OsACOS12, an orthologue of Arabidopsis acyl-CoA synthetase5, plays an important role in pollen exine formation and anther development in rice

Sporopollenin is a major component of the pollen exine pattern. In Arabidopsis, acyl-CoA synthetase5 (ACOS5) is involved in sporopollenin precursor biosynthesis. In this study, we identified its orthologue, OsACOS12, in rice (Oryza sativa) and compared the functional conservation of ACOS in rice to Arabidopsis. Sequence analysis showed that OsACOS12 shares 63.9 % amino acid sequence identity with ACOS5. The osacos12 mutation caused by a pre-mature stop codon in LOC_Os04g24530 exhibits defective sexine resulting in a male sterile phenotype in rice. In situ hybridization shows that OsACOS12 is expressed in tapetal cells and microspores at the transcript level. The localization of OsACOS12-GFP demonstrated that OsACOS12 protein is accumulated in tapetal cells and anther locules. OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis. OsACOS12 is an orthologue of ACOS5 that is essential for sporopollenin synthesis in rice. ACOS5 and OsACOS12 are conserved for pollen wall formation in monocot and dicot species.


Background
Male reproductive development is an essential biological process for the propagation of flowering plants. Pollen development is the major event of male reproduction. Developmental defects leading to male sterility are widely used for hybrid production in agriculture [1]. During pollen development, pollen wall formation is a key process required for pollen viability and male fertility. The pollen wall structure divides into the outer exine and the inner intine. The exine is further divided into a species-specific sexine and a flat nexine [2]. The major composition of the sexine is sporopollenin [3], while the nexine is mainly composed of glycoproteins [4]. The biological function of the sexine layer is to provide an external barrier for adapting the terrestrial environment to ensure microgamete survival in land plants to resist various environmental stresses and microbial attacks [5,6]. The sexine patterning also acts as an important feature of plant taxonomic classifications [7].
Sporopollenin, the major constituent of the sexine, was considered to be a complex polymer primarily composed of long-chain fatty acids, oxygenated aromatic rings and phenylpropanoic acids [8]. The tapetal layer is an essential tissue required for normal sexine development and pollen maturity [9]. Based on cytological and molecular evidence, the material of sporopollenin precursors originate from tapetal cells [10,11]. The sporopollenin precursors are initially deposited at the mould of the sexine to form probaculae and protectum structures. After a microspore is released, the exine structure increases in size with continuous deposits and polymerization of sporopollenin until the decorated sexine pattern is formed [12]. However, the exact composition of sporopollenin precursors is not clear. In Arabidopsis, several genes have been reported to be involved in the complex biochemical pathways of sporopollenin precursor formation, including CYP703A2, CYP704B1 and MS2 for fatty-acid-derived compound metabolism, PKSA/B and TKPR1/2 for phenylpropanoids synthesis, and ABCG26 for transportation [13][14][15][16][17][18][19]. All these genes are expressed in the tapetal layer at the transcript level. On the protein level, MS2 is localized in tapetal cells, while CYP703A2 is in both tapetal cells and the anther locule [19,20]. It is likely that the last several steps of sporopollenin precursor synthesis occur in the locule.
The sporopollenin is a general constituent that has been widely found in moss, ferns, gymnosperms and angiosperms. Sporopollenin synthesis seems to share common metabolic pathways in various species. The tapetum directly provides materials for pollen wall formation. The genetic pathway for tapetum development is generally conserved between rice and Arabidopsis [21]. In rice, several sporopollenin enzymes have been identified [22][23][24][25]. The biological functions of these enzymes and the metabolic pathways for sporopollenin synthesis were very conserved between rice and Arabidopsis. However, the anther cuticle had defects in mutations of the rice genes discussed above, whereas homologous mutants in Arabidopsis did not show obvious morphological changes in the anther walls. It was suggested that the lipidic pathway was diversified in rice [22,25].
ACOS5 encodes a fatty acyl-CoA synthetase (ACOS) for sporopollenin precursor synthesis in Arabidopsis [26,27]. There are nine fatty acyl-CoA synthetase encoding genes in rice [28]. In this study, we characterized an orthologue of Arabidopsis ACOS5 in rice, OsACOS12 (LOC_Os04g24530). A knockout of this gene led to a male sterile phenotype in rice with a defective sexine layer and anther cuticle. The tapetal and anther locule localization of the OsACOS12 protein suggested that there was synchronous biosynthesis and transportation of sporopollenin precursors. The expression of the OsA-COS12 gene in the Arabidopsis acos5 mutant partially restored the male sterile phenotype, which indicated that the acyl-CoA synthetase gene is a conserved function between Arabidopsis and rice.

