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  <cit> .
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  <cit> .
the major composition of the sexine is sporopollenin  <cit> , while the nexine is mainly composed of glycoproteins  <cit> .
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  <cit> .
the sexine patterning also acts as an important feature of plant taxonomic classifications  <cit> .
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  <cit> .
the tapetal layer is an essential tissue required for normal sexine development and pollen maturity  <cit> .
based on cytological and molecular evidence, the material of sporopollenin precursors originate from tapetal cells  <cit> .
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  <cit> .
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 cyp703a <dig>  cyp704b <dig> and ms <dig> for fatty-acid-derived compound metabolism, pksa/b and tkpr1/ <dig> for phenylpropanoids synthesis, and abcg <dig> for transportation .
all these genes are expressed in the tapetal layer at the transcript level.
on the protein level, ms <dig> is localized in tapetal cells, while cyp703a <dig> is in both tapetal cells and the anther locule  <cit> .
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  <cit> .
in rice, several sporopollenin enzymes have been identified .
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  <cit> .
acos <dig> encodes a fatty acyl-coa synthetase  for sporopollenin precursor synthesis in arabidopsis  <cit> .
there are nine fatty acyl-coa synthetase encoding genes in rice  <cit> .
in this study, we characterized an orthologue of arabidopsis acos <dig> in rice, osacos <dig> .
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 osacos <dig> protein suggested that there was synchronous biosynthesis and transportation of sporopollenin precursors.
the expression of the osacos <dig> gene in the arabidopsis acos <dig> mutant partially restored the male sterile phenotype, which indicated that the acyl-coa synthetase gene is a conserved function between arabidopsis and rice.
RESULTS
osacos <dig> in o. sativa is an orthologue of acos <dig> in a. thaliana
blastp analysis using arabidopsis acos <dig> protein sequence yield the loc_os04g <dig> in rice encoding a fatty acyl-coa synthetase .
the sequence of loc_os04g <dig> also showed the highest sequence similarity with acos <dig> in arabidopsis genome .
loc_os04g <dig> was designated osacos <dig> previously  <cit> .
the large superfamily of acyl-activating enzymes contains putative motifs for amp-binding and fatty acid-binding  <cit> .
these motifs are highly conserved between osacos <dig> and acos <dig> .
the homologues of osacos <dig> 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 osacos <dig> 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 .
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.fig.
<dig>
osacos <dig> in o. sativa is an orthologue of acos <dig> in arabidopsis.
a amino acid sequences alignment of osacos <dig> and acos <dig>  the sequences were aligned using clustal w and displayed using boxshade.
b a neighbour-joining phylogenetic tree of osacos <dig> and its orthologues in different species.
bootstrap values are the percentage of  <dig>  replicates.
the conserved amp binding domain and fatty acid binding domain are indicated
osacos <dig> mutant shows complete male sterility
to characterize the function of osacos <dig>  we obtained an allele of loc_os04g <dig> using targeting induced local lesions in genomes  technology from the ethyl methane sulfonate-induced population of rice zhonghua <dig>   <cit> .
sequence analysis revealed a point mutation from a to t, in the 1000th base downstream of the start codon of the loc_os04g <dig> genomic sequence in the mutant.
this transition caused a premature termination  at the first exon of loc_os04g <dig> .
the osacos <dig> mutant exhibited normal vegetative and spikelet development .
however, the mutant anthers had a white colour without pollen grains inside, which led to complete male sterility .
reciprocal crosses with the wild type indicted that female fertility was not affected in the osacos <dig> mutant.
the fertile and sterile plants of the f <dig> population segregated with a 3: <dig> ratio  indicated that there was a single recessive sporophytic mutation for osacos <dig>  to complement the osacos <dig> mutant phenotype, the osacos <dig> genomic fragment fused with gfp driven by its own promoter  was constructed and transformed into heterozygous osacos <dig> seeds.
of the  <dig> transgenic lines,  <dig> were identified to have a homozygous osacos <dig> mutant background .
all of these homozygous lines exhibited normal fertility .
these results demonstrate that osacos <dig> was responsible for the male fertility of the osacos <dig> mutant.fig.
