BACKGROUND
the dead box rna helicase family, the largest family of rna helicases, belongs to helicase superfamily  <dig>  each dead box rna helicase contains nine conserved amino acid motifs that constitute the helicase core domain.
besides these conserved motifs within dead box proteins, there are also n- and c-terminal extension sequences in each dead box rna family member that varies in terms of their length and composition; they have been proposed to provide substrate binding specificity, and to act as signals for subcellular localization or as domains that interact with accessory components .
dead box proteins are found in most prokaryotes and all eukaryotes, including plants .
rice is an important staple food crop and is also valuable as a model plant for studies in cereal functional genomics.
although predicted protein sequences in the rice genome database as determined by silico analysis to indicate that there are at least  <dig> dead box proteins in rice  <cit> , the functional characterizations of most of them remain unknown.
eukaryotic initiation factor 4aiii , a dead box rna helicase, is a core component of the exon junction complex  that also contains mago/nashi , y14/tsunagi/rna-binding protein 8a, and barentsz/metastatic lymph node  <dig> .
the ejc is formed in both the nucleus and the cytoplasm, and plays important roles in gene expression, including the following:  it assembles 20– <dig> bases upstream of each exon of pre-mrna for its involvement in mrna splicing  <cit> .
it is involved in nonsense-mediated decay, a surveillance mechanism that degrades mrna containing premature termination codons  <cit> .
it is involved in the regulation of gene expression at the translational level  <cit> .
it has a role in mrna subcellular localization  <cit> .
although most research has been undertaken in mammals, genes encoding core ejc components have been found in plants  <cit> , suggesting that there is structural and functional conservation in the ejc complex among plant and mammalian.
however, only limited evidence has been reported on the physiological role of the ejc in plants.
in arabidopsis, eif4aiii interacts with an ejc component, aly/ref, and colocalizes with other ejc components, such as mago, y <dig>  and rnps <dig>  <cit> .
in o. sativa, two forms of mago, osmago <dig> and osmago <dig>  and two forms of y <dig>  osy14a and osy14b, were analyzed .
osmago <dig> and osmago <dig> double-knockdown rice plants displayed dwarfism and abnormal flowers in which the endothecium and tapetum of the stamen were maintained  <cit> .
osy14b may function in embryogenesis, while the down-regulation of osy14b resulted in a failure to induce plantlets  <cit> .
osy14a knockdown plants also displayed phenotypes similar to those of osmago <dig> and osmago <dig> double-knockdown rice plants  <cit> .
moreover, osmago <dig> and osmago <dig> double-knockdown, and osy14a knockdown transgenic plants showed abnormal accumulation of the pre-mrna of undeveloped tapetum  <dig> , a key regulator of stamen development  <cit> .
these findings indicate that the ejc participates in the regulation of pre-mrna splicing in rice.
despite the fact that the functions of homologs of mago and y <dig> have been demonstrated in rice, it is still unknown whether eif4aiii is essential for ejc function in rice.
in this study, two putative rice dead box rna helicase genes, osrh <dig>  and osrh <dig> , were therefore characterized.
both osrh <dig> and osrh <dig> are homologous to eif4aiii, which is a member of the eif4a family, and their gene expression patterns were similar in various rice tissues, but the level of osrh <dig> mrna was about 58-fold higher than that of osrh <dig> mrna in seedlings.
the results from bimolecular fluorescence complementation  analysis showed that both osrh <dig> and osrh <dig> can interact with osmago <dig> and osy14b.
transgenic plants with both osrh <dig> and osrh <dig> knocked down by rna interference displayed phenotypes that resembled those of mutants with gibberellin-related developmental defects.
moreover, these osrh <dig> and osrh <dig> double-knockdown plants exhibited severe defects in terms of pollen and seed development.
the accumulation of osudt <dig> pre-mrna was also detected in the osrh <dig> and osrh <dig> double-knockdown transgenic lines.
our data demonstrate that both osrh <dig> and osrh <dig> are core components of the ejc and play critical roles in regulation of plant height, pollen, and seed development in rice.
RESULTS
osrh <dig> and osrh <dig> are putative dead box rna helicases
to identify rice eif4aiii homologs, human eif4aiii protein sequences were used as queries to search protein databases at phytozome and national center for biotechnology information .
two eif4aiii-like putative proteins, encoded by osrh <dig>  and osrh <dig>  were identified in rice .
the osrh <dig> is located on rice chromosome  <dig> and has eight exons.
the deduced amino acid sequence of osrh <dig> cdna consists of nine conserved rna helicase domains  and the characteristic amino acid residues d-e-a-d in motif ii.
besides, the osrh <dig> gene has eight exons and is located on chromosome  <dig>  the levels of identity between osrh <dig> and osrh <dig> in terms of the dna sequence and the deduced amino acid sequence were found to be  <dig> and 99 %, respectively.
phylogenetic relationships were established using amino acid sequences from the eif4a families of dicots, monocots, green algae, vertebrates, invertebrates, and yeast , which showed that osrh <dig> and osrh <dig> are closely related to eif4aiii and can be clustered into the monocot group .fig.
<dig> amino acid sequences and domain structures of the osrh <dig> and osrh <dig> proteins.
a. the amino acid sequences of osrh <dig> and osrh <dig> were compared using the clustal w program.
identical amino acid residues are labeled in black.
different amino acid residues are marked by asterisks.
the conserved helicase motif is highlighted by a line above it and includes motifs q, i, ia, ib, ii, iii, iv, v, and vi
fig.
<dig> phylogenetic relationships of eif4aiii family members.
a phylogenetic tree for eif4aiii in dicots, monocots, green algae, vertebrates, invertebrates, and yeast was generated using mega  <dig>  eif4aiii members from rice, maize, sorghum, and brachypodium are categorized into the monocot group with at least 50 % bootstrap support.
accession numbers of the genes listed here are shown in additional file 2
expression patterns of osrh <dig> and osrh34
to determine the relative expression levels of osrh <dig> and osrh <dig> in rice, total rna was isolated from a variety of vegetative and reproductive tissues and was subjected to qrt-pcr with specific primers .
the osrh <dig> transcript was expressed in all selected tissues and organs, including roots, stems, leaves, sheaths, panicles, and seedlings .
relatively high levels of osrh <dig> mrna were detected in vegetative leaf blades, flag leaves, and panicles before heading .
expression of osrh <dig> was relatively abundant in vegetative leaf blades, flag leaves, and seedlings, whereas its expression was rarely detected in roots, stems, and panicles .
these results indicate that these two paralogous genes are coexpressed in most selected tissues and organs in rice.
to compare the levels of osrh <dig> and osrh <dig> mrna in rice plants, absolute qrt-pcr was performed.
standard curves were used with a serial dilution of either osrh <dig> cdna- or osrh <dig> cdna-containing plasmids.
as shown in fig.
3b, the level of osrh <dig> mrna was 58-fold higher than that of osrh <dig> mrna in rice seedlings at the three-leaf stage.fig.
<dig> expression of osrh <dig> and osrh <dig>
a qrt-pcr analysis of osrh <dig> and osrh <dig> gene expression in rice.
total rna was isolated from seedlings , roots , stems , leaves , sheaths , flag leaves , booting panicles , heading panicles , flowering panicles , and pollinated panicles .
the rice act <dig> gene was used as an internal control.
b absolute quantitative rt-pcr analysis of osrh <dig> and osrh <dig>  in which plasmid dna was applied as a control to compare the mrna levels of osrh <dig> and osrh34
osrh <dig> and osrh <dig> were colocalized in nucleus and cytoplasm
to determine the subcellular localization of osrh <dig> and osrh <dig>  plasmids containing an osrh2–gfp fusion gene and osrh34–gfp under the control of the camv 35s promoter were generated and introduced into onion epidermal cells.
fluorescent signals were emitted from both osrh2–gfp  and osrh34–gfp  in both the nucleus and the cytoplasm.
similar results were obtained in onion cells for the expression of either gfp–osrh <dig>  or mcherry–osrh <dig> .
to confirm the subcellular localization of osrh <dig> and osrh <dig>  onion cells were cotransformed with gfp–osrh <dig> and mcherry–osrh <dig>  gfp and mcherry signals were colocalized in the nucleus and the cytoplasm .
these results suggest that the osrh <dig> and osrh <dig> proteins are localized in both the nucleus and the cytoplasm.fig.