Results
OsACOS12 in O. sativa is an orthologue of ACOS5 in A. thaliana BLASTP analysis using Arabidopsis ACOS5 protein sequence yield the LOC_Os04g24530 in rice encoding a fatty acyl-CoA synthetase (http://rice.plantbiology.msu.edu/). The sequence of LOC_Os04g24530 also showed the highest sequence similarity with ACOS5 in Arabidopsis genome (63.9 % amino acid sequence identity, Fig. 1a).
LOC_Os04g24530 was designated OsACOS12 previously [26]. The large superfamily of acyl-activating enzymes contains putative motifs for AMP-binding and fatty acidbinding [29,30]. These motifs are highly conserved between OsACOS12 and ACOS5 (Fig. 1a). The homologues of OsACOS12 protein have been identified in various plant species by a BLASTP search according to the GenBank database. No orthologue could be identified in the genome of the green alga. Phylogenetic analysis shows OsACOS12 and its homologues formed four distinct clades. The homologues from Physcomitrella and Selaginella formed two distinct clades that diverged early in land plant evolution. The homologues from dicotyledoneae and monocotyledon species form two other clades (Fig. 1b). Sequence analysis demonstrated that ACOS enzymes are apparently present in land plants, which supports a possible role for them in the biosynthesis of sporopollenin, which is the demand for protecting gametophytes to adapt to a land environment.

osacos12 mutant shows complete male sterility
To characterize the function of OsACOS12, we obtained an allele of LOC_Os04g24530 using Targeting Induced Local Lesions In Genomes (Tilling) technology from the ethyl methane sulfonate-induced population of rice Zhonghua11 (O. sativa ssp. Japonica) [31]. Sequence analysis revealed a point mutation from A to T, in the 1000th base downstream of the start codon of the LOC_Os04g24530 genomic sequence in the mutant. This transition caused a premature termination (AAG-TAG) at the first exon of LOC_Os04g24530 (Fig. 2a). The osa-cos12 mutant exhibited normal vegetative and spikelet development (Fig. 2c, e). However, the mutant anthers had a white colour without pollen grains inside, which led to complete male sterility (Fig. 2c, f-h). Reciprocal crosses with the wild type indicted that female fertility was not affected in the osacos12 mutant. The fertile and sterile plants of the F 2 population segregated with a 3:1 ratio (86:23) indicated that there was a single recessive sporophytic mutation for osacos12. To complement the osacos12 mutant phenotype, the OsACOS12 genomic fragment fused with GFP driven by its own promoter (1428 bp) was constructed and transformed into heterozygous OsACOS12 seeds. Of the 32 transgenic lines, 7 were identified to have a homozygous osacos12 mutant background ( Fig. 2i-j). All of these homozygous lines exhibited normal fertility (Fig. 2d). These results demonstrate that OsACOS12 was responsible for the male fertility of the osacos12 mutant.
Pollen sexine formation and anther cuticle are defective in osacos12 Scanning electron microscopy (SEM) was used to elucidate the abnormal morphological defects of anther development in the osacos12 plant. The osacos12 anther was much shorter and smaller compared to that of the wild type ( Fig. 3a-b). In the wild type, the anther surface was covered by cuticle (Fig. 3c), and orbicules were intensively distributed on the inner surface (Fig. 3d). However, osacos12 anther surface was lack of these materials and looks smooth (Fig. 3g), and orbicules were barely detected in the inner surface (Fig. 3h). The wild type anther was filled with mature pollen grains ( Fig. 3e-f). In contrast, only a few remnants of degenerated pollen could be observed in the osacos12 anther ( Fig. 3i-j). In the complementary transgenic lines, the anther surface and pollen exine were restored (Additional file 1: Figure S1).
We subsequently obtained semi-thin transverse sections to understand the detailed defects of pollen development in osacos12. There were no detectable differences between wild type and osacos12 during early anther development. The microspore mother cells (MMCs) and tetrads of osa-cos12 appeared to be comparable with the wild type ( Fig. 3k-l, p-q). In the wild type, newly released microspores of the wild type were angular in shape (Fig. 3m) and became enlarged and vacuolated (Fig. 3n). In osacos12 plants, the released microspores contained much less cytoplasm (Fig. 3r) and were degenerated before/during volume enlargement (Fig. 3s). No mature pollen grains were observed in the locule of osacos12 at later stages of anther development (Fig. 3t). The defective phenotype of osacos12 was similar to acos5 in Arabidopsis.
To further clarify the details of the abnormal exine development of osacos12 pollen, anther samples were investigated using transmission electron microscopy (TEM). During the tetrad stage, primexine is formed between callose wall and plasma membrane. It is critical for pollen wall pattern. The primexine formation in osacos12 is consistent with that in wild type at tetrad stage ( Fig. 3u, x). At stage 9, the pollen exine in osacos12 was not as deeply stained as that in the wild type, indicating an abnormal sporopollenin deposition (Fig. 3v, y). At stage 10, the exine layer of microspore was formed with gradually deposition of sporopollenin precursors in the wild type (Fig. 3w). However, in osacos12, no sporopollenin precursor accumulated on the microspore surface resulted in absent exine layer phenotype of collapsed pollen grains (Fig. 3z). Additionally, there were no obvious aberrations in the appearance of the tapetum in the osacos12 mutant (Additional file 2: Figure S2). These observations revealed that the exine formation and cuticle structures of anther epidermis were abnormal in osacos12.

