white‐brown complex (WBC) (Ewart and Howells 1998), with more than 40 members in both Arabidopsis and rice, respectively (Borst et al. 2000). The functions of plant WBC homologs were obscure until the discovery that AtABCG11 (AtWBC11) and AtABCG12 (AtWBC12, CER5) are required for cuticular lipid export (Pighin et al. 2004). In Arabidopsis, AtABCG12 transports the very long‐chain fatty acids that form wax precursors, and ABCG11 is involved in the transport of both long‐chain and very‐long‐chain fatty acids, i.e. precursors of cutin and wax, respectively (Panikashvili et al. 2007). ABCG19 confers kanamycin resistance to tobacco (Mentewab and Stewart 2005). ABCG25 and ABCG40 mediate ABA efflux and influx, respectively, as part of the drought resistance response in plants (Kang et al. 2010; Kuromori et al. 2010). Recently, AtABCG26 was reported to transport sporopollenin precursors across the tapetum plasma membrane into the locule for polymerization on developing microspore walls, playing a critical role in exine formation and pollen development (Quilichini et al. 2010; Choi et al. 2011; Dou et al. 2011). However, the function of ABCG subfamily in rice remains poorly understood. In this study, we identified a new male sterility mutant that shows a defect in the pollen maturation. Map‐based cloning revealed that this mutant is caused by a 1,479‐bp deletion in an ABC transporter gene OsABCG15. Overexpression of OsABCG15 in the mutants recovered the male fertility. OsABCG15 is expressed specifically in anther tapetum during microspore development. The key regulators required for sporopollenin biosynthesis, CYP704B2 and WDA1 were downregulated in this mutant, suggesting that OsABCG15 is a key regulator for rice postmeiotic anther development.

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Results Identification and phenotypic analyses of the male sterile mutant We generated a rice mutant library of a rice cultivar 02428 (O. sativa ssp. japonica) by 60Co g‐ray irradiation (Y‐G Liu, unpubl. data, 2008). One of the mutants produced more tillers (Figure 1A), and exhibited collapsed anthers with smaller size and white color, and no pollen grains, thus leading to complete spikelet sterility (Figure 1B–D). Genetic analysis indicated that the male sterility of this mutant is sporophytic and is controlled by a single recessive locus (Table S1). We named this mutant osabcg15 because it was caused by a deletion‐mutation in the ABC transporter gene OsABCG15 revealed by the following map‐based cloning (see below).

Map‐based cloning of the gene confers to the male sterile mutant We used a map‐based cloning approach to identify the gene for this male sterile mutant. InDel and simple sequence repeat (SSR) markers distributed over the rice genome were designed based on sequence difference between indica cultivar 93‐11 and japonica cultivar Nipponbare, and applied for mapping of the male‐sterile locus. Using 190 F2 sterile plants from a cross of this mutant with an indica variety Huanghuazhan (HHZ), the target locus was primarily mapped to a 286‐kb region on chromosome 6. This locus was then fine‐mapped to a 43‐kb region defined by two flanking InDel markers M4 and M6 with an additional 156 recombinant individuals derived from a large F2 population consisting of about 10 000 plants (Figure 2A, Table S2). A BAC clone P0029C06 covers this target region containing two predicted open reading frames (ORFs), Os06g0607700 and Os06g0607800 (Figure 2B). To determine which ORF in the target region confers to male sterility in the mutant, we amplified and sequenced the mapped region from four male‐sterile F2 plants and three fertile cultivars, 02428, HHZ and Zhonghua11 (ZH11). A 1,479‐bp deletion was detected in Os06g0607700 in these four male‐sterile plants, while this gene was intact in all three wild‐type cultivars, indicating that Os06g0607700 is the candidate gene for the male sterility mutant (Figure 2B). According to the deletion in Os06g0607700, two primers F1 and R1 were designed for association analysis (Figure 2B). The polymerase chain reaction (PCR) results were consistent with the sequencing data, demonstrating that the deletion was co‐segregated with the sterile phenotype in these recombinant individuals (Figure 2C). Bioinformatic analysis showed that Os06g0607700 consists of seven exons and six introns, and encodes an ABC transpoter OsABCG15 with unknown function (Verrier et al. 2008).