<dig> subcellular localization of osrh <dig> and osrh <dig>  a and b osrh <dig> fluorescence fusion protein was localized in the nucleus and the cytoplasm.
onion epidermal cells were transformed with either 35s::osrh2–gfp  or 35s::gfp–osrh <dig> .
c and d onion epidermal cells were transformed with either 35s::osrh34–gfp  or 35s::mcherry–osrh <dig> .
e colocalization of gfp–osrh <dig> and mcherry–osrh <dig> in the nucleus and the cytoplasm.
onion epidermal cells were cotransformed with 35s::gfp–osrh <dig> and 35s::mcherry–osrh <dig>  bars = 100 μm
both osrh <dig> and osrh <dig> are components of the ejc core complex
eif4aiii can interact with y <dig> and mago to form the ejc core complex in eukaryotic cells  <cit> .
gong and he  <cit>  have also reported that rice mago and y <dig> can form heterodimers.
to determine whether osrh <dig> and osrh <dig> were components of the ejc in rice, interactions among rice mago, y <dig>  and eif4aiii were examined by bifc.
the n-terminus  of yellow fluorescent protein  was fused at the downstream end of osrh <dig> and osrh <dig>  the c-terminus  of yfp was fused at the downstream end of osy14b and osmago <dig>  coexpression of osrh2-yn and yc, osrh34-yn and yc, yn and osmago1-yc, yn and osy14b-yc in onion epidermal cells were used as negative controls for interaction tests among osrh <dig>  osmago <dig>  and osy <dig>  and no fluorescent signals were detected .
the interaction between osmago <dig> and osy14b was used as a positive control that exhibited remarkable fluorescent signals in onion cells .
these two fusion proteins, osrh2-yn and osy14b-yc, were coexpressed in onion cells and the yfp fluorescence was observed .
osrh2-yn and osmago1-yc coexpressed in onion cells also displayed the yfp signal .
meanwhile, yfp fluorescence was also detected upon the coexpression of osrh34-yn with osy14b-yc  and osrh34-yn with osmago1-yc , respectively.
these results indicate that both osrh <dig> and osrh <dig> directly interact with osy14b and osmago <dig>  demonstrating that they are indeed a component of the ejc core complex in rice.fig.
<dig> bifc analysis of the interaction among rice mago, y <dig>  and eif4aiii in onion epidermal cells.
n- and c-terminal fragments of yfp  were fused to the c-terminus of osrh <dig>  osrh <dig>  osmago <dig>  and osy14b, respectively.
onion epidermal cells were cotransformed with combinations of 35s:: osrh2–yn and 35s::yc, 35s::osrh34–yn and 35s::yc, 35s::yn and 35s::y14b–yc, and 35s::yn and 35s::mago1–yc as negative controls  onion epidermal cells were cotransformed with 35s::osmago1–yn and 35s::osy14b–yc , 35s::osrh2–yn and 35s::osy14b–yc , 35s::osrh2–yn and 35s::osmago1–yc , 35s::osrh34–yn and 35s::osy14b–yc , 35s::osrh34–yn and 35s::osmago1–yc.
bars = 100 μm
the osrh <dig> and the osrh <dig> were colocalized , so protein interaction between these two isoforms was further examined by the bifc analysis.
the yfp fluorescent signals were not be observed in onion cells coexpressed with either combinations of osrh2-yn and osrh2-yc, osrh34-yn and osrh34-yc, osrh2-yn and osrh34-yc, or osrh34-yn and osrh2-yc .
these results indicated that proteins of osrh <dig> and osrh <dig> were not able to interact to form homomer or heteromer.
characterization of double knockdown of osrh <dig> and osrh <dig> transgenic lines
to unravel the physiological functions of osrh <dig> and osrh <dig>  a rna interference mediated genes silencing approach was performed.
because osrh <dig> and osrh <dig> shared extremely high sequence identity, it was difficult to achieve specific gene silencing.
thus, double knockdown of osrh <dig> and osrh <dig> was carried out in rice.
to minimize the potential off-target gene silencing, the sequences of 271-bp rnai designed region at the 3´ end of osrh <dig> cdna and osrh <dig> cdna were used as queries to search rice mrna databases at ncbi.
none of region identical of around or more than  <dig> nucleotides was obtained.
further, a public web-based computational tool developed for identification of potential off-targets, sirna scan  <cit> , was applied to search rice mrna databases, and no potential off-target was detected in the rnai designed region.
inverted repeat of the 271-bp region was fused at the up- and downstream ends of a gfp coding sequence, and the fusion construct was expressed under the control of the maize ubiquitin gene  promoter  in transgenic rice.
several independent t <dig> transgenic plants were obtained, and the levels of osrh <dig> mrna and osrh <dig> were determined by qrt-pcr.
as results showed in fig.
6b, both osrh <dig> mrna and osrh <dig> mrna were barely detectable in three independent t <dig> transgenic lines, rh2ri 2b, rh2ri  <dig>  and rh2ri 14b, indicating that both osrh <dig> and osrh <dig> were knocked down.
therefore, rh2ri 2b, rh2ri  <dig>  and rh2ri 14b lines were selected to address roles of osrh <dig> and osrh <dig> in rice.fig.
<dig> characterization of osrh <dig> and osrh <dig> double-knockdown transgenic lines.
a schematic presentation of the double silencing of osrh <dig> and osrh <dig> of the rna interference construct.
a 271-bp fragment at the 3′ end of osrh <dig> and osrh <dig> conserved region was ligated in sense and antisense orientations to the gfp cdna and fused downstream of the ubi promoter.
b expression of osrh <dig> and osrh <dig> in t <dig> transgenic rice seedlings.
total rna was isolated from 14-day-old seedlings and subjected to qrt-pcr using osrh2- and osrh34-specific primers.
rice act <dig> was used as an internal control.
error bars indicate the standard deviations  of triplicate experiments.
gene expression was related to wild-type plants, as  <dig>  * is significantly different from the wild-type plants .
osrh <dig> and osrh <dig> double-knockdown lines are named as rh2ri 2b,  <dig>  and 14b.
wild-type line is indicated by wt
reduced plant height in transgenic rice double knockdown of osrh <dig> and osrh34
significant differences in the height of plants in the t <dig> transgenic lines were observed.
rh2ri 2b, rh2ri  <dig>  and rh2ri 14b showed a dwarf phenotype; their seedlings were  <dig> to 44 % shorter than those of wild-type plants at 2 weeks old .
moreover, rh2ri transgenic plants were shorter than wild-type plants at following growth stage.
one example was shown in fig.
7c, the plant height of rh2ri 2b t <dig> plant was  <dig> and 26 % shorter than wild-type plants at 78-day-old and 147-day-old stages, respectively.
plant height was further compared between wild-type and rh2ri transgenic plants at the reproductive stage.
the culm of wild-type plants contained five internodes, named i to v from top to bottom.
culm lengths of the rh2ri transgenic plants also appeared to be reduced in each internode region compared with those in the wild-type plants .
the dwarf phenotype of rh2ri transgenic plants was also observed in a paddy field.
significant differences in plant heights between wild-type plants and rh2ri plants of the three transgenic t1-t <dig> generation were observed .
in addition, the leaves of the rh2ri transgenic plants were a deeper green and they had a greater number of tillers than the wild-type plants .fig.
<dig> phenotype of osrh <dig> and osrh <dig> double-knockdown t <dig> transgenic rice.
a wt and three independent osrh <dig> and osrh <dig> double-knockdown lines, rh2ri 2b, rh2ri  <dig>  and rh2ri 14b, seedlings were grown on ½ ms agar medium for 10 days and transferred to hydroponic cultures for 7 days.
bar = 1 cm.
b quantification of plant height at seedling stages.
the plant height of 17-day-old seedlings was measured.
error bars indicate the sd of ten individual plants for each line.