Defective wax components in osacos12 anthers
The defective anther cuticle and sporopollenin in osa-cos12 suggested that the lipidic mechanisms were aberrant in the mutant. To confirm this point, we performed gas chromatography-mass spectrometry (GC-MS) to quantify wax extracts from whole anthers of both the wild type and osacos12 mutants. The results showed that the total cuticular wax amount was reduced by approximately 42.6 % in the mutant (Fig. 4a), which contributed to the significant reduction of most wax constituents. The components of wax, including long-chain fatty acids (C14 to C26), alkanes (C30 to C36) and alcohols (C28 to C30) were significantly decreased in the osacos12 mutant (Fig. 4b). Therefore, chemical analysis indicated that OsACOS12 was involved in the synthesis of lipidic compounds during rice anther development.

OsACOS12 is located in the tapetum and anther locule
In Arabidopsis, ACOS5 is expressed in tapetal cells and microspores from late stage 5 to stage 8, as shown through in situ hybridization [26]. To analyse the expression of OsACOS12 in rice, semi-quantitative RT-PCR analysis was performed. OsACOS12 expression was detected in the anthers with glume lengths of 2.5 mm to 4.0 mm but was not detected in roots, shoots, leaves and palea/lemma (Fig. 5a). This result was further confirmed by quantitative real-time PCR analysis (Fig. 5b). Spatial and temporal expressions of OsACOS12 were detected by an RNA in situ hybridization analysis using an OsA-COS12-specific probe. The OsACOS12 was initially expressed in the tapetal layer and microspore mother cells at the beginning of meiosis (Fig. 5c-d). The signal was increased significantly and reached the highest level during the tetrad stage ( Fig. 5e-f ). At microspores stage, the signal of OsACOS12 transcripts was obviously decreased in the tapetum and microspores (Fig. 5g-h). In the control, only background signal was detected using a sense probe at tetrad stage ( Fig. 5i-j).
To understand the expression of OsACOS12 at the protein level, we analysed the GFP signal in the  (Fig. 2d). In this transgenic line, OsACOS12-GFP can complement the osa-cos12 phenotype. Thus, the GFP signal in this line represented the OsACOS12-GFP protein level. The GFP signal was detected within the tapetal cells, which formed a circle in the anther (Fig. 5m-o). In the late stages of anther development, the fluorescence signal transferred into the locule (Fig. 5p). However, the GFP Fig. 3 The defective anther cuticle and pollen sexine formation in osacos12. a and b SEM images for the WT and osacos12 anthers. c-j SEM observation for the epidermal surface of the WT (c) and osacos12 (g) anthers, the inner surface of WT (d) and osacos12 (h) anthers, and the pollen grains in WT (e and f) and osacos12 (i and j) anthers. Or, orbicule; Bars = 500 μm in a and b, 100 μm in e, i, 10 μm in c, g, f, j and 5 μm in h, d. k-t Semi-thin cross-sectional analysis of anther development of WT (k-o) and the osacos12 mutant (p-t) during the anther development stages. E, epidermis; En, endothecium; ML, middle layer; T, tapetum; MMC, microspore mother cell; Tds, tetrads; Msp, microspore. Bars = 20 μm. u-z TEM observation for WT (u-w) and osacos12 (x-z) pollen development from stages 8-10. The boxed image on the right