BACKGROUND
drought is a major environmental stress factor that reduces agricultural productivity.
rice is one of the most important crops worldwide, and it consumes a large amount of fresh water resources, e.g.
about 50 % in china.
developing water-saving and drought resistant rice varieties is an effective strategy to achieve food security and prevent the detrimental effects of drought and water deficit  <cit> .
elucidating the hereditary basis and molecular mechanism that underlies the drought resistance in rice is indispensable and vital for the development of new rice varieties with improved drought resistance  <cit> .
drought and water deficit can decrease photosynthetic capacity, result in oxidative damage to chloroplasts, limit metabolic reactions, and reduce dry matter accumulation and partitioning  <cit> .
to cope with drought stress, plants have developed various strategies, which include developing larger and deeper root systems to increase water absorption from the deep soil, regulating stomata closure to reduce water loss, accumulation of compatible solutes and protective proteins, and increasing the level of antioxidants  <cit> .
on exposure of plants to drought stresses, a series of genes are induced, the products of which would then participate in the stress responses.
transcription of these stress-response genes is largely controlled by transcription factors  <cit> .
a number of transcription factors have been identified in the past few years that have been demonstrated to play an essential role in regulating plant responses to stresses  <cit> .
for instance, ap <dig> transcription factors including dreb and cbf proteins bind to the dehydration response element and control expression of stress-responsive genes  <cit> .
overexpression of dreb1b and dreb1a in arabidopsis enhanced freezing tolerance and dehydration/salt tolerance, respectively  <cit> .
in rice, ap <dig> transcription activators such as osdreb1a have been isolated.
osdreb1a was induced by dehydration and high salinity stress  <cit> , and overexpression of osdreb1a in transgenic arabidopsis improved stress tolerance.
recently, several other types of transcription factors in rice including snca <dig>  <cit> , dst  <cit> , myb  <cit> , and zip  <cit>  have been identified to play important roles in drought resistance through regulating stomata closure, reactive oxygen species  scavenging, or other physiological processes.
although the transcription factor genes have been extensively studied, further studies are still needed to identify other novel transcription factors that are involved in stress responses.
gras proteins are plant specific proteins, and homologues have been found in many higher plants such as arabidopsis, tomato, petunia, rice, and barley.
the name is derived from the three initially identified members, gibberellin-acid insensitive , repressor of ga <dig>  and scarecrow   <cit> .
gras proteins are typically composed of 400– <dig> amino acid residues and exhibit considerable sequence homology to each other in their respective c-terminal domains, whereas the n-terminal amino acid sequences are highly divergent  <cit> .
gras family proteins are divided into several sub-families such as della, shr, scr, pat, liscl, and scl <dig>  <cit> .
in the past few years, studies have demonstrated that gras proteins play diverse roles in gibberellin signal transduction, root development, meristem development, light signaling, biotic stress, and abiotic stress responses  <cit> .
della proteins are one of the most extensively studied gras subfamilies, and they function as repressors of gibberellin  responsive plant growth and are key regulatory targets in the ga signaling pathway .
dellas have also been revealed to participate in the regulation of plant responses to jasmonic acid  signaling and light signaling through interactions with the jaz <dig> protein  and the light-responsive transcription factor pifs, respectively .
in general, gras proteins have been hypothesized to be transcription factors; however, there are only a few reports, such as those about liscl and nsp1/nsp <dig> , that show gras proteins acting as classic transcription factors, which have transcription activation activity and can directly bind to dna.
there are at least  <dig> gras genes in rice, among which, several genes, such as moc <dig>  slr <dig>  scr, dlt, and osgras <dig> , have been well characterized, whereas the functions of other gras genes in rice are unknown.
in this work, osgras <dig>  a gras gene that is localized in a rice drought resistant qtl interval, was isolated.
its expression pattern and function in rice drought resistance were investigated.
it was found that osgras <dig> was induced by osmotic stress, and overexpression of this gene enhanced the drought resistance of transgenic rice plants.
the protein possessed trans-activation activity, and it could bind to the promoter of the putative target genes.
these results reveal that the osgras <dig> protein acts as a transcription factor and is involved in the drought stress response.
RESULTS
osgras <dig> encodes a gras protein that belongs to the liscl subfamily
in our previous work, rice drought resistance qtls were mapped using a ril population derived from the cross between upland rice irat <dig> and paddy rice zhenshan97b  <cit> .
a qtl interval on chromosome  <dig>  which contains several drought resistance qtls, was selected for further study.
some key candidate genes in this qtl interval were chosen through bioinformatics and gene expression profile analysis  <cit> ; among which, one candidate gene coding a gras transcription factor was isolated from the upland rice and identified as osgras <dig>   <cit> .
the amino acid sequence alignment analysis showed that osgras <dig> possesses a typical gras domain that included the leucine heptad, vhiid, pfyre, and saw motifs in its c-terminus .
phylogenetic analysis indicated that osgras <dig> groups with the liscl branch contained arabidopsis homologs atscl <dig> and atscl <dig> .fig.