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Figure 1. The phenotype of osabcg15 mutant. (A) Phenotypic difference between the wild type (WT) and osabcg15 mutant after heading. The osabcg15 mutant produced more tillers with similar plant height. Bars ¼ 10 cm. (B) Spikelets of WT and osabcg15. The anthers in WT (upper) were yellow color, while the anthers in osabcg15 (lower) were whitish. Bars ¼ 2 mm. (C) The anthers of WT and osabcg15 stained with I2–KI. The WT anther (left) was black for the staining of starch of pollen grains by I2–KI, while the anther of osabcg15 (right) remained whitish for lack of pollen grains. Bars ¼ 0.5 mm. (D) No seed‐setting in osabcg15. Bars ¼ 2 cm.

Therefore, this sterile mutant was designated as osabcg15. To test the function of OsABCG15, a binary construct of OsABCG15 driven by the CaMV35S promoter (P35S::OsABCG15) was transformed into the homozygous osabcg15 mutants (derived from heterozygotes). The expression level of the transgene was examined by quantitative reverse transcription‐PCR (qRT‐PCR) and the fertility phenotype was demonstrated by I2–KI staining in T1 progenies (Figure 2D, E). The pollen fertility of independent transgenic plants (P35S::OsABCG15) was restored to 50–90% (Figure 2E) and the tiller number of these transgenic lines recovered to normal as wild type (data not shown). These results demonstrated that OsABCG15 is required for the anther development and pollen fertility in rice.

OsABCG15, there is a P‐loop NTPase domain (aa 81–289), which has been demonstrated to bind and hydrolyze ATP (Figure 3B). In addition, there is an ABC transporter signature motif within this region. In the C‐terminal region OsABCG15 possesses a typical ABC2 trans‐membrane domain (aa 417–626) (Figure 3B). The 1,459‐bp deletion in osabcg15, including the 6th exon, resulted in a 131‐aa truncated protein excluding most of the trans‐membrane domain of OsABCG15 (Figures 2B, 3B). OsABCG15 shares high similarity in amino acid sequences with the known fertility‐related Arabidopsis orthologs such as AtABCG26 (76%), AtABCG11 (23.6%), and AtABCG12 (23.7%) (Garcia et al. 2004) (Figure 3C).

The temporal‐special expression of OsABCG15 OsABCG15 is conserved in plants OsABCG15 belongs to the G subfamily of ABC transporter with size of 688 amino acid (aa) and is highly conserved in plants (Figure 3A, Figure S1, Table S3). In the N‐terminal region of

To know the temporal‐spatial expression of OsABCG15, we investigated the expression profiles of OsABCG15 in different tissues and the developmental anthers. In rice, anther development has been divided into 14 stages, according to


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Figure 2. Map‐based cloning of OsABCG15. (A) OsABCG15 was fine‐mapped into a 43‐kb region on chromosome 6. The key InDel markers, marker positions and recombinants were indicated accordingly. (B) Two candidate genes Os06g0607700 (black) and Os06g0607800 (gray) were predicted in the mapped 43‐kb region. Sequence analysis revealed a 1,479‐bp deletion in Os06g0607700. F1 and R1 were primers for detection of the deletion in (C). (C) Association analysis by PCR of the 1,479‐bp deletion in rice materials with different genotype. The specific primers for association analysis were indicated in (B). PCR product from homozygotic fertile lines 02428, HHZ, ZH11 contained an intact 2‐kb fragment, while PCR product from the homozygous sterile lines F3‐8, R13‐4 was a 0.6‐kb fragment. Both 2‐ and 0.6‐kb fragments were detected in the heterozygous (male‐fertile) F1 and recombinant (R15‐3) plants. (D) Overexpression of P35S::OsABCG15 in anthers of transgenic T1 plants. The construct was transferred into calli from seeds of fertile lines with heterozygous mutant locus. OX‐5 and OX‐9 were two male‐fertile T1 plants that were homozygous for osabcg15. The primers (osabcg15‐ OX) for the qRT‐PCR were located in the deleted exon. Actin1 gene served as the reference. Data are shown as means SD (n ¼ 3). (E) Pollen fertility of a T1 plant shown by I2–KI staining.

morphological landmarks of cellular events (Zhang et al. 2011). Our quantitative reverse transcription PCR (qRT‐PCR) analysis demonstrated that OsABCG15 specifically expressed in the anthers (Figure 4A). During the development of anthers, OsABCG15 is highly expressed from stage 8 (meiosis II stage) to stage 10 (late microspore stage), and reached the peak at stage 9 (early microspore stage; Figure 4B). Furthermore, in situ hybridization was performed to determine the tissue specificity of OsABCG15 at a cellular level during the anther development. Consistently, the expression of OsABCG15 started from stage 7

and reached the maximum at stage 9 in the tapetal cells and microspores, followed by decreasing onwards (Figure 4C). These data suggested that OsABCG15 may function specifically in the anther and pollen development. Since osabcg15 mutant produced more tillers (Figure 1A), we examined the expression of OsABCG15 in the stem and shoot apical meristem (SAM). The results indicated that the transcriptional level of OsABCG15 was undetectable in the stem and SAM, while the ORF Os06g0607800 expressed in all tested tissues/organs (Figure S2).