<dig> isolation of the rice osacos <dig> mutant with complete male sterility.
a the gene structure and position of the nucleotide change in osacos <dig>  the black boxes indicate exons.
b-d the wild-type  plant, osacos <dig> mutant and complementation plant after the heading stage.
e comparison of the wt plant  and osacos <dig> mutant panicles  at the heading stage.
f the spikelets of the wt plant  and an osacos <dig> mutant  after removing the palea.
g the anther of a wt plant  and an osacos <dig> mutant .
h alexander staining of the wt plant  and an osacos <dig> mutant anther .
i and j identification of the osacos <dig> gene in a wt plant and osacos <dig> mutant by sequencing .
bars = 100 μm in e-g and 200 μm in h
pollen sexine formation and anther cuticle are defective in osacos12
scanning electron microscopy  was used to elucidate the abnormal morphological defects of anther development in the osacos <dig> plant.
the osacos <dig> anther was much shorter and smaller compared to that of the wild type .
in the wild type, the anther surface was covered by cuticle , and orbicules were intensively distributed on the inner surface .
however, osacos <dig> anther surface was lack of these materials and looks smooth , and orbicules were barely detected in the inner surface .
the wild type anther was filled with mature pollen grains .
in contrast, only a few remnants of degenerated pollen could be observed in the osacos <dig> anther .
in the complementary transgenic lines, the anther surface and pollen exine were restored .fig.
<dig> the defective anther cuticle and pollen sexine formation in osacos <dig>  a and b sem images for the wt and osacos <dig> anthers.
c-j sem observation for the epidermal surface of the wt  and osacos <dig>  anthers, the inner surface of wt  and osacos <dig>  anthers, and the pollen grains in wt  and osacos <dig>  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  and the osacos <dig> mutant  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  and osacos <dig>  pollen development from stages 8– <dig>  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
we subsequently obtained semi-thin transverse sections to understand the detailed defects of pollen development in osacos <dig>  there were no detectable differences between wild type and osacos <dig> during early anther development.
the microspore mother cells  and tetrads of osacos <dig> appeared to be comparable with the wild type .
in the wild type, newly released microspores of the wild type were angular in shape  and became enlarged and vacuolated .
in osacos <dig> plants, the released microspores contained much less cytoplasm  and were degenerated before/during volume enlargement .
no mature pollen grains were observed in the locule of osacos <dig> at later stages of anther development .
the defective phenotype of osacos <dig> was similar to acos <dig> in arabidopsis.
to further clarify the details of the abnormal exine development of osacos <dig> pollen, anther samples were investigated using transmission electron microscopy .
during the tetrad stage, primexine is formed between callose wall and plasma membrane.
it is critical for pollen wall pattern.
the primexine formation in osacos <dig> is consistent with that in wild type at tetrad stage .
at stage  <dig>  the pollen exine in osacos <dig> was not as deeply stained as that in the wild type, indicating an abnormal sporopollenin deposition .
at stage  <dig>  the exine layer of microspore was formed with gradually deposition of sporopollenin precursors in the wild type .
however, in osacos <dig>  no sporopollenin precursor accumulated on the microspore surface resulted in absent exine layer phenotype of collapsed pollen grains .
additionally, there were no obvious aberrations in the appearance of the tapetum in the osacos <dig> mutant .
these observations revealed that the exine formation and cuticle structures of anther epidermis were abnormal in osacos <dig>
defective wax components in osacos <dig> anthers
the defective anther cuticle and sporopollenin in osacos <dig> suggested that the lipidic mechanisms were aberrant in the mutant.
to confirm this point, we performed gas chromatography–mass spectrometry  to quantify wax extracts from whole anthers of both the wild type and osacos <dig> mutants.
the results showed that the total cuticular wax amount was reduced by approximately  <dig>  % in the mutant , which contributed to the significant reduction of most wax constituents.
the components of wax, including long-chain fatty acids , alkanes  and alcohols  were significantly decreased in the osacos <dig> mutant .
therefore, chemical analysis indicated that osacos <dig> was involved in the synthesis of lipidic compounds during rice anther development.fig.
<dig> anther cuticle wax constitutions in wt and osacos <dig>  a the total amount of anther wax per unit of anther surface area.
b the amounts of anther wax per unit of anther surface area.
compound names are abbreviated as follows: c <dig>  myristic acid; c <dig>  stearic acid; c18: <dig>  linolenic acid; c <dig>  arachidic acid; c <dig>  lignoceric acid; c <dig>  hexacosanoic acid; c <dig>  hexacosane; c <dig>  heptacosane; c <dig>  triacontane; c <dig>  dotriacontane; c <dig>  tricosane; c <dig>  pentatriacontane; c <dig>  hexatriacontane; c <dig>  1-heptacosanol; c <dig>  24-epicampesterol; c <dig>  sitosterol; c <dig>  1-triacontanol.
values are the mean ± sd .
*, p <  <dig> ; **, p <  <dig>
osacos <dig> is located in the tapetum and anther locule
in arabidopsis, acos <dig> is expressed in tapetal cells and microspores from late stage  <dig> to stage  <dig>  as shown through in situ hybridization  <cit> .
to analyse the expression of osacos <dig> in rice, semi-quantitative rt-pcr analysis was performed.
osacos <dig> expression was detected in the anthers with glume lengths of  <dig>  mm to  <dig>  mm but was not detected in roots, shoots, leaves and palea/lemma .
this result was further confirmed by quantitative real-time pcr analysis .
spatial and temporal expressions of osacos <dig> were detected by an rna in situ hybridization analysis using an osacos12-specific probe.
the osacos <dig> was initially expressed in the tapetal layer and microspore mother cells at the beginning of meiosis .
the signal was increased significantly and reached the highest level during the tetrad stage .
at microspores stage, the signal of osacos <dig> transcripts was obviously decreased in the tapetum and microspores .
in the control, only background signal was detected using a sense probe at tetrad stage .fig.