* is significantly different from the wild-type plants .
c comparison of plant height between wt and rh2ri 2b in 78-day-old plants and 147-day-old plants.
bars = 19 cm.
d comparison of internode distance of 4-month-old rice plants among wt, rh2ri 2b, rh2ri  <dig>  and rh2ri 14b.
bars = 5 cm.
e determination of internode distance of rh2ri 2b, rh2ri  <dig>  rh2ri 14b, and wild-type plants.
error bars show ± sd , * is significantly different from the wild-type plants
± indicates standard deviation, n =  <dig> for each line
* is significantly different from the wild-type plants
severe defects in pollen and seed development in double knockdowns of osrh <dig> and osrh34
the rh2ri transgenic plants had 30 ~ 40 % fewer seeds than the wild-type plants .
this marked reduction in the number of seeds suggested that double knockdown of osrh <dig> and osrh <dig> may cause defects in fertilization or seed development.
aborted pollen was previously identified in osmago <dig> and osmago <dig> double-knockdown plants and osy14a knockdown plants  <cit> .
to address whether osrh <dig> and osrh <dig> function in the male gametophyte development, pollen viability of rh2ri transgenic plants was determined by the alexander staining.
in fig.
8c, aborted pollens were more in rh2ri transgenic plants than that in wild type, suggesting that double knockdowns of osrh <dig> and osrh <dig> affected male gametophyte development.fig.
<dig> seed setting rate and seed development in osrh <dig> and osrh <dig> double-knockdown transgenic rice.
a and b a low seed setting rate was observed in osrh <dig> and osrh <dig> double-knockdown plants.
a spikelet phenotype of three independent osrh <dig> and osrh <dig> double-knockdown lines, rh2ri 2b, rh2ri  <dig>  and rh2ri 14b.
bars = 5 cm.
b determination of the numbers of mature and aborted seeds in osrh <dig> and osrh <dig> double-knockdown lines.
error bars show ± sd , * is significantly different from the wild-type plants .
c the osrh <dig> and osrh <dig> double-knockdown lines showed defects in pollen development.
bars = 200 μm.
d–f the osrh <dig> and osrh <dig> double-knockdown lines showed defects in embryonic development.
d micrographs of husked of wild-type rice seeds at various developmental stages.
rice seeds were harvested at  <dig>   <dig>   <dig>   <dig>  and 30 days after pollination .
e the internal seed stages of the rh2ri 2b line at  <dig> dap.
f determination of the numbers of the seeds at different stages in the rh2ri 2b transgenic and wild-type plants
on the other hand, the levels of seed development normally seen at  <dig>   <dig>   <dig>   <dig>  and 30 days after pollination  were set as stage i to stage v, respectively .
most seeds in the wild-type plants had developed to stage v at  <dig> dap .
however, in the rh2ri 2b transgenic plants, the level of seed development at  <dig> dap varied, from stage i to v; about one-third of the plants remained at stage i, one-third were at stages ii, iii, or iv, and one-third formed mature seeds  .
these phenotypes suggested that the osrh <dig> and osrh <dig> genes play critical roles in the development of rice seeds.
exogenous gibberellic acid  partially rescues the phenotype of rh2ri transgenic plants and double knockdown of osrh <dig> and osrh <dig> influences on ga biosynthesis and ga signaling genes
phenotypes of the osrh <dig> rnai transgenic plants included dwarf, reduced internode length, deep green in the leaf color, increased tiller number, abnormal seed development and reduced seed germination rate, are similar to mutants deficient in ga biosynthesis or ga signaling pathway.
to investigate whether osrh <dig> and osrh <dig> are involved in the ga biosynthesis or signaling pathway, rice seedlings were treated with  <dig>  and 1 μm ga <dig>  elongation of the dwarf phenotype of 10-day-old rh2ri seedlings was recovered partially by ga <dig> treatment .
to further characterize of ga sensing in rh2ri transgenic plants, starch plate assay for activity of α-amylase from aleurone layer cells was conducted.
the embryoless half-seeds were placed on starch plates with or without 1 μm ga <dig> for 2 days, and then starch plates were stained with iodine.
activity of α-amylase was not detected in rh2ri 2b and wild-type embryoless half seeds without treatment of ga <dig> .
cleared zone was detected both in ga <dig> treatment of half seeds, and no difference in cleared zone size was observed between wild-type and rh2ri 2b transgenic lines .
these results demonstrated that rh2ri transgenic plants were responsive to exogenously supplied ga <dig> fig.
<dig> effect of exogenous ga on the osrh <dig> and osrh <dig> double-knockdown t <dig> transgenic rice.
a three-day-old rice seedlings were incubated in water containing  <dig>   <dig> , and 1 μm ga <dig> for 7 days.
bars = 1 cm.
b a starch plate assay of α-amylase activity.
embryoless half seeds were incubated on starch plates with 10− <dig> m ga <dig> for 2 days.
c quantification of clear zone diameter on starch plates with 10− <dig> and 10− <dig> m ga <dig>  error bars show ± sd .
d expression of osga20ox <dig> and osgamyb in the osrh <dig> and osrh <dig> double-knockdown seedlings.
total rnas were isolated from three-leaf-stage seedling and subjected to qrt-pcr.
rice act <dig> as an internal control.
error bars indicate the sd of four replicate experiments with two biological replicates.
gene expression was related to wild-type plants, as  <dig>  * is significantly different from the wild-type plants
to investigate the role of osrh <dig> and osrh <dig> in ga biosynthesis and ga signaling, the expression levels of the osga20ox <dig>  a gene encoded for ga biosynthesis, and the osgamyb, a transcription factor in ga signaling, were determined.
total rnas were isolated from three-leaf-stage of rh2ri transgenic seedlings and subjected to qrt-pcr analyses.
the mrna levels of the osga20ox <dig> and the osgamyb were significantly decreased in various rh2ri lines, compared to the wild type .
this result suggested that osrh <dig> and osrh <dig> participate the regulation of ga biosynthesis and ga signaling pathways.
double knockdown of osrh <dig> and osrh <dig> transgenic plants exhibit accumulation of unspliced osudt <dig> mrna
it has been demonstrated that osmago <dig>  osmago <dig>  and osy14b are involved in the splicing of osudt <dig> mrna  <cit> .
both osrh <dig> and osrh <dig> are one component of the ejc core complex, suggesting that double knockdowns of osrh <dig> and osrh <dig> may affect osudt <dig> mrna maturation.
total rna was isolated from inflorescence of plants and subjected to rt-pcr using specific primers  for amplifying fragments of osudt <dig> mrna.
four fragments, namely type i, mature, type ii, and type iii, were amplified using udt1r and udt1f primers .
the accumulated levels of the type i, type ii, and type iii were higher in three independent osrh <dig> and osrh <dig> double-knockdown lines than wild type .
using the udtin1f and udtin1r primer pair to specifically amplify the type i fragments , more accumulated unspliced type i osudt <dig> mrnas were detected in these three independent osrh <dig> and osrh <dig> double-knockdown transgenic lines, as compared to wild type .
these results indicate that osrh <dig> and osrh <dig> play critical roles in the accurate splicing of osudt <dig> pre-mrna.fig.