of each panel was enlarged from the left region. AEX, abnormal exine; Ba, bacula; E, epidermis; En, endothecium; Ex, exine; Msp, microspore; Ne, nexine, PE, primexine; Se, sexine; T, tapetum; Tds, tetrads. Bars = 5 μm and 500 nm in u, x, 2 μm and 500 nm in v, y, 5 μm and 1 μm in w, 2 μm and 1 μm in z signal was not expressed inside the microspores (Fig. 5p). The wild type anther of rice was used as a negative control (Fig. 5k). These results indicate that OsACOS12 is accumulated in tapetal cells and anther locules according to different anther stages.
OsACOS12 could partially fulfil the function of ACOS5 for pollen development in Arabidopsis To investigate whether the OsACOS12 and ACOS5 were functionally conserved, genetic complementation of the Arabidopsis acos5 mutant (cs919318, Additional file 3: Figure S3) with the OsACOS12 genomic sequence was performed. We generated two constructs, proACOS5:OsA-COS12 and proOsACOS12:OsACOS12, with OsACOS12 driven by ACOS5 and OsACOS12 promoters, respectively. After the constructs were introduced into acos5/+ heterozygous plants (Fig. 6a-b), we obtained 12 and 14 transgenic lines with a homozygous acos5 background for these constructs (Additional file 4: Figure S4). All 12 transgenic lines for proACOS5:OsACOS12 exhibited partial fertility compared with the complete sterility of acos5 (Fig. 6e). RT-PCR demonstrated that OsACOS12 was highly expressed in the transgenic lines (Fig. 6o). Alexander staining showed that these transgenic plants contained mature grains that were similar to the grains of wild type plants (Fig. 6i). However, SEM analysis showed these pollen grains still had slight morphology defects (Fig. 6m). This result showed that the expression of OsACOS12 partially rescued the fertility of the acos5 mutant, which suggested OsACOS12 can fulfil the function of ACOS5 in Arabidopsis. All the transgenic lines for proOsACOS12:OsACOS12 have complete male sterility (Fig. 6f). RT-PCR demonstrated that the expression of OsACOS12 was low in these transgenic lines (Fig. 6o). Alexander staining showed that all pollen grains in the locule were aborted during the late stages of anther development (Fig. 6j). However, SEM showed that many pollen remnants could be formed in the anthers of proOsACO-S12:OsACOS12 transgenic plants although these pollen grains were still defective (Fig. 6n). These results suggested that the OsACOS12 promoter was not strong enough to drive the expression of the fatty acyl-CoA synthetase gene in Arabidopsis.  . c-j In situ hybridization of OsACOS12 in WT anthers. The anthers at the MMC stage (c), early meiosis stage (d), tetrad stage (e and f), microspore release stage (g), and microspore vacuolate stage (h) hybridized with an OsACOS12 antisense probe. The anthers at the tetrad stage (i-j) hybridized with an OsACOS12 sense probe. Msp, microspore; T, tapetum; Tds, tetrads. MMC, microspore mother cell; MC, meiotic cell; Dy, dyad cell. Bars = 50 μm. k-p Fluorescence confocal images of the OsACOS12-GFP fusion proteins at different stages. The green channel shows the GFP expression (530 nm), and the red channel shows the chlorophyll autofluorescence (>560 nm). The bright-field images of (p) show that these fusion proteins are not localized to the microspores. Bars = 10 μm; 100 μm