<dig> osacos <dig> is specifically expressed in the anther.
a rt-pcr analysis of rna isolated from various tissues using osacos <dig> and osactin primer sets.
le, lemma; pa, palea; l <dig> , glumes length  <dig>  mm; l <dig> , glumes length  <dig>  mm; l <dig> , glumes length  <dig>  mm; l <dig> , glumes length  <dig>  mm; l <dig> , glumes length  <dig>  mm.
b quantitative real-time pcr analysis of osacos <dig>  the osactin gene served as the reference.
data are shown as the mean ± sd .
c-j in situ hybridization of osacos <dig> in wt anthers.
the anthers at the mmc stage , early meiosis stage , tetrad stage , microspore release stage , and microspore vacuolate stage  hybridized with an osacos <dig> antisense probe.
the anthers at the tetrad stage  hybridized with an osacos <dig> 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 , and the red channel shows the chlorophyll autofluorescence .
the bright-field images of  show that these fusion proteins are not localized to the microspores.
bars = 10 μm; 100 μm
to understand the expression of osacos <dig> at the protein level, we analysed the gfp signal in the complemented transgenic lines .
in this transgenic line, osacos12-gfp can complement the osacos <dig> 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 .
in the late stages of anther development, the fluorescence signal transferred into the locule .
however, the gfp signal was not expressed inside the microspores .
the wild type anther of rice was used as a negative control .
these results indicate that osacos <dig> is accumulated in tapetal cells and anther locules according to different anther stages.
osacos <dig> could partially fulfil the function of acos <dig> for pollen development in arabidopsis
to investigate whether the osacos <dig> and acos <dig> were functionally conserved, genetic complementation of the arabidopsis acos <dig> mutant  with the osacos <dig> genomic sequence was performed.
we generated two constructs, proacos5:osacos <dig> and proosacos12:osacos <dig>  with osacos <dig> driven by acos <dig> and osacos <dig> promoters, respectively.
after the constructs were introduced into acos5/+ heterozygous plants , we obtained  <dig> and  <dig> transgenic lines with a homozygous acos <dig> background for these constructs .
all  <dig> transgenic lines for proacos5:osacos <dig> exhibited partial fertility compared with the complete sterility of acos <dig> .
rt-pcr demonstrated that osacos <dig> was highly expressed in the transgenic lines .
alexander staining showed that these transgenic plants contained mature grains that were similar to the grains of wild type plants .
however, sem analysis showed these pollen grains still had slight morphology defects .
this result showed that the expression of osacos <dig> partially rescued the fertility of the acos <dig> mutant, which suggested osacos <dig> can fulfil the function of acos <dig> in arabidopsis.
all the transgenic lines for proosacos12:osacos <dig> have complete male sterility .
rt-pcr demonstrated that the expression of osacos <dig> was low in these transgenic lines .
alexander staining showed that all pollen grains in the locule were aborted during the late stages of anther development .
however, sem showed that many pollen remnants could be formed in the anthers of proosacos12:osacos <dig> transgenic plants although these pollen grains were still defective .
these results suggested that the osacos <dig> promoter was not strong enough to drive the expression of the fatty acyl-coa synthetase gene in arabidopsis.fig.
<dig> the osacos <dig> could partially restore acos <dig> fertility.
a and b structural representation of the proacos5:osacos <dig> and the proosacos12:osacos <dig> constructs.
c-f the main stem of the col , acos <dig> , proacos5:osacos <dig> with a acos5/acos <dig> background , and proosacos12:osacos <dig> with a acos5/acos <dig> background  plants.
g-j alexander staining of the anthers from the col , acos <dig> , proacos5:osacos <dig> , and proosacos12:osacos <dig> transgenic lines .
bars = 100 μm.
k-n sem examination of the dehiscent pollen grains of the wild type , acos <dig> , proacos5:osacos <dig> , and proosacos12:osacos <dig> transgenic lines .
bars = 5 μm.
o rt-pcr analysis of acos <dig> or osacos <dig> expression in the flower buds of the col, acos <dig> plants and the proacos5:osacos <dig> and proosacos12:osacos <dig> transgenic lines.
tubulin was used to monitor the cdna yield and integrity of the samples
discussion
osacos <dig> is an orthologue of arabidopsis acos <dig> 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  converted palmitoyl-acyl carrier protein  to palmitoyl alcohol.