<dig> accumulation of abnormal osudt <dig> transcripts in osrh <dig> and osrh <dig> double-knockdown plants.
a illustration of gene structure and abnormal transcript structures of osudt <dig>  <cit> , and the positions of primers used for rt-pcr analysis.
gray rectangles, utrs; white rectangles, exons ; lines, introns .
b and c accumulation of osudt <dig> abnormal transcripts.
total rnas were isolated from inflorescence of wt, rh2ri 2b, rh2ri  <dig>  and rh2ri 14b plants at vegetative stage.
unspliced osudt <dig> pre-mrnas were detected by rt-pcr analysis with specific primers .
act <dig> mrnas were used as internal control.
c relative level of abnormal  and mature osudt <dig> mrnas were determined by image j with normalization relative to the wt.
error bars indicated the sd of four replicate experiments with two biological replicates.
level of dna fragment was related to wild-type plants, as  <dig>  * is significantly different from the wild-type plants
discussion
in this study, two dead box rna helicase genes, osrh <dig> and osrh <dig>  were characterized in rice.
amino acid sequence analysis indicated that osrh <dig> and osrh <dig> share 99 % identity and 100 % similarity, suggesting that these two dead box rna helicases might have similar biochemical properties in rice.
both osrh <dig> and osrh <dig> are homologous to eif4aiii, which is a member of the eif4a family.
eif4aiii is a core component of the ejc, which is one of the fundamental factors involved in post-transcriptional processes in eukaryotes  <cit> .
besides eif4aiii, the ejc also contains three other subunits, mago, y <dig>  and btz  <cit> .
the results obtained in the present study demonstrate that both osrh <dig> and osrh <dig> interact physically with osmago <dig> and osy14b.
three independent osrh <dig> and osrh <dig> double-knockdown transgenic lines showed phenotypes that were similar to those of plants in which the osy14a gene had been knocked down or both osmago <dig> and osmago <dig> had been knocked down, namely, reduced plant height and abnormal endothecium and tapetum in flowers  <cit> .
thus, osrh <dig> and osrh <dig> are a core component of the ejc in rice.
immunofluorescence microscopy indicated that eif4aiii was localized to the nucleoplasm  <cit>  in hela cells; a similar localization pattern of eif4aiii was observed for transiently expressed myc-eif4aiii  <cit> .
however, excessive eif4aiii were found in the cytoplasm by subcellular fractionation analysis  <cit> .
these studies indicated that eif4aiii is localized in the nucleus and the cytoplasm.
in terms of the results of the subcellular localization of the osrh2–gfp and gfp–osrh <dig> fusion protein, fluorescence was detected in the nucleus and the cytoplasm.
similarly, the osrh34–gfp and mcherry–osrh <dig> fluorescence was detected in the nucleus and cytoplasm.
these results indicate that both osrh <dig> and osrh <dig> proteins are localized in the nucleus and the cytoplasm, and also suggest that they can shuttle between these two locations.
indeed, arabidopsis eif4aiii is mainly localized in the nucleoplasm under normal growth conditions, but is located in the nucleolus and forms splicing speckles under hypoxic stress  <cit> .
the function of the ejc in rice is poorly understood.
however, recently, the ejc core subunits osmago <dig>  osmago <dig>  osy14a, and osy14b were identified.
it has been identified that there are different types of mago-y <dig> complex, and variation in their specific functions has been proposed  <cit> .
knockdown of a single mago did not lead to any visible phenotype, while the double knockdown of mago genes in rice plants led to dwarfism with abnormal flowers  <cit> , suggesting that osmago <dig> and osmago <dig> are functionally redundant.
the phenotype of osy14a knockdown rice plants matched that of osmago <dig> and osmago <dig> double-knockdown plants, while the knockdown of osy14b led to failure of the induction of plantlets  <cit> , suggesting the functional specialization of osy14b in embryogenesis.
the amino acid sequences of osrh <dig> and osrh <dig> were found to be highly conserved, and their gene expression patterns were also similar in various rice tissues.
however, the abundance of osrh <dig> mrna was about 58-fold higher than that of osrh <dig> mrna in seedlings.
these results suggest that osrh <dig> and osrh <dig> may be functionally redundant, and that osrh <dig> plays a major role in rice.
since dna sequence of osrh <dig> and osrh <dig> are too similar to make specific gene silencing in rice, we therefore cannot rule out a possibility whether each of them has specific functions in rice.
we introduced osrh <dig> mrna-based interfering rna into rice and knocked down both the osrh <dig> and the osrh <dig> genes in the same transgenic line.
the osrh <dig> and osrh <dig> double-knockdown transgenic lines showed a dwarf phenotype.
the number of nodes and the internode distance in osrh <dig> and osrh <dig> double-knockdown lines were less than in the wild type.
in addition, the leaves of the osrh <dig> and osrh <dig> double-knockdown transgenic plants were a deeper green and they had a greater number of tillers than the wild-type plants.
these phenotypes are similar to those of mutants with defects in gibberellin signaling  or gibberellin biosynthesis .
exogenous ga was able to partially rescue the dwarf phenotype and induce α-amylase activities in aleurone layers of the osrh <dig> and osrh <dig> double-knockdown transgenic lines.
the expression levels of the osga20ox <dig> were decreased in the osrh <dig> and osrh <dig> double-knockdown transgenic lines as compared to wt.
these results suggested that the dwarf phenotype of the osrh <dig> and osrh <dig> double-knockdown transgenic lines may due to the decreased level of ga. however, low levels of osgamyb mrna were detected in the osrh <dig> and osrh <dig> double-knockdown transgenic lines.
in a previous study, osy14a knockdown rice plants and those with double knockdown with mago genes also exhibited dwarfism with abnormal flowers, and low level of the osga20ox <dig> and the osgamyb mrna  <cit> .
thus, the function of ejc was suggested to be strongly correlated with the gibberellin action in rice.
there was a discrepancy in the seed setting rate between the wild-type and osrh <dig> and osrh <dig> double-knockdown transgenic lines.
the ratio of mature seeds to total seeds in the osrh <dig> and osrh <dig> double-knockdown transgenic lines was lower than in the wild type.
the aborted pollen phenotype observed in the osrh <dig> and osrh <dig> double-knockdown plants was consistent with previously identified in osmago <dig> and osmago <dig> double-knockdown plants and osy14a knockdown plants  <cit> .
these results suggest that the low seed setting rate may be caused by a defective ejc, which affects pollen development.
however, it is necessary to address whether the ejc is also involved in female gametophyte development.
alternatively, seed development was compared between the wild type and the osrh <dig> and osrh <dig> double-knockdown transgenic lines.
after pollination, 90 % of the seeds of the wild type developed to the mature stage.
in contrast, in the transgenic lines, one-third of the seeds developed to maturity, one-third remained at an intermediate stage, and one-third did not progress beyond a very early stage.
these results suggested that the double knockdown of osrh <dig> and osrh <dig> impaired seed development.
taking these findings together, the double knockdown of the osrh <dig> and osrh <dig> genes may cause defects in pollen and seed development.
in eukaryotic cells, nonsense-mediated mrna decay , a surveillance mechanism, eliminates mrna that contains nonsense mutations or has acquired premature termination codons because of aberrant splicing  <cit> .
it is thus an effective safeguard for eliminating aberrant gene expression .
the ejc has been demonstrated to be involved in the post-transcriptional processing of mrna, including mrna splicing and nmd, in eukaryotes  <cit> .
in rice, osmago <dig> and osmago <dig> double-knockdown plants and osy14a knockdown plants exhibited abnormal splicing of osudt <dig> transcripts.
multiple types of osudt <dig> mrna were detected in osmago <dig> and osmago <dig> double-knockdown plants and osy14a knockdown plants  <cit> .
in the present study, double knockdown of the osrh <dig> and osrh <dig> genes also led to abnormal osudt <dig> pre-mrna splicing accumulation.
thus, the knockdown of one component of the ejc causes defects of ejc function, which is strongly correlated to the accumulation of certain abnormal pre-mrna.
however, this type of intron retained pre-mrna accumulation may be due to an ejc dependent-splicing defect or an ejc dependent-nmd defect.
future studies to identify the proteins that interact with osrh <dig> or osrh <dig> might provide more information on the specificity of the function of ejc in rice.