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Figure 3. OsABCG15 is a conserved protein. (A) Amino acid sequences of OsABCG15; Italic amino acid (site 81–289) indicates P‐loop NTPase domain, bold amino acid (site 417–626) indicates trans‐membrane domain (ABC2 domain). The underlined region (site 469–599) was deleted in osabcg15 mutant. (B) The conserved domains of OsABCG15. (C) Phylogenetic relationship of OsABCG15 and three fertility‐associated ABC transporters in Arabidopsis.

OsABCG15 may affect anther development by affecting expression of genes involved in biosynthesis of long‐ chain fatty acid To reveal the regulatory relationship between OsABCG15 and other known regulators for the anther development, the expression of six anther development‐associated genes WDA1, CYP704B2, UDT1, OsCP1, GAMYB, and TDR was analyzed by qRT‐PCR (Kaneko et al. 2004; Lee et al. 2004; Tsuji et al. 2006; Aya et al. 2009, 2011a; Zhang et al. 2010). No obvious changes were observed in the transcriptional level of OsCP1, GAMYB in the osabcg15 mutant, suggesting that the male sterility in osabcg15 mutant is independent from the pathway regulated by OsCP1 and GAMYB (Figure 5). However, the expression of two genes for long‐chain fatty acid biosynthesis, WDA1 and CYP704B2, was dramatically decreased in osabcg15 mutant plants (Figure 5). In contrast, the TDR was upregulated in osabcg15 mutants (Figure 5).

The osabcg15 mutant is defective in tapetum function failing to produce mature pollen To determine at which stage the osabcg15 mutant showed abnormal morphological defects during anther development leading to male sterility, semi‐thin transverse sections of anthers were performed at different developmental stages in osabcg15 mutant plants. The microspore mother cells (MMCs) of osabcg15 mutant appeared to undergo meiosis (S7) and form tetrads (stage 8), similar to those of wild‐type plants (Figure 6A). The obvious aberration of anther development in osabcg15 occurred during the microspore development, in which tapetal cells exhibited hill‐shape structure at stage 10 and degenerated more rapidly than wild‐type (Figure 6B). At the anther developmental stage 10, the microspore of the WT normally vacuolated with an increase of volume, resulting in a round shape, and the tapetal cells degenerated or disappeared, whereas in the osabcg15 mutant the microspores were


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degraded from stage 9, therefore, no mature pollen grains were produced (Figure 6B).

Discussion

Figure 4. The temporal‐special expression of OsABCG15. (A) OsABCG15 is specifically expressed in the anther. Actin1 gene served as the reference. Data are shown as means SD (n ¼ 3). (B) The expression of OsABCG15 during the developmental anthers. OsABCG15 could be detected in stages 8–10 with a maximum level in stage 9. Actin1 gene served as the reference. Data are shown as means SD (n ¼ 3). (C) In situ hybridization of OsABCG15. OsABCG15 is expressed in the tapetum cells and microspores with a maximum level at stage 9. Sense RNA of OsABCG15 served as negative control at stage 9. Bars ¼ 50 mm.