the wda <dig> gene participated in the biosynthesis of very long chain fatty acids.
cyp704b <dig> and cyp703a <dig> could catalyse the production of w-hydroxylated fatty acids with lauric, palmitic and oleic acid .
in addition to these catalytic reactions, the fatty acyl-coas were indispensable for sporopollenin monomer synthesis.
in arabidopsis, the acos <dig> gene esterifies medium- to long-chain fatty acids to the corresponding fatty acyl-coas for the biosynthesis of sporopollenin  <cit> .
in this study, we identified that osacos <dig> is the orthologue of acos <dig> in rice .
knockout of osacos <dig> led to the defective exine layer of the microspore and male sterility .
these results were consistent with the phenotype of acos <dig> in arabidopsis  <cit> .
these results suggest that osacos <dig> is involved in the fatty acyl-coas synthesis required for sporopollenin precursors.
rice anthers have obvious orbicules and reticulate anther cuticle  <cit> .
the cuticle is essential for rice anther development because it resists abiotic and biotic pressure  <cit> .
orbicules have been proposed to play a role in the translocation of sporopollenin constituents.
the orbicules were barely detected in the inner surface , and the wax components of the osacos <dig> anther were aberrant .
this outcome suggested that osacos <dig> was not only essential for pollen wall formation but also involved in anther cuticle lipid metabolism.
the mutants of sporopollenin-related genes including dpw, cyp703a <dig>  cyp704b <dig>  abcg <dig> in rice also exhibited the defective exine and anther epicuticle formation  <cit> .
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 acos <dig> mutants .
this result suggests there was a divergence in the lipid pathway between arabidopsis and rice.
osacos <dig> 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  <cit> .
the acos <dig> is an essential enzyme for sporopollenin synthesis in arabidopsis.
it transcribes components of the tapetum, meiocytes and microspores  <cit> .
osacos <dig> is an orthologue of acos <dig>  it exhibits a similar expression pattern to acos <dig> 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 .
during anther development, all tapetal cellular degradation products, including proteins, entered the anther locule after it underwent the pcd process  <cit> .
given the secretary function of the tapetum, osacos <dig> is likely to be translated in the tapetum and secreted into the locule.
therefore, the osacos <dig> expression pattern at the protein level was different from the transcription level.
this trend was also observed for dyt <dig>  an essential regulator for early tapetum development.
its transcript was detected in meiocytes, the tapetum and microspores.
however, dyt <dig> protein was only detected in tapetal cells  <cit> .
the expression pattern of osacos <dig> was consistent with the genes involved in the biosynthesis and transportation processes of sporopollenin precursors.
cyp703a <dig> catalyses the conversion of medium-chain saturated fatty acids to the corresponding monohydroxylated fatty acids in arabidopsis  <cit> .
it was expressed in the tapetum cells and secreted into the locule at the late stage of anther development  <cit> .
several lipid transfer proteins  have been noted for their secretory accumulation pattern  <cit> .
the ltps bound or unbound to exine precursors are secreted from the tapetal cells to become exine layer constituents.
the protein localization of osacos <dig> and cyp703a <dig> was different from ms <dig>  which is another enzyme for sporopollenin synthesis in arabidopsis.
it was only localized in tapetal cells  <cit> .
osacos <dig> might be secreted by the tapetum along with sporopollenin transportation.
the sporopollenin biosynthesis pathway was conserved between rice and arabidopsis
in arabidopsis, approximately  <dig> genes were reported to be involved in sporopollenin biosynthesis and transportation  <cit> .
the orthologues for several genes, including cyp704b <dig>  cyp703a <dig>  ospks <dig>  ostkpr <dig>  dpw and abcg <dig>  have been identified in rice .
in this study, osacos <dig> was identified as an orthologue of acos <dig>  osacos <dig> driven by the acos <dig> promoter was able to partially restore the fertility of the male sterile acos <dig> mutant , 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 ms <dig> mutant  <cit> .
ppascl in physcomitrella patens could produce hydroxyalkyl a-pyrones, which was consistent with the results from the arabidopsis orthologue pksa  <cit> .
it is likely that the functions of the sporopollenin biosynthesis genes were very conservative in land plants.
in the transgenic line of proacos5:osacos <dig>  the expression of osacos <dig> was comparable to that of acos <dig> in wild type .
however, the sexine layer of pollen grains in this transgenic line was still defective .
this result suggests that the enzyme activity of osacos <dig> might be lower than that of acos <dig> 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  <cit> .
the slightly different substrates and products derived from osacos <dig> 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  <cit> .
for the ms <dig> mutant, ppms <dig> driven by the ms <dig> promoter could not rescue its fertility.
however, dpw driven by the ms <dig> promoter could rescue its fertility with normal pollen wall formation  <cit> .
these results suggested that dpw and ms <dig> have a very similar function, while ppms <dig> and ms <dig> have evolutionary divergence.
in the proosacos12:osacos <dig> line, osacos <dig> is driven by its own promoter, and the transgenic plants exhibited a male sterile phenotype.
the expression of osacos <dig> in the acos <dig> mutant was detected.
however, its expression level was lower than acos <dig> in wild type .
this result suggests that the upstream regulators for sporopollenin synthesis in arabidopsis could recognize the osacos <dig> promoter.
however, the activation efficiency was lower compared to that of the acos <dig> promoter.