CONCLUSIONS
the ejc contains four core components, eukaryotic initiation factor 4aiii , mago/nashi, y14/tsunagi/rna-binding protein 8a, and barentsz/metastatic lymph node  <dig>  and plays important roles in gene regulation.
genes encoding core ejc components have been found in rice, and currently.the functional characterizations of mago and y <dig> homologs have been demonstrated in economically important crop, rice.
however, little is known about how important of eif4aiii in rice.
in this study, two rice eif4aiii homologous genes, osrh <dig> and osrh <dig>  were identified.
deduced amino acid sequence of osrh <dig> and osrh <dig> share 99 % identity and 100 % similarity.
both rice eif4aiii fluorescent fusion proteins were localized in the cytoplasm and the nucleus.
moreover, osrh <dig> and osrh <dig> can interact with rice mago and y <dig>  indicating that osrh <dig> and osrh <dig> are core components of the ejc.
osrh <dig> and osrh <dig> may be functionally redundant, but the abundantly expressed osrh <dig> may play a major role in rice.
double-knockdown of osrh <dig> and osrh <dig> exhibited severe defects in terms of plant height, pollen, and seed development.
moreover, double knockdown of the osrh <dig> and osrh <dig> genes led to decrease in expression levels of osga20ox <dig> and the osgamyb and abnormal accumulation of osudt <dig> pre-mrna.
these visible and molecular phenotypes caused by osrh <dig> and osrh <dig> double-knockdown are similar to osmago <dig> and osmago <dig> double-knockdown plants and osy14a knockdown plants.
collectively, our findings demonstrate the eif4aiii proteins, osrh <dig> and osrh <dig>  play critical roles in the functional rice ejc.

 the dead box rna helicase family, the largest family of rna helicases, belongs to helicase superfamily each dead box rna helicase contains nine conserved amino acid motifs that constitute the helicase core domain. besides these conserved motifs within dead box proteins, there are also n- and c-terminal extension sequences in each dead box rna family member that varies in terms of their length and composition; they have been proposed to provide substrate binding specificity, and to act as signals for subcellular localization or as domains that interact with accessory components. dead box proteins are found in most prokaryotes and all eukaryotes, including plants. rice is an important staple food crop and is also valuable as a model plant for studies in cereal functional genomics. although predicted protein sequences in the rice genome database as determined by silico analysis to indicate that there are at least dead box proteins in rice, the functional characterizations of most of them remain unknown. eukaryotic initiation factor 4aiii, a dead box rna helicase, is a core component of the exon junction complex that also contains mago/nashi, y14/tsunagi/rna-binding protein 8a, and barentsz/metastatic lymph node. the ejc is formed in both the nucleus and the cytoplasm, and plays important roles in gene expression, including the following: it assembles 20– bases upstream of each exon of pre-mrna for its involvement in mrna splicing. it is involved in nonsense-mediated decay, a surveillance mechanism that degrades mrna containing premature termination codons. it is involved in the regulation of gene expression at the translational level. it has a role in mrna subcellular localization. although most research has been undertaken in mammals, genes encoding core ejc components have been found in plants, suggesting that there is structural and functional conservation in the ejc complex among plant and mammalian. however, only limited evidence has been reported on the physiological role of the ejc in plants. in arabidopsis, eif4aiii interacts with an ejc component, aly/ref, and colocalizes with other ejc components, such as mago, y and rnps. in o. sativa, two forms of mago, osmago and osmago and two forms of y osy14a and osy14b, were analyzed. osmago and osmago double-knockdown rice plants displayed dwarfism and abnormal flowers in which the endothecium and tapetum of the stamen were maintained. osy14b may function in embryogenesis, while the down-regulation of osy14b resulted in a failure to induce plantlets. osy14a knockdown plants also displayed phenotypes similar to those of osmago and osmago double-knockdown rice plants. moreover, osmago and osmago double-knockdown, and osy14a knockdown transgenic plants showed abnormal accumulation of the pre-mrna of undeveloped tapetum, a key regulator of stamen development. these findings indicate that the ejc participates in the regulation of pre-mrna splicing in rice. despite the fact that the functions of homologs of mago and y have been demonstrated in rice, it is still unknown whether eif4aiii is essential for ejc function in rice. in this study, two putative rice dead box rna helicase genes, osrh and osrh, were therefore characterized. both osrh and osrh are homologous to eif4aiii, which is a member of the eif4a family, and their gene expression patterns were similar in various rice tissues, but the level of osrh mrna was about 58-fold higher than that of osrh mrna in seedlings. the results from bimolecular fluorescence complementation analysis showed that both osrh and osrh can interact with osmago and osy14b. transgenic plants with both osrh and osrh knocked down by rna interference displayed phenotypes that resembled those of mutants with gibberellin-related developmental defects. moreover, these osrh and osrh double-knockdown plants exhibited severe defects in terms of pollen and seed development. the accumulation of osudt pre-mrna was also detected in the osrh and osrh double-knockdown transgenic lines. our data demonstrate that both osrh and osrh are core components of the ejc and play critical roles in regulation of plant height, pollen, and seed development in rice.  osrh and osrh are putative dead box rna helicases to identify rice eif4aiii homologs, human eif4aiii protein sequences were used as queries to search protein databases at phytozome and national center for biotechnology information. two eif4aiii-like putative proteins, encoded by osrh and osrh were identified in rice. the osrh is located on rice chromosome and has eight exons. the deduced amino acid sequence of osrh cdna consists of nine conserved rna helicase domains and the characteristic amino acid residues d-e-a-d in motif ii. besides, the osrh gene has eight exons and is located on chromosome the levels of identity between osrh and osrh in terms of the dna sequence and the deduced amino acid sequence were found to be and 99 %, respectively. phylogenetic relationships were established using amino acid sequences from the eif4a families of dicots, monocots, green algae, vertebrates, invertebrates, and yeast, which showed that osrh and osrh are closely related to eif4aiii and can be clustered into the monocot group.fig. amino acid sequences and domain structures of the osrh and osrh proteins. a. the amino acid sequences of osrh and osrh were compared using the clustal w program. identical amino acid residues are labeled in black. different amino acid residues are marked by asterisks. the conserved helicase motif is highlighted by a line above it and includes motifs q, i, ia, ib, ii, iii, iv, v, and vi fig. phylogenetic relationships of eif4aiii family members. a phylogenetic tree for eif4aiii in dicots, monocots, green algae, vertebrates, invertebrates, and yeast was generated using mega eif4aiii members from rice, maize, sorghum, and brachypodium are categorized into the monocot group with at least 50 % bootstrap support. accession numbers of the genes listed here are shown in additional file 2 expression patterns of osrh and osrh34 to determine the relative expression levels of osrh and osrh in rice, total rna was isolated from a variety of vegetative and reproductive tissues and was subjected to qrt-pcr with specific primers. the osrh transcript was expressed in all selected tissues and organs, including roots, stems, leaves, sheaths, panicles, and seedlings. relatively high levels of osrh mrna were detected in vegetative leaf blades, flag leaves, and panicles before heading. expression of osrh was relatively abundant in vegetative leaf blades, flag leaves, and seedlings, whereas its expression was rarely detected in roots, stems, and panicles. these results indicate that these two paralogous genes are coexpressed in most selected tissues and organs in rice. to compare the levels of osrh and osrh mrna in rice plants, absolute qrt-pcr was performed. standard curves were used with a serial dilution of either osrh cdna- or osrh cdna-containing plasmids. as shown in fig. 3b, the level of osrh mrna was 58-fold higher than that of osrh mrna in rice seedlings at the three-leaf stage.fig. expression of osrh and osrh a qrt-pcr analysis of osrh and osrh gene expression in rice. total rna was isolated from seedlings, roots, stems, leaves, sheaths, flag leaves, booting panicles, heading panicles, flowering panicles, and pollinated panicles. the rice act gene was used as an internal control. b absolute quantitative rt-pcr analysis of osrh and osrh in which plasmid dna was applied as a control to compare the mrna levels of osrh and osrh34 osrh and osrh were colocalized in nucleus and cytoplasm to determine the subcellular localization of osrh and osrh plasmids containing an osrh2–gfp fusion gene and osrh34–gfp