In rice, a number of genes involved in anther differentiation and development have been identified (Wilson and Zhang 2009; Zhang et al. 2011; Guo and Liu 2012). For the transcriptional levels of OsCP1, GAMYB and UDT1 in osabcg15 remained similar to those of WT (Figure 5), the male sterility of osabcg15 mutant may be independent from the pathway regulated by OsCP1, GAMYB and UDT1. CYP703A3, CYP704B2, and Osc6 have roles in sporopollenin biosynthesis or transport (Zhang et al. 2010; Aya et al. 2011b). CYP704B2 catalyzes the production of omega‐ hydroxylated fatty acids essential for the formation of both cuticle and exine during plant male reproductive and spore development (Li et al. 2010). WDA1 is homologous to Arabidopsis CER1, which mainly expresses in the epidermal cells of anther walls, and is proposed to participate in the biosynthesis of very‐long‐ chain fatty acids for the establishment of the anther cuticle and pollen exine (Aarts et al. 1995; Jung et al. 2006). Previous biochemical analyses have shown that the exine layer consists mainly of sporopollenin, a polymer of phenylpropanoid and lipidic monomers covalently linked by ether and ester linkages (Ahlers et al. 2003). In the latest model of the sporopollenin biosynthetic pathway, fatty acid precursors are esterified to CoA by ACOS5, and then hydroxylated by CYP703A and CYP704B to produce substrates of the subsequent ACOS5 reaction. Subsequently, ACOS5, PKSs, TKPRs, and MS2 mediate the biochemical reactions to produce sporopollenin precursors via fatty alcohols (Wilson and Zhang 2009; Ariizumi and Toriyama 2011; Aya et al. 2011b). It was proposed that OsABCG15 may transfer sporopollenin from tapetal cells to anther locules as a transporter in the processes related to sporopollenin biosynthesis (Aya et al. 2011b). Based on the morphological investigations of the anther development, the morphologies of primary sporogenous cells and the four layers of the anther wall are normal in the osabcg15 mutants till stage 8. However, in the anthers of osabcg15, the tapetum degeneration is abnormal during the microspore developmental stages, resulting in degradation of the microspores. On the other hand, our RT‐PCR analysis also revealed the defective osabcg15 resulted in the dramatically downregulated expression of CYP704B2 and WDA1. Therefore, OsABCG15 may function coordinately with CYP704B2 and WDA1 for pollen development. Arabidopsis and rice may use similar regulatory pathways for pollen development (Wilson and Zhang 2009; Zhang et al. 2011). Many rice genes involved in pollen development are orthologous or functionally equivalent to those genes in Arabidopsis. Recently, several ABC transporters have been shown to be

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Figure 5. Expression of known regulators of anther development in osabcg15 mutant. The expression of six anther development related regulators WDA1, CYP704B2, UDT1, CP1, GAMYB, TDR1 were examined in the WT and the osabcg15 mutant. WDA1, CYP704B2 were downregulated dramatically in the osabcg15 mutant. Actin1 gene served as the reference. Data are shown as means SD (n ¼ 3).

involved in male sterility in Arabidopsis (Panikashvili et al. 2007; Quilichini et al. 2010; Choi et al. 2011; Dou et al. 2011), whether ABC homologs in rice also function in fertility and anther development was unknown. AtAMS binds the E‐boxes in the AtABCG26 promoter and thereby regulates AtABCG26 expression (Xu et al. 2010). This study suggested that the AtABCG26 ortholog OsABCG15 in rice is one of the important components for sporopollenin synthesis and secretion, functioning as transporter of sporopollenin precursors, translocating its substrates from tapetal cells to the developmental microspores. The increase of tiller number in osabcg15 mutants may be a side‐effect of the loss‐of‐function mutation in OsABCG15. We reasoned that mutated OsABCG15 causes rice to fail to complete the reproductive processes, the extra nutrition and energy turn into vegetative growth producing more tillers; the overexpression of OsABCG15 in the transgenic plants produced normal seeds, and the tiller number recovered to normal. This possibility needs to be confirmed by further experimental evidence. Most recently, during the preparation of our manuscript, Qin et al. (2013) reported an allelic mutant of OsABCG15 due to a 12‐ bp deletion in the gene and revealed similar function in microspore development. Our results provided valuable information to understand the function of OsABCG15 in the anther development and male sterility in rice.

Materials and Methods Growth conditions of plant materials Mutant osabcg15 was obtained from a 60Co‐g‐ray‐treated rice (Oryza sativa L.) mutant library. All rice materials were grown in the paddy field of South China Agricultural University, Guangzhou. The F2 mapping population was generated from a cross between the mutant osabcg15 (02428, japonica) and Huanghuazhan (HHZ, indica). In the F2 population, male sterile plants were selected for gene mapping. Then seedlings of F2 were planted in 96‐well plates for screening recombinants in a high‐ throughput way (Wang et al. 2013).