CONCLUSIONS
in this study, we functionally identified the osacos <dig> gene in rice, which is an orthologue of arabidopsis acos <dig>  our results provided genetic evidence to suggest that osacos <dig> was involved in the lipidic metabolism for sporopollenin and cuticle synthesis in rice.
the accumulation of osacos <dig> in tapetal cells and the anther locule suggested the processes of sporopollenin biosynthesis and transportation occurred synchronously.
genetic complementation assays indicated that acos <dig> and osacos <dig> 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.

 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. 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. the major composition of the sexine is sporopollenin, while the nexine is mainly composed of glycoproteins. 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. the sexine patterning also acts as an important feature of plant taxonomic classifications. 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. the tapetal layer is an essential tissue required for normal sexine development and pollen maturity. based on cytological and molecular evidence, the material of sporopollenin precursors originate from tapetal cells. 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. 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 cyp703a cyp704b and ms for fatty-acid-derived compound metabolism, pksa/b and tkpr1/ for phenylpropanoids synthesis, and abcg for transportation. all these genes are expressed in the tapetal layer at the transcript level. on the protein level, ms is localized in tapetal cells, while cyp703a is in both tapetal cells and the anther locule. 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. in rice, several sporopollenin enzymes have been identified. 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. acos encodes a fatty acyl-coa synthetase for sporopollenin precursor synthesis in arabidopsis. there are nine fatty acyl-coa synthetase encoding genes in rice. in this study, we characterized an orthologue of arabidopsis acos in rice, osacos. 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 osacos protein suggested that there was synchronous biosynthesis and transportation of sporopollenin precursors. the expression of the osacos gene in the arabidopsis acos mutant partially restored the male sterile phenotype, which indicated that the acyl-coa synthetase gene is a conserved function between arabidopsis and rice.  osacos in o. sativa is an orthologue of acos in a. thaliana blastp analysis using arabidopsis acos protein sequence yield the loc_os04g in rice encoding a fatty acyl-coa synthetase. the sequence of loc_os04g also showed the highest sequence similarity with acos in arabidopsis genome. loc_os04g was designated osacos previously. the large superfamily of acyl-activating enzymes contains putative motifs for amp-binding and fatty acid-binding. these motifs are highly conserved between osacos and acos. the homologues of osacos 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 osacos 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. 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.fig. osacos in o. sativa is an orthologue of acos in arabidopsis. a amino acid sequences alignment of osacos and acos the sequences were aligned using clustal w and displayed using boxshade. b a neighbour-joining phylogenetic tree of osacos and its orthologues in different species. bootstrap values are the percentage of replicates. the conserved amp binding domain and fatty acid binding domain are indicated osacos mutant shows complete male sterility to characterize the function of osacos we obtained an allele of loc_os04g using targeting induced local lesions in genomes technology from the ethyl methane sulfonate-induced population of rice zhonghua. sequence analysis revealed a point mutation from a to t, in the 1000th base downstream of the start codon of the loc_os04g genomic sequence in the mutant. this transition caused a premature termination at the first exon of loc_os04g. the osacos mutant exhibited normal vegetative and spikelet development. however, the mutant anthers had a white colour without pollen grains inside, which led to complete male sterility. reciprocal crosses with the wild type indicted that female fertility was not affected in the osacos mutant. the fertile and sterile plants of the f population segregated with a 3: ratio indicated that there was a single recessive sporophytic mutation for osacos to complement the osacos mutant phenotype, the osacos genomic fragment fused with gfp driven by its own promoter was constructed and transformed into heterozygous osacos seeds. of the transgenic lines, were identified to have a homozygous osacos mutant background. all of these homozygous lines exhibited normal fertility. these results demonstrate that osacos was responsible for the male fertility of the osacos mutant.fig. isolation of the rice osacos mutant with complete male sterility. a the gene structure and position of the nucleotide change in osacos the black boxes indicate exons. b-d the wild-type plant, osacos mutant and complementation plant after the heading stage. e comparison of the wt plant and osacos mutant panicles at the heading stage. f the spikelets of the wt plant and an osacos mutant after removing the palea. g the anther of a wt plant and an osacos mutant. h alexander staining of the wt plant and an osacos mutant anther. i and j identification of the osacos gene in a wt plant and osacos mutant by sequencing. bars = 100 μm in e-g and 200 μm in h pollen sexine formation and anther cuticle are defective in osacos12 scanning electron microscopy was used to elucidate the abnormal morphological defects of anther development in the osacos plant. the