under the control of the camv 35s promoter were generated and introduced into onion epidermal cells. fluorescent signals were emitted from both osrh2–gfp and osrh34–gfp in both the nucleus and the cytoplasm. similar results were obtained in onion cells for the expression of either gfp–osrh or mcherry–osrh. to confirm the subcellular localization of osrh and osrh onion cells were cotransformed with gfp–osrh and mcherry–osrh gfp and mcherry signals were colocalized in the nucleus and the cytoplasm. these results suggest that the osrh and osrh proteins are localized in both the nucleus and the cytoplasm.fig. subcellular localization of osrh and osrh a and b osrh fluorescence fusion protein was localized in the nucleus and the cytoplasm. onion epidermal cells were transformed with either 35s::osrh2–gfp or 35s::gfp–osrh. c and d onion epidermal cells were transformed with either 35s::osrh34–gfp or 35s::mcherry–osrh. e colocalization of gfp–osrh and mcherry–osrh in the nucleus and the cytoplasm. onion epidermal cells were cotransformed with 35s::gfp–osrh and 35s::mcherry–osrh bars = 100 μm both osrh and osrh are components of the ejc core complex eif4aiii can interact with y and mago to form the ejc core complex in eukaryotic cells. gong and he have also reported that rice mago and y can form heterodimers. to determine whether osrh and osrh were components of the ejc in rice, interactions among rice mago, y and eif4aiii were examined by bifc. the n-terminus of yellow fluorescent protein was fused at the downstream end of osrh and osrh the c-terminus of yfp was fused at the downstream end of osy14b and osmago coexpression of osrh2-yn and yc, osrh34-yn and yc, yn and osmago1-yc, yn and osy14b-yc in onion epidermal cells were used as negative controls for interaction tests among osrh osmago and osy and no fluorescent signals were detected. the interaction between osmago and osy14b was used as a positive control that exhibited remarkable fluorescent signals in onion cells. these two fusion proteins, osrh2-yn and osy14b-yc, were coexpressed in onion cells and the yfp fluorescence was observed. osrh2-yn and osmago1-yc coexpressed in onion cells also displayed the yfp signal. meanwhile, yfp fluorescence was also detected upon the coexpression of osrh34-yn with osy14b-yc and osrh34-yn with osmago1-yc, respectively. these results indicate that both osrh and osrh directly interact with osy14b and osmago demonstrating that they are indeed a component of the ejc core complex in rice.fig. bifc analysis of the interaction among rice mago, y and eif4aiii in onion epidermal cells. n- and c-terminal fragments of yfp were fused to the c-terminus of osrh osrh osmago and osy14b, respectively. onion epidermal cells were cotransformed with combinations of 35s:: osrh2–yn and 35s::yc, 35s::osrh34–yn and 35s::yc, 35s::yn and 35s::y14b–yc, and 35s::yn and 35s::mago1–yc as negative controls onion epidermal cells were cotransformed with 35s::osmago1–yn and 35s::osy14b–yc, 35s::osrh2–yn and 35s::osy14b–yc, 35s::osrh2–yn and 35s::osmago1–yc, 35s::osrh34–yn and 35s::osy14b–yc, 35s::osrh34–yn and 35s::osmago1–yc. bars = 100 μm the osrh and the osrh were colocalized, so protein interaction between these two isoforms was further examined by the bifc analysis. the yfp fluorescent signals were not be observed in onion cells coexpressed with either combinations of osrh2-yn and osrh2-yc, osrh34-yn and osrh34-yc, osrh2-yn and osrh34-yc, or osrh34-yn and osrh2-yc. these results indicated that proteins of osrh and osrh were not able to interact to form homomer or heteromer. characterization of double knockdown of osrh and osrh transgenic lines to unravel the physiological functions of osrh and osrh a rna interference mediated genes silencing approach was performed. because osrh and osrh shared extremely high sequence identity, it was difficult to achieve specific gene silencing. thus, double knockdown of osrh and osrh was carried out in rice. to minimize the potential off-target gene silencing, the sequences of 271-bp rnai designed region at the 3´ end of osrh cdna and osrh cdna were used as queries to search rice mrna databases at ncbi. none of region identical of around or more than nucleotides was obtained. further, a public web-based computational tool developed for identification of potential off-targets, sirna scan, was applied to search rice mrna databases, and no potential off-target was detected in the rnai designed region. inverted repeat of the 271-bp region was fused at the up- and downstream ends of a gfp coding sequence, and the fusion construct was expressed under the control of the maize ubiquitin gene promoter in transgenic rice. several independent t transgenic plants were obtained, and the levels of osrh mrna and osrh were determined by qrt-pcr. as results showed in fig. 6b, both osrh mrna and osrh mrna were barely detectable in three independent t transgenic lines, rh2ri 2b, rh2ri and rh2ri 14b, indicating that both osrh and osrh were knocked down. therefore, rh2ri 2b, rh2ri and rh2ri 14b lines were selected to address roles of osrh and osrh in rice.fig. characterization of osrh and osrh double-knockdown transgenic lines. a schematic presentation of the double silencing of osrh and osrh of the rna interference construct. a 271-bp fragment at the 3′ end of osrh and osrh conserved region was ligated in sense and antisense orientations to the gfp cdna and fused downstream of the ubi promoter. b expression of osrh and osrh in t transgenic rice seedlings. total rna was isolated from 14-day-old seedlings and subjected to qrt-pcr using osrh2- and osrh34-specific primers. rice act was used as an internal control. error bars indicate the standard deviations of triplicate experiments. gene expression was related to wild-type plants, as * is significantly different from the wild-type plants. osrh and osrh double-knockdown lines are named as rh2ri 2b, and 14b. wild-type line is indicated by wt reduced plant height in transgenic rice double knockdown of osrh and osrh34 significant differences in the height of plants in the t transgenic lines were observed. rh2ri 2b, rh2ri and rh2ri 14b showed a dwarf phenotype; their seedlings were to 44 % shorter than those of wild-type plants at 2 weeks old. moreover, rh2ri transgenic plants were shorter than wild-type plants at following growth stage. one example was shown in fig. 7c, the plant height of rh2ri 2b t plant was and 26 % shorter than wild-type plants at 78-day-old and 147-day-old stages, respectively. plant height was further compared between wild-type and rh2ri transgenic plants at the reproductive stage. the culm of wild-type plants contained five internodes, named i to v from top to bottom. culm lengths of the rh2ri transgenic plants also appeared to be reduced in each internode region compared with those in the wild-type plants. the dwarf phenotype of rh2ri transgenic plants was also observed in a paddy field. significant differences in plant heights between wild-type plants and rh2ri plants of the three transgenic t1-t generation were observed. in addition, the leaves of the rh2ri transgenic plants were a deeper green and they had a greater number of tillers than the wild-type plants.fig. phenotype of osrh and osrh double-knockdown t transgenic rice. a wt and three independent osrh and osrh double-knockdown lines, rh2ri 2b, rh2ri and rh2ri 14b, seedlings were grown on ½ ms agar medium for 10 days and transferred to hydroponic cultures for 7 days. bar = 1 cm. b quantification of plant height at seedling stages. the plant height of 17-day-old seedlings was measured. error bars indicate the sd of ten individual plants for each line. * is significantly different from the wild-type plants. c comparison of plant height between wt and rh2ri 2b in 78-day-old plants and 147-day-old plants. bars = 19 cm. d comparison of internode distance of 4-month-old rice plants among wt, rh2ri 2b, rh2ri and rh2ri 14b. bars = 5 cm. e determination of internode distance of rh2ri 2b, rh2ri rh2ri 14b, and wild-type plants. error bars show ± sd, * is significantly different from the wild-type plants ± indicates standard deviation, n =  for each line * is significantly different from the wild-type plants severe defects in pollen and seed development in double knockdowns of osrh and osrh34 the rh2ri transgenic plants had 30 ~ 40 % fewer seeds than the wild-type plants. this marked reduction in the number of seeds suggested that double knockdown of osrh and osrh may cause defects in fertilization or seed development. aborted pollen was previously identified in osmago and osmago double-knockdown plants and osy14a knockdown plants. to address whether osrh and osrh function in the male gametophyte development, pollen viability of rh2ri transgenic plants was determined by the alexander staining. in fig. 8c, aborted pollens were more in rh2ri transgenic plants than that in wild type, suggesting that double knockdowns of osrh and osrh affected male gametophyte development.fig. seed setting rate and seed development in osrh and osrh double-knockdown transgenic rice. a and b a low seed setting rate was observed in osrh and osrh double-knockdown plants. a spikelet phenotype of three independent osrh and osrh double-knockdown lines, rh2ri 2b, rh2ri and rh2ri 14b. bars = 5 cm. b determination of the numbers of mature and aborted seeds in osrh and osrh double-knockdown lines. error bars show ± sd, * is significantly different from the wild-type plants. c the osrh and osrh double-knockdown lines showed defects in pollen development. bars = 200 μm. d–f the osrh and osrh double-knockdown lines showed defects in embryonic development. d micrographs of husked of wild-type rice seeds at various developmental stages. rice seeds were harvested at and 30 days after pollination. e the internal seed stages of the rh2ri 2b line at dap. f determination of the numbers of the seeds at different stages in the rh2ri 2b transgenic and wild-type plants on the other hand, the levels of seed development normally seen at and 30 days after pollination were set as stage i to stage v, respectively. most seeds in the wild-type plants had developed to stage v at dap. however, in the rh2ri 2b transgenic plants, the level of seed development at dap varied, from stage i to v; about one-third of the plants remained at stage i, one-third were at stages ii, iii, or iv, and one-third formed mature seeds. these phenotypes suggested that the osrh and osrh genes play critical roles in the development of rice seeds. exogenous gibberellic acid partially rescues the phenotype of rh2ri transgenic plants and double knockdown of osrh and osrh influences on ga biosynthesis and ga signaling genes phenotypes of the osrh rnai transgenic plants included dwarf, reduced internode length, deep green in the leaf color, increased tiller number, abnormal seed development and reduced seed germination rate, are similar to mutants deficient in ga biosynthesis or ga signaling pathway. to investigate whether osrh and osrh are involved in the ga biosynthesis or signaling pathway, rice seedlings were treated with and 1 μm ga elongation of the dwarf phenotype of 10-day-old rh2ri seedlings was recovered partially by ga treatment. to further characterize of ga sensing in rh2ri transgenic plants, starch plate assay for activity of α-amylase from aleurone layer cells was conducted. the embryoless half-seeds were placed on starch plates with or without 1 μm ga for 2 days, and then starch plates were stained with iodine. activity of α-amylase was not detected in rh2ri 2b and wild-type embryoless half seeds without treatment of ga. cleared zone was detected both in ga treatment of half seeds, and no difference in cleared zone size was observed between wild-type and rh2ri 2b transgenic lines. these results demonstrated that rh2ri transgenic plants were responsive to exogenously supplied ga fig. effect of exogenous ga on the osrh and osrh double-knockdown t transgenic rice. a three-day-old rice seedlings were incubated in water containing, and 1 μm ga for 7 days. bars = 1 cm. b a starch plate assay of α-amylase activity. embryoless half seeds were incubated on starch plates with 10− m ga for 2 days. c quantification of clear zone diameter on starch plates with 10− and 10− m ga error bars show ± sd. d expression of osga20ox and osgamyb in the osrh and osrh double-knockdown seedlings. total rnas were isolated from three-leaf-stage seedling and subjected to qrt-pcr. rice act as an internal control. error bars indicate the sd of four replicate experiments with two biological replicates. gene expression was related to wild-type plants, as * is significantly different from the wild-type plants to investigate the role of osrh and osrh in ga biosynthesis and ga signaling, the expression levels of the osga20ox a gene encoded for ga biosynthesis, and the osgamyb, a transcription factor in ga signaling, were determined. total rnas were isolated from three-leaf-stage of rh2ri transgenic seedlings and subjected to qrt-pcr analyses. the mrna levels of the osga20ox and the osgamyb were significantly decreased in various rh2ri lines, compared to the wild type. this result suggested that osrh and osrh participate the regulation of ga biosynthesis and ga signaling pathways. double knockdown of osrh and osrh transgenic plants exhibit accumulation of unspliced osudt mrna it has been demonstrated that osmago osmago and osy14b are involved in the splicing of osudt mrna. both osrh and osrh are one component of the ejc core complex, suggesting that double knockdowns of osrh and osrh may affect osudt mrna maturation. total rna was isolated from inflorescence of plants and subjected to rt-pcr using specific primers for amplifying fragments of osudt mrna. four fragments, namely type i, mature, type ii, and type iii, were amplified using udt1r and udt1f primers. the accumulated levels of the type i, type ii, and type iii were higher in three independent osrh and osrh double-knockdown lines than wild type. using the udtin1f and udtin1r primer pair to specifically amplify the type i fragments, more accumulated unspliced type i osudt mrnas were detected in these three independent osrh and osrh double-knockdown transgenic lines, as compared to wild type. these results indicate that osrh and osrh play critical roles in the accurate splicing of osudt pre-mrna.fig. accumulation of abnormal osudt transcripts in osrh and osrh double-knockdown plants. a illustration of gene structure and abnormal transcript structures of osudt, and the positions of primers used for rt-pcr analysis. gray rectangles, utrs; white rectangles, exons; lines, introns. b and c accumulation of osudt abnormal transcripts. total rnas were isolated from inflorescence of wt, rh2ri 2b, rh2ri and rh2ri 14b plants at vegetative stage. unspliced osudt pre-mrnas were detected by rt-pcr analysis with specific primers. act mrnas were used as internal control. c relative level of abnormal and mature osudt mrnas were determined by image j with normalization relative to the wt. error bars indicated the sd of four replicate experiments with two biological replicates. level of dna fragment was related to wild-type plants, as * is significantly different from the wild-type plants discussion in this study, two dead box rna helicase genes, osrh and osrh were characterized in rice. amino acid sequence analysis indicated that osrh and osrh share 99 % identity and 100 % similarity, suggesting that these two dead box rna helicases might have similar biochemical properties in rice. both osrh and osrh are homologous to eif4aiii, which is a member of the eif4a family. eif4aiii is a core component of the ejc, which is one of the fundamental factors involved in post-transcriptional processes in eukaryotes. besides eif4aiii, the ejc also contains three other subunits, mago, y and btz. the results obtained in the present study demonstrate that both osrh and osrh interact physically with osmago and osy14b. three independent osrh and osrh double-knockdown transgenic lines showed phenotypes that were similar to those of plants in which the osy14a gene had been knocked down or both osmago and osmago had been knocked down, namely, reduced plant height and abnormal endothecium and tapetum in flowers. thus, osrh and osrh are a core component of the ejc in rice. immunofluorescence microscopy indicated that eif4aiii was localized to the nucleoplasm in hela cells; a similar localization pattern of eif4aiii was observed for transiently expressed myc-eif4aiii. however, excessive eif4aiii were found in the cytoplasm by subcellular fractionation analysis. these studies indicated that eif4aiii is localized in the nucleus and the cytoplasm. in terms of the results of the subcellular localization of the osrh2–gfp and gfp–osrh fusion protein, fluorescence was detected in the nucleus and the cytoplasm. similarly, the osrh34–gfp and mcherry–osrh fluorescence was detected in the nucleus and cytoplasm. these results indicate that both osrh and osrh proteins are localized in the nucleus and the cytoplasm, and also suggest that they can shuttle between these two locations. indeed, arabidopsis eif4aiii is mainly localized in the nucleoplasm under normal growth conditions, but is located in the nucleolus and forms splicing speckles under hypoxic stress. the function of the ejc in rice is poorly understood. however, recently, the ejc core subunits osmago osmago osy14a, and osy14b were identified. it has been identified that there are different types of mago-y complex, and variation in their specific functions has been proposed. knockdown of a single mago did not lead to any visible phenotype, while the double knockdown of mago genes in rice plants led to dwarfism with abnormal flowers, suggesting that osmago and osmago are functionally redundant. the phenotype of osy14a knockdown rice plants matched that of osmago and osmago double-knockdown plants, while the knockdown of osy14b led to failure of the induction of plantlets, suggesting the functional specialization of osy14b in embryogenesis. the amino acid sequences of osrh and osrh were found to be highly conserved, and their gene expression patterns were also similar in various rice tissues. however, the abundance of osrh mrna was about 58-fold higher than that of osrh mrna in seedlings. these results suggest that osrh and osrh may be functionally redundant, and that osrh plays a major role in rice. since dna sequence of osrh and osrh are too similar to make specific gene silencing in rice, we therefore cannot rule out a possibility whether each of them has specific functions in rice. we introduced osrh mrna-based interfering rna into rice and knocked down both the osrh and the osrh genes in the same transgenic line. the osrh and osrh double-knockdown transgenic lines showed a dwarf phenotype. the