Characterization of the mutant phenotype with microscopy Plants and flowers at mature stage were photographed with a Nikon digital camera. The wild‐type and mutant anthers were immersed into I2–KI solution and centrifuged for a short time to make the I2–KI solution to enter into the anther for pollen starch staining, and photographed with a DMLB microscope (Leica, Germany). Transverse sections of anthers were obtained using the plastic embedded sectioning method and were carried out according to the manufacturer instructions (Leica His toresin Kit). Flowers from WT and osabcg15 mutant at different stages were


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Figure 6. The osabcg15 mutant had abnormal tapetum degeneration and degradation of microspores. (A) Transverse sections of the anther development were performed in wild type (WT) and the osabcg15 mutant. There was no obvious difference between WT and mutant in stages 5–8. (B) In osabcg15 the tapetal cells showed hill‐shape structure at stage 10 and degenerated more rapidly than WT; the microspores were degraded from stage 9, thus, no mature pollen grains were produced. The mutant showed less stained and condensed tapetal cells at stage 9; the mutant displayed less cytoplasm and obvious degraded remnants in the locule, microspores showed irregular shape and degraded at stage 10; microspores further degraded in the mutant at stage 11; collapsed anther without pollen grains showed in the mutant at stage 12. E, epidermis; En, endothecium; ML, middle layer; Mp, mature pollen; Msp, mi crospore; T, tapetum. Bars ¼ 20 mm.

fixed overnight in FAA (38% formaldehyde 5 mL, acetic acid 5 mL, 50% alcohol 90 mL), dehydrated through an ethanol series (from 85% to 100%), embedded into 7100 (Heraeus Kulzer GmbH) and sectioned with Microtome (Leica, Germany). The 1–3 mm thick sections on slides were stained with 0.25% toluidine blue O, observed under microscope (Leica, Germany).

Molecular mapping The F2 seeds were collected from F1 heterozygous mutants and the F2 generation seedlings were used for segregation analysis. In the F2 population, male sterile plants with smaller and white anther were selected and used for gene mapping. The rice

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genomic DNA samples were prepared from the fresh leaf tissues of the field grown F2 plants using 1% sodium dodecyl sulfate (SDS). The polymorphic InDel markers were obtained by comparison of the sequences in japonica rice Nipponbare and indica rice 93‐11 (http://www.ncbi.nlm.nih.gov/; http://www.rgp.dna.affrc. go.jp/ for japonica), and applied for linkage analysis (Table S2). One hundred and fifty‐six recombinants screened from 10,000 individuals of F2 population were used for primary mapping and fine‐mapping.

qRT‐PCR Total RNA of anthers at different stages was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as described by the supplier. The isolated RNA was treated with DNase (Promega, Madison, WI, USA), 0.3 mg RNA was used for the oligo (dT) primed first‐strand cDNA synthesis using the first strand cDNA synthesis kit (Promega). Three microliters of the RT products were then used as the template of PCR reactions. All the primers for RT‐PCR are listed in Table S2. Quantitative RT‐PCR analyses were performed using the CFX96 Real‐Time PCR System (Bio‐Rad Laboratories, CA, USA). Reactions contained the Brilliant SYBR Green QPCR Master Mix (TaKaRa, Otsu, Japan) in a final volume of 25 mL with 2 pmole of the appropriate primers (Table S2) and 5 mL of cDNA. PCR cycling conditions for amplification were 95 °C for 5 min followed by 40 cycles of 95 °C for 15 s, 55 °C for 15 s and 72 °C for 15 s. Each experiment was biologically repeated three times, each with three replicates. Data acquisition and analyses were performed using the Bio‐ Rad CFX Manger 2.1 software. Samples were normalized using Actin1 expression. Relative expression levels were measured using the 2DC t analysis method.

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polymerase, using the DIG RNA labeling kit (Roche, Basel, Switzerland). In situ hybridization was performed according to Yong et al. (2003).

Acknowledgements This work was supported by a grant from the National High Technology Research and Development Program of China (2012AA10A303). Received 31 Jan. 2013

Accepted 27 Mar. 2013

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Figure S1. Phylogenetic relationship of ABCG15 homologs in soybean (Glycine max), poplar (Populus trichocarpa), Arabidopsis (Arabidopsis thaliana), cotton (Gossypium raimondii), cucumber (Cucumis stativus), rice (Oryza sativa), and maize (Zea mays). Figure S2. Expression profiles of Os06g0607800 in rice. Os06g0607800 expressed in the leaf, panicle (Stage 5), leaf

2013

sheath, stem, shoot apical meristem (SAM), and root, while the transcript of OsABCG15 was not detectable in the above tissues/ organs tested. Table S1. Genetic analysis of locus controls the phenotype of osabcg15. Table S2. Primers used in this study. Table S3. ABCG15 in different plant species.