osacos anther was much shorter and smaller compared to that of the wild type. in the wild type, the anther surface was covered by cuticle, and orbicules were intensively distributed on the inner surface. however, osacos anther surface was lack of these materials and looks smooth, and orbicules were barely detected in the inner surface. the wild type anther was filled with mature pollen grains. in contrast, only a few remnants of degenerated pollen could be observed in the osacos anther. in the complementary transgenic lines, the anther surface and pollen exine were restored.fig. the defective anther cuticle and pollen sexine formation in osacos a and b sem images for the wt and osacos anthers. c-j sem observation for the epidermal surface of the wt and osacos anthers, the inner surface of wt and osacos anthers, and the pollen grains in wt and osacos 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 and the osacos mutant 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 and osacos pollen development from stages 8– 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 we subsequently obtained semi-thin transverse sections to understand the detailed defects of pollen development in osacos there were no detectable differences between wild type and osacos during early anther development. the microspore mother cells and tetrads of osacos appeared to be comparable with the wild type. in the wild type, newly released microspores of the wild type were angular in shape and became enlarged and vacuolated. in osacos plants, the released microspores contained much less cytoplasm and were degenerated before/during volume enlargement. no mature pollen grains were observed in the locule of osacos at later stages of anther development. the defective phenotype of osacos was similar to acos in arabidopsis. to further clarify the details of the abnormal exine development of osacos pollen, anther samples were investigated using transmission electron microscopy. during the tetrad stage, primexine is formed between callose wall and plasma membrane. it is critical for pollen wall pattern. the primexine formation in osacos is consistent with that in wild type at tetrad stage. at stage the pollen exine in osacos was not as deeply stained as that in the wild type, indicating an abnormal sporopollenin deposition. at stage the exine layer of microspore was formed with gradually deposition of sporopollenin precursors in the wild type. however, in osacos no sporopollenin precursor accumulated on the microspore surface resulted in absent exine layer phenotype of collapsed pollen grains. additionally, there were no obvious aberrations in the appearance of the tapetum in the osacos mutant. these observations revealed that the exine formation and cuticle structures of anther epidermis were abnormal in osacos defective wax components in osacos anthers the defective anther cuticle and sporopollenin in osacos suggested that the lipidic mechanisms were aberrant in the mutant. to confirm this point, we performed gas chromatography–mass spectrometry to quantify wax extracts from whole anthers of both the wild type and osacos mutants. the results showed that the total cuticular wax amount was reduced by approximately  % in the mutant, which contributed to the significant reduction of most wax constituents. the components of wax, including long-chain fatty acids, alkanes and alcohols were significantly decreased in the osacos mutant. therefore, chemical analysis indicated that osacos was involved in the synthesis of lipidic compounds during rice anther development.fig. anther cuticle wax constitutions in wt and osacos a the total amount of anther wax per unit of anther surface area. b the amounts of anther wax per unit of anther surface area. compound names are abbreviated as follows: c myristic acid; c stearic acid; c18: linolenic acid; c arachidic acid; c lignoceric acid; c hexacosanoic acid; c hexacosane; c heptacosane; c triacontane; c dotriacontane; c tricosane; c pentatriacontane; c hexatriacontane; c 1-heptacosanol; c 24-epicampesterol; c sitosterol; c 1-triacontanol. values are the mean ± sd. *, p < ; **, p <  osacos is located in the tapetum and anther locule in arabidopsis, acos is expressed in tapetal cells and microspores from late stage to stage as shown through in situ hybridization. to analyse the expression of osacos in rice, semi-quantitative rt-pcr analysis was performed. osacos expression was detected in the anthers with glume lengths of  mm to  mm but was not detected in roots, shoots, leaves and palea/lemma. this result was further confirmed by quantitative real-time pcr analysis. spatial and temporal expressions of osacos were detected by an rna in situ hybridization analysis using an osacos12-specific probe. the osacos was initially expressed in the tapetal layer and microspore mother cells at the beginning of meiosis. the signal was increased significantly and reached the highest level during the tetrad stage. at microspores stage, the signal of osacos transcripts was obviously decreased in the tapetum and microspores. in the control, only background signal was detected using a sense probe at tetrad stage.fig. osacos is specifically expressed in the anther. a rt-pcr analysis of rna isolated from various tissues using osacos and osactin primer sets. le, lemma; pa, palea; l, glumes length  mm; l, glumes length  mm; l, glumes length  mm; l, glumes length  mm; l, glumes length  mm. b quantitative real-time pcr analysis of osacos the osactin gene served as the reference. data are shown as the mean ± sd. c-j in situ hybridization of osacos in wt anthers. the anthers at the mmc stage, early meiosis stage, tetrad stage, microspore release stage, and microspore vacuolate stage hybridized with an osacos