number of nodes and the internode distance in osrh and osrh double-knockdown lines were less than in the wild type. in addition, the leaves of the osrh and osrh double-knockdown transgenic plants were a deeper green and they had a greater number of tillers than the wild-type plants. these phenotypes are similar to those of mutants with defects in gibberellin signaling or gibberellin biosynthesis. exogenous ga was able to partially rescue the dwarf phenotype and induce α-amylase activities in aleurone layers of the osrh and osrh double-knockdown transgenic lines. the expression levels of the osga20ox were decreased in the osrh and osrh double-knockdown transgenic lines as compared to wt. these results suggested that the dwarf phenotype of the osrh and osrh double-knockdown transgenic lines may due to the decreased level of ga. however, low levels of osgamyb mrna were detected in the osrh and osrh double-knockdown transgenic lines. in a previous study, osy14a knockdown rice plants and those with double knockdown with mago genes also exhibited dwarfism with abnormal flowers, and low level of the osga20ox and the osgamyb mrna. thus, the function of ejc was suggested to be strongly correlated with the gibberellin action in rice. there was a discrepancy in the seed setting rate between the wild-type and osrh and osrh double-knockdown transgenic lines. the ratio of mature seeds to total seeds in the osrh and osrh double-knockdown transgenic lines was lower than in the wild type. the aborted pollen phenotype observed in the osrh and osrh double-knockdown plants was consistent with previously identified in osmago and osmago double-knockdown plants and osy14a knockdown plants. these results suggest that the low seed setting rate may be caused by a defective ejc, which affects pollen development. however, it is necessary to address whether the ejc is also involved in female gametophyte development. alternatively, seed development was compared between the wild type and the osrh and osrh double-knockdown transgenic lines. after pollination, 90 % of the seeds of the wild type developed to the mature stage. in contrast, in the transgenic lines, one-third of the seeds developed to maturity, one-third remained at an intermediate stage, and one-third did not progress beyond a very early stage. these results suggested that the double knockdown of osrh and osrh impaired seed development. taking these findings together, the double knockdown of the osrh and osrh genes may cause defects in pollen and seed development. in eukaryotic cells, nonsense-mediated mrna decay, a surveillance mechanism, eliminates mrna that contains nonsense mutations or has acquired premature termination codons because of aberrant splicing. it is thus an effective safeguard for eliminating aberrant gene expression. the ejc has been demonstrated to be involved in the post-transcriptional processing of mrna, including mrna splicing and nmd, in eukaryotes. in rice, osmago and osmago double-knockdown plants and osy14a knockdown plants exhibited abnormal splicing of osudt transcripts. multiple types of osudt mrna were detected in osmago and osmago double-knockdown plants and osy14a knockdown plants. in the present study, double knockdown of the osrh and osrh genes also led to abnormal osudt pre-mrna splicing accumulation. thus, the knockdown of one component of the ejc causes defects of ejc function, which is strongly correlated to the accumulation of certain abnormal pre-mrna. however, this type of intron retained pre-mrna accumulation may be due to an ejc dependent-splicing defect or an ejc dependent-nmd defect. future studies to identify the proteins that interact with osrh or osrh might provide more information on the specificity of the function of ejc in rice.  the ejc contains four core components, eukaryotic initiation factor 4aiii, mago/nashi, y14/tsunagi/rna-binding protein 8a, and barentsz/metastatic lymph node and plays important roles in gene regulation. genes encoding core ejc components have been found in rice, and currently.the functional characterizations of mago and y homologs have been demonstrated in economically important crop, rice. however, little is known about how important of eif4aiii in rice. in this study, two rice eif4aiii homologous genes, osrh and osrh were identified. deduced amino acid sequence of osrh and osrh share 99 % identity and 100 % similarity. both rice eif4aiii fluorescent fusion proteins were localized in the cytoplasm and the nucleus. moreover, osrh and osrh can interact with rice mago and y indicating that osrh and osrh are core components of the ejc. osrh and osrh may be functionally redundant, but the abundantly expressed osrh may play a major role in rice. double-knockdown of osrh and osrh exhibited severe defects in terms of plant height, pollen, and seed development. moreover, double knockdown of the osrh and osrh genes led to decrease in expression levels of osga20ox and the osgamyb and abnormal accumulation of osudt pre-mrna. these visible and molecular phenotypes caused by osrh and osrh double-knockdown are similar to osmago and osmago double-knockdown plants and osy14a knockdown plants. collectively, our findings demonstrate the eif4aiii proteins, osrh and osrh play critical roles in the functional rice ejc. 
both osrh and osrh are core components of the ejc, and participate in regulating of plant height, pollen, and seed development in rice. the exon junction complex, which contains four core components, eukaryotic initiation factor 4aiii, mago/nashi, y14/tsunagi/rna-binding protein 8a, and barentsz/metastatic lymph node is formed in both nucleus and cytoplasm, and plays important roles in gene expression. amino acid sequence analysis indicated that osrh and osrh had 99 % identity and 100 % similarity, and their gene expression patterns were similar in various rice tissues, but the level of osrh mrna was about 58-fold higher than that of osrh mrna in seedlings. from bimolecular fluorescence complementation results, osrh and osrh interacted physically with osmago and osy14b, respectively, which indicated that both of osrh and osrh were core components of the ejc in rice. dead box rna helicaseeukaryotic initiation factor 4aiii exon junction complex rice ministry of science and technology of the republic of china, taiwan103-231-b-008-001-104-2321-b-008-001-lu chung-an issue-copyright-statement© the author 2016 this study investigated two dead box rna helicases, osrh and osrh which are homologous to eif4aiii, in rice. genes encoding core ejc components have been found in plants, including rice. the phenotypes of three independent osrh and osrh double-knockdown transgenic lines included dwarfism, a short internode distance, reproductive delay, defective embryonic development, and a low seed setting rate. to study the biological roles of osrh and osrh in rice, transgenic rice plants were generated by rna interference. these phenotypes resembled those of mutants with gibberellin-related developmental defects. currently, the functional characterizations of mago and y homologs have been demonstrated in rice.
 the exon junction complex, which contains four core components, eukaryotic initiation factor 4aiii, mago/nashi, y14/tsunagi/rna-binding protein 8a, and barentsz/metastatic lymph node is formed in both nucleus and cytoplasm, and plays important roles in gene expression. genes encoding core ejc components have been found in plants, including rice. currently, the functional characterizations of mago and y homologs have been demonstrated in rice. however, it is still unknown whether eif4aiii is essential for the functional ejc in rice.  this study investigated two dead box rna helicases, osrh and osrh which are homologous to eif4aiii, in rice. amino acid sequence analysis indicated that osrh and osrh had 99 % identity and 100 % similarity, and their gene expression patterns were similar in various rice tissues, but the level of osrh mrna was about 58-fold higher than that of osrh mrna in seedlings. from bimolecular fluorescence complementation results, osrh and osrh interacted physically with osmago and osy14b, respectively, which indicated that both of osrh and osrh were core components of the ejc in rice. to study the biological roles of osrh and osrh in rice, transgenic rice plants were generated by rna interference. the phenotypes of three independent osrh and osrh double-knockdown transgenic lines included dwarfism, a short internode distance, reproductive delay, defective embryonic development, and a low seed setting rate. these phenotypes resembled those of mutants with gibberellin-related developmental defects. in addition, the osrh and osrh double-knockdown transgenic lines exhibited the accumulation of unspliced rice undeveloped tapetum mrna.  rice contains two eif4aiii paralogous genes, osrh and osrh the abundance of osrh mrna was about 58-fold higher than that of osrh mrna in seedlings, suggesting that the osrh is major eif4aiii in rice. both osrh and osrh are core components of the ejc, and participate in regulating of plant height, pollen, and seed development in rice. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords dead box rna helicaseeukaryotic initiation factor 4aiii exon junction complex rice ministry of science and technology of the republic of china, taiwan103-231-b-008-001-104-2321-b-008-001-lu chung-an issue-copyright-statement© the author 2016 