Discussion
OsACOS12 is an orthologue of Arabidopsis ACOS5 for pollen exine formation and the anther wall in rice The biosynthesis pathway of sporopollenin precursors has been identified in rice through several genetic studies. The fatty acyl reductase (DPW) converted palmitoylacyl carrier protein (ACP) to palmitoyl alcohol. The WDA1 gene participated in the biosynthesis of very long chain fatty acids. CYP704B2 and CYP703A3 could catalyse the production of w-hydroxylated fatty acids with lauric, palmitic and oleic acid [22-25, 32, 33]. In addition to these catalytic reactions, the fatty acyl-CoAs were indispensable for sporopollenin monomer synthesis. In Arabidopsis, the ACOS5 gene esterifies mediumto long-chain fatty acids to the corresponding fatty acyl-CoAs for the biosynthesis of sporopollenin [26]. In this study, we identified that OsACOS12 is the orthologue of ACOS5 in rice (Fig. 1). Knockout of OsACOS12 led to the defective exine layer of the microspore and male sterility (Figs. 2 and 3). These results were consistent with the phenotype of acos5 in Arabidopsis [26]. These results suggest that OsACOS12 is involved in the fatty acyl-CoAs synthesis required for sporopollenin precursors. Rice anthers have obvious orbicules and reticulate anther cuticle [24]. The cuticle is essential for rice anther development because it resists abiotic and biotic pressure [34]. Orbicules have been proposed to play a role in the translocation of sporopollenin constituents. The orbicules were barely detected in the inner surface (Fig. 3), and the wax components of the osacos12 anther were aberrant (Fig. 4). This outcome suggested that OsACOS12 was not only essential for pollen wall formation but also involved in anther cuticle lipid metabolism. The mutants of sporopollenin-related genes including DPW, CYP703A3, CYP704B2, ABCG15 in rice also exhibited the defective exine and anther epicuticle formation [22,24,25,32]. This result suggests that the anther cuticle and sporopollenin synthesis share a common lipid metabolism pathway in rice. However, the reticulate anther cuticle was not affected in Arabidopsis  Figure S5). This result suggests there was a divergence in the lipid pathway between Arabidopsis and rice.
OsACOS12 was expressed in the tapetum and secreted into the locule during pollen development The tapetum cell likely synthesizes the sporopollenin precursors and subsequently transports them into the locule to be assembled on the pollen surface and form the sexine layer [5,8,10,11]. The ACOS5 is an essential enzyme for sporopollenin synthesis in Arabidopsis. It transcribes components of the tapetum, meiocytes and microspores [17,26,35]. OsACOS12 is an orthologue of ACOS5. It exhibits a similar expression pattern to ACOS5 at the transcript level. In the complementary line, the OsACOS12-GFP signal was initially detected in the tapetal cells. At later stages, the signal occupied the free space in the locule but not in the microspores (Fig. 5). During anther development, all tapetal cellular degradation products, including proteins, entered the anther locule after it underwent the PCD process [36]. Given the secretary function of the tapetum, OsACOS12 is likely to be translated in the tapetum and secreted into the locule. Therefore, the OsACOS12 expression pattern at the protein level was different from the transcription level. This trend was also observed for DYT1, an essential regulator for early tapetum development. Its transcript was detected in meiocytes, the tapetum and microspores. However, DYT1 protein was only detected in tapetal cells [37]. The expression pattern of OsA-COS12 was consistent with the genes involved in the biosynthesis and transportation processes of sporopollenin precursors. CYP703A2 catalyses the conversion of medium-chain saturated fatty acids to the corresponding monohydroxylated fatty acids in Arabidopsis [13]. It was expressed in the tapetum cells and secreted into the locule at the late stage of anther development [20]. Several lipid transfer proteins (LTPs: LTPC6, LTPC14, OsC6) have been noted for their secretory accumulation pattern [38,39]. The LTPs bound or unbound to exine precursors are secreted from the tapetal cells to become exine layer constituents. The protein localization of OsA-COS12 and CYP703A2 was different from MS2, which is another enzyme for sporopollenin synthesis in Arabidopsis. It was only localized in tapetal cells [19]. OsA-COS12 might be secreted by the tapetum along with sporopollenin transportation.