antisense probe. the anthers at the tetrad stage hybridized with an osacos 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, and the red channel shows the chlorophyll autofluorescence. the bright-field images of show that these fusion proteins are not localized to the microspores. bars = 10 μm; 100 μm to understand the expression of osacos at the protein level, we analysed the gfp signal in the complemented transgenic lines. in this transgenic line, osacos12-gfp can complement the osacos 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. in the late stages of anther development, the fluorescence signal transferred into the locule. however, the gfp signal was not expressed inside the microspores. the wild type anther of rice was used as a negative control. these results indicate that osacos is accumulated in tapetal cells and anther locules according to different anther stages. osacos could partially fulfil the function of acos for pollen development in arabidopsis to investigate whether the osacos and acos were functionally conserved, genetic complementation of the arabidopsis acos mutant with the osacos genomic sequence was performed. we generated two constructs, proacos5:osacos and proosacos12:osacos with osacos driven by acos and osacos promoters, respectively. after the constructs were introduced into acos5/+ heterozygous plants, we obtained and transgenic lines with a homozygous acos background for these constructs. all transgenic lines for proacos5:osacos exhibited partial fertility compared with the complete sterility of acos. rt-pcr demonstrated that osacos was highly expressed in the transgenic lines. alexander staining showed that these transgenic plants contained mature grains that were similar to the grains of wild type plants. however, sem analysis showed these pollen grains still had slight morphology defects. this result showed that the expression of osacos partially rescued the fertility of the acos mutant, which suggested osacos can fulfil the function of acos in arabidopsis. all the transgenic lines for proosacos12:osacos have complete male sterility. rt-pcr demonstrated that the expression of osacos was low in these transgenic lines. alexander staining showed that all pollen grains in the locule were aborted during the late stages of anther development. however, sem showed that many pollen remnants could be formed in the anthers of proosacos12:osacos transgenic plants although these pollen grains were still defective. these results suggested that the osacos promoter was not strong enough to drive the expression of the fatty acyl-coa synthetase gene in arabidopsis.fig. the osacos could partially restore acos fertility. a and b structural representation of the proacos5:osacos and the proosacos12:osacos constructs. c-f the main stem of the col, acos, proacos5:osacos with a acos5/acos background, and proosacos12:osacos with a acos5/acos background plants. g-j alexander staining of the anthers from the col, acos, proacos5:osacos, and proosacos12:osacos transgenic lines. bars = 100 μm. k-n sem examination of the dehiscent pollen grains of the wild type, acos, proacos5:osacos, and proosacos12:osacos transgenic lines. bars = 5 μm. o rt-pcr analysis of acos or osacos expression in the flower buds of the col, acos plants and the proacos5:osacos and proosacos12:osacos transgenic lines. tubulin was used to monitor the cdna yield and integrity of the samples discussion osacos is an orthologue of arabidopsis acos 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 converted palmitoyl-acyl carrier protein to palmitoyl alcohol. the wda gene participated in the biosynthesis of very long chain fatty acids. cyp704b and cyp703a could catalyse the production of w-hydroxylated fatty acids with lauric, palmitic and oleic acid. in addition to these catalytic reactions, the fatty acyl-coas were indispensable for sporopollenin monomer synthesis. in arabidopsis, the acos gene esterifies medium- to long-chain fatty acids to the corresponding fatty acyl-coas for the biosynthesis of sporopollenin. in this study, we identified that osacos is the orthologue of acos in rice. knockout of osacos led to the defective exine layer of the microspore and male sterility. these results were consistent with the phenotype of acos in arabidopsis. these results suggest that osacos is involved in the fatty acyl-coas synthesis required for sporopollenin precursors. rice anthers have obvious orbicules and reticulate anther cuticle. the cuticle is essential for rice anther development because it resists abiotic and biotic pressure. orbicules have been proposed to play a role in the translocation of sporopollenin constituents. the orbicules were barely detected in the inner surface, and the wax components of the osacos anther were aberrant. this outcome suggested that osacos was not only essential for pollen wall formation but also involved in anther cuticle lipid metabolism. the mutants of sporopollenin-related genes including dpw, cyp703a cyp704b abcg in rice also exhibited the defective exine and anther epicuticle formation. 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 acos mutants. this result suggests there was a divergence in the lipid pathway between arabidopsis and rice. osacos 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. the acos is an essential enzyme for sporopollenin synthesis in arabidopsis. it transcribes components of the tapetum, meiocytes and microspores. osacos is an orthologue of acos it exhibits a similar expression pattern to acos 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. during anther development, all tapetal cellular degradation products, including proteins, entered the anther locule after it underwent the pcd process. given the secretary function of the tapetum, osacos is likely to be translated in the tapetum and secreted into the locule. therefore, the osacos expression pattern at the protein level was different from the transcription level. this trend was also observed for dyt an essential regulator for early tapetum development. its transcript was detected in meiocytes, the tapetum and microspores. however, dyt protein was only detected in tapetal cells. the expression pattern of osacos was consistent with the genes involved in the biosynthesis and transportation processes of sporopollenin precursors. cyp703a catalyses the conversion of medium-chain saturated fatty acids to the corresponding monohydroxylated fatty acids in arabidopsis. it was expressed in the tapetum cells and secreted into the locule at the late stage of anther development. several lipid transfer proteins have been noted for their secretory accumulation pattern. the ltps bound or unbound to exine precursors are secreted from the tapetal cells to become exine layer constituents. the protein localization of osacos and cyp703a was different from ms which is another enzyme for sporopollenin synthesis in arabidopsis. it was only localized in tapetal cells. osacos might be secreted by the tapetum along with sporopollenin transportation. the sporopollenin biosynthesis pathway was conserved between rice and arabidopsis in arabidopsis, approximately genes were reported to be involved in sporopollenin biosynthesis and transportation. the orthologues for several genes, including cyp704b cyp703a ospks ostkpr dpw and abcg have been identified in rice. in this study, osacos was identified as an orthologue of acos osacos driven by the acos promoter was able to partially restore the fertility of the male sterile acos mutant, 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 ms mutant. ppascl in physcomitrella patens could produce hydroxyalkyl a-pyrones, which was consistent with the results from the arabidopsis orthologue pksa. it is likely that the functions of the sporopollenin biosynthesis genes were very conservative in land plants. in the transgenic line of proacos5:osacos the expression of osacos was comparable to that of acos in wild type. however, the sexine layer of pollen grains in this transgenic line was still defective. this result suggests that the enzyme activity of osacos might be lower than that of acos 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. the slightly different substrates and products derived from osacos 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. for the ms mutant, ppms driven by the ms promoter could not rescue its fertility. however, dpw driven by the ms promoter could rescue its fertility with normal pollen wall formation. these results suggested that dpw and ms have a very similar function, while ppms and ms have evolutionary divergence. in the proosacos12:osacos line, osacos is driven by its own promoter, and the transgenic plants exhibited a male sterile phenotype. the expression of osacos in the acos mutant was detected. however, its expression level was lower than acos in wild type. this result suggests that the upstream regulators for sporopollenin synthesis in arabidopsis could recognize the osacos promoter. however, the activation efficiency was lower compared to that of the acos promoter.  in this study, we functionally identified the osacos gene in rice, which is an orthologue of arabidopsis acos our results provided genetic evidence to suggest that osacos was involved in the lipidic metabolism for sporopollenin and cuticle synthesis in rice. the accumulation of osacos in tapetal cells and the anther locule suggested the processes of sporopollenin biosynthesis and transportation occurred synchronously. genetic complementation assays indicated that acos and osacos 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. 
the localization of osacos12-gfp demonstrated that osacos protein is accumulated in tapetal cells and anther locules. acos and osacos are conserved for pollen wall formation in monocot and dicot species. in situ hybridization shows that osacos is expressed in tapetal cells and microspores at the transcript level. osacos driven by the acos promoter could partially restore the male fertility of the acos mutant in arabidopsis. sequence analysis showed that osacos shares  % amino acid sequence identity with acos the osacos mutation caused by a pre-mature stop codon in loc_os04g exhibits defective sexine resulting in a male sterile phenotype in rice. osacos is an orthologue of acos that is essential for sporopollenin synthesis in rice.
 sporopollenin is a major component of the pollen exine pattern. in arabidopsis, acyl-coa synthetase is involved in sporopollenin precursor biosynthesis. in this study, we identified its orthologue, osacos in rice and compared the functional conservation of acos in rice to arabidopsis.  sequence analysis showed that osacos shares  % amino acid sequence identity with acos the osacos mutation caused by a pre-mature stop codon in loc_os04g exhibits defective sexine resulting in a male sterile phenotype in rice. in situ hybridization shows that osacos is expressed in tapetal cells and microspores at the transcript level. the localization of osacos12-gfp demonstrated that osacos protein is accumulated in tapetal cells and anther locules. osacos driven by the acos promoter could partially restore the male fertility of the acos mutant in arabidopsis.  osacos is an orthologue of acos that is essential for sporopollenin synthesis in rice. acos and osacos are conserved for pollen wall formation in monocot and dicot species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords oryza sativaosacos12male sterilitypollen exineanther cuticleissue-copyright-statement© the author 2016 