The sporopollenin biosynthesis pathway was conserved between rice and Arabidopsis
In Arabidopsis, approximately 9 genes were reported to be involved in sporopollenin biosynthesis and transportation [3]. The orthologues for several genes, including CYP704B2, CYP703A3, OsPKS1, OsTKPR1, DPW and ABCG15, have been identified in rice [22][23][24][25]32]. In this study, OsACOS12 was identified as an orthologue of ACOS5. OsACOS12 driven by the ACOS5 promoter was able to partially restore the fertility of the male sterile acos5 mutant (Fig. 6e), which suggested that the functions of acyl-CoA synthetases were mainly conserved between monocot and dicot species. Previous studies showed that the rice DPW gene can completely rescue the sexine defects in the ms2 mutant [32]. PpASCL in Physcomitrella patens could produce hydroxyalkyl apyrones, which was consistent with the results from the Arabidopsis orthologue PKSA [40]. It is likely that the functions of the sporopollenin biosynthesis genes were very conservative in land plants. In the transgenic line of proACOS5:OsACOS12, the expression of OsACOS12 was comparable to that of ACOS5 in wild type (Fig. 6o). However, the sexine layer of pollen grains in this transgenic line was still defective (Fig. 6e, m). This result suggests that the enzyme activity of OsACOS12 might be lower than that of ACOS5 in Arabidopsis or that there may be some functional divergence for fatty acid metabolism. The pollen wall patterning for a specific plant species was a conserved and elaborate process [41]. The slightly different substrates and products derived from OsACOS12 probably led to the defective pollen surface in the transgenic line. A recent study also showed that several lipid metabolic enzymes for sporopollenin formation were conserved in tobacco and rice, while some products were different [23]. For the ms2 mutant, PpMS2 driven by the MS2 promoter could not rescue its fertility. However, DPW driven by the MS2 promoter could rescue its fertility with normal pollen wall formation [32,42]. These results suggested that DPW and MS2 have a very similar function, while PpMS2 and MS2 have evolutionary divergence. In the proOsACO-S12:OsACOS12 line, OsACOS12 is driven by its own promoter, and the transgenic plants exhibited a male sterile phenotype. The expression of OsACOS12 in the acos5 mutant was detected. However, its expression level was lower than ACOS5 in wild type (Fig. 6o). This result suggests that the upstream regulators for sporopollenin synthesis in Arabidopsis could recognize the OsACOS12 promoter. However, the activation efficiency was lower compared to that of the ACOS5 promoter.

Conclusion
In this study, we functionally identified the OsACOS12 gene in rice, which is an orthologue of Arabidopsis ACOS5. Our results provided genetic evidence to suggest that OsA-COS12 was involved in the lipidic metabolism for sporopollenin and cuticle synthesis in rice. The accumulation of OsACOS12 in tapetal cells and the anther locule suggested the processes of sporopollenin biosynthesis and transportation occurred synchronously. Genetic complementation assays indicated that ACOS5 and OsACOS12 were functionally conserved in general for pollen wall formation in rice and Arabidopsis. These findings provide new insights to illustrate fatty acyl-CoA synthetase function in the sporopollenin synthesis pathway of rice and provide a potential male sterile line for the utilization of heterosis in crops.

Plant materials and growth conditions
The osacos12 mutant was obtained from Tilling technology [31]. Rice accessions, including osacos12 and wild type Zhonghua11 (O. sativa ssp. Japonica), were grown in a paddy field at Shanghai Normal University (Shanghai, China). The Arabidopsis ecotype Columbia-0 and acos5 plants were grown in a growth chamber at DATs of 22°C under a 16 h light and 8 h dark photoperiod, unless specifically indicated.

Characterization of the mutant phenotype
The male sterility mutant was crossed with Zhonghua11 to produce the F 1 generation. The homozygous male sterility mutant was obtained in the F 2 generation accompanied by the co-segregation assay. The Arabidopsis and rice florets were photographed with a digital camera (Nikon D7000) and an dissecting microscope (Olympus, SZX10). The wild type and osacos12 mutant anthers were stained with alexander's solution and observed with a Leica microscope (Leica, USA). For a semi-thin section, SEM and TEM observation, spikelets and anthers of wild-type and osacos12 mutants at different stages were dissected to avoid experimental deviation. The embedding and observation procedures were performed as described by Lou et al [2].

Complementation of the osacos12 and acos5 mutants
For functional complementation, a genomic DNA fragment including the OsACOS12 coding region and 1428 bp promoter sequence were amplified from Zhonghua11 genomic DNA using gene-specific primers (Additional file 6: Table S1). The DNA fragment was subcloned into the pCAMBIA1300 binary vector using the pEASY-Uni seamless cloning and assembly kit with the BamHI restriction enzyme. The construct was introduced into Agrobacterium tumefaciens EHA105 and transformed into the calli induced from osacos12 heterozygous seeds (Biorun, China). The T 1 transgenic lines were genotyped to confirm the homozygous male sterility mutant by PCR and then verified by DNA sequence. OsACOS12-GFP florescence was detected using an LSM 5 PASCAL confocal laser scanning microscope (ZEISS, German).
For the transgenic rescue assay, the coding sequences of the 1047 bp promoter region of ACOS5, 1428 bp promoter region of OsACOS12 and the genomic region of OsACOS12 were generated by PCR amplification using primer star DNA polymerase (TaKaRa, Japan) and genespecific primers (Additional file 6: Table S1). These sequences were subsequently cloned into the pCAM-BIA1300 binary vector (CAMBIA, Australia). Constructs were transformed into fertile heterozygous Arabidopsis acos5 plants. The transgenic lines were screened using 20 mg · L -1 hygromycin. Genotypic and phenotypic analysis of the segregation populations was then performed in the T 1 generation.

Phylogenetic analysis
The OsACOS12 protein sequence was used to search for orthologues from the plant species using the basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ ). Multiple sequence alignments of the full-length protein sequences were performed using ClustalW and displayed using BOXSHADE (http://www.ch.embnet.org/software/ ClustalW.html). The phylogenetic tree was generated by the MEGA6.0 program using the Neighbour-Joining method with default parameters with 1000 bootstrap replicates.

RT-PCR and quantitative real-time PCR assay
Total RNA from roots, shoots, leaves, paleas and anthers at different developmental stages were extracted using a TRIzol kit (Life Technologies, USA). Subsequently, a 2 μg aliquot of total RNA was used as template for reverse transcription by AMV reverse transcriptase with a poly (dT12-18) primer (TOYOBO, Japan). Quantitative PCR analyses were performed with three repeats for each sample using SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) and utilizing an ABI 7300 system (Life Technologies, USA). The quantitative PCR procedure and conditions were described previously by Lou et al [2]. The cDNA levels of the target genes were normalized to the internal standard gene OsACTIN. Three replicates of each sample were used for gene expression analysis. The relevant primer sequences are listed in Additional file 6: Table S1.

In situ hybridization analyses
Fresh Zhonghua11 young panicles from different developmental stages were fixed in FAA immediately, embedded in paraffin, and sectioned at a thickness of 7 μm. A 443 bp fragment of OsACOS12 cDNA was amplified from the wild type DNA with its specific primers (Additional file 6: Table S1). The PCR product was cloned into a pBluescript-SK vector (Stratagene) and then digested with BamHI or EcoRI to obtain the templates, respectively. These templates were transcribed in vitro by the T7 or T3 RNA polymerases to produce antisense or sense probes