<dig> molecular phylogenetic tree of representative members of gras family and osgras <dig>  proteins are mainly from arabidopsis and oryza sativa, among others.
the sequence alignment and phylogenetic tree construction were performed using the mega <dig> software.
the della and liscl clades are indicated by gray boxes.
the protein accession numbers in the arabidopsis information resource  and genbank database are as follows: atscl <dig>  at1g214520; atscl <dig>  at1g50420; atscl <dig>  at1g50600; atscl <dig>  at4g00150; atscl <dig>  at3g50650; atscl <dig>  at5g52510; atscl <dig>  at2g37650; atscl <dig>  at5g59450; atscl <dig>  at4g17230; atscl <dig>  at1g07530; atscl <dig>  at2g04890; atshr, at4g37450; atscr, at3g54220; atlas, at1g55580; atpat <dig>  at5g48150; atrga, at2g01570; atrgl <dig>  at1g66350; atrgl <dig>  at3g03450; atrgl <dig>  at5g17490; osshr <dig>  a2yn <dig> ; osshr <dig>  a2xia <dig> ; osslr <dig>  ac <dig> ; osscr <dig>  a2zax <dig> ; osscr <dig>  a2zhl <dig> ; osgras <dig>  np_001053706; liscl, bac77269; and ntgras <dig>  abe <dig>
expression pattern of osgras23
to investigate and predict the function of osgras <dig>  its expression patterns under various stresses and phytohormone treatments were investigated by real-time quantitative rt-pcr.
as shown in fig.
<dig>  the expression of osgras <dig> was significantly induced by polyethylene glycol , dehydration, salt, ga, and ja treatment in the rice leaves.fig.
<dig> expression patterns of osgras <dig>  a relative expression level of osgras <dig> under hormone treatment including aba , ga , and ja .
b relative expression level of osgras <dig> under stresses include peg <dig> , dehydration, and nacl.
c relative expression of osgras <dig> in different tissues  of rice plants under normal condition.
all the expression levels of osgars- <dig> were examined by quantitative real-time pcr.
the data represent the mean ± se .
d osgras <dig> promoter: gus expression pattern in transgenic rice plants.
gus staining in the leaves, sheath , spike , stem , root , and root tip
the tissue expression pattern of osgras <dig> was further investigated by transforming rice with a β-glucuronidase  construct driven by the osgras <dig> promoter.
after staining the transgenic rice plants, strong gus signals were detected in the root tips and spikes.
there were also gus signals in the stem and leaves, but the signals were relatively weak .
real-time pcr further confirmed that osgras <dig> expression was higher in rice panicles and roots than in the stems and leaves .
molecular characterization of osgras23-overexpressing transgenic rice plants
to investigate the biological function of osgras <dig>  transgenic rice plants over-expressing osgras <dig> under the control of the constitutive camv35s promoter were produced .
real-time pcr analysis showed that the expression levels of osgras <dig> in these transgenic rice plants were significantly higher than in the wild type  zhonghua <dig> plants .
no significant differences in morphological  and agronomic traits  between the wt and transgenic plants in the adult plant stage were observed.fig.
<dig> molecular identification of osgras23-overexpressing transgenic lines.
a schematic diagram of overexpression construct.
lb, left border; hpt, hygromycin phosphotransferase; pcamv35s, cauliflower mosaic virus 35s promoter; tnos, nopaline synthase gene terminator; and rb, right border.
b relative expression levels of osgras <dig> in transgenic rice lines.
wt: wild type, oe1-oe58: transgenic lines.
the gene expression level was analyzed by quantitative real-time pcr.
the data represent the mean ± se .
c phenotypes of transgenic rice plants and wt plants grown in pvc pipes
overexpression of osgras <dig> improved tolerance of transgenic rice plants to drought and oxidative stresses
to further determine the function of osgras <dig> on the plant stress responses, various stress treatments on the transgenic rice and wt plants were performed.
under the dehydration stress condition, osgras23-overexpressing rice plants showed a lower degree of wilting and better recovery compared with wt plants , and 37-45 % of osgras23-overexpressing plants recovered, which was significantly more than that of the wt plants .
when grown in a paddy field under drought stress during the later tillering stage, the seed setting rate and yield of osgras23-overexpressing plants  were significantly higher than those of the wt plants .
these results indicated that overexpression of osgras <dig> could improve the drought resistance of the transgenic rice plants.fig.
<dig> drought resistance assay of osgras23-overexpressing transgenic rice.
a three-week-old transgenic rice plants and wt plants grown in the 96-well plates and cultivated with culture solution, and exposed to dehydration stress for one day before being transferred to the culture solution.
b survival rate of wt and transgenic lines after dehydration stress.
the data represent the mean ± sd ,*p ≤  <dig> .
c, d seed setting rate and yield of transgenic rice plants under drought stress at the reproductive stage.
the data represent the mean ± sd ,*p ≤  <dig> , ** p ≤  <dig> , t-test
the seedlings of the wt and osgras23-overexpressing plants were also treated with 30 μm methyl viologen , which is an oxidative stress inducer, to determine the tolerance to oxidative stress.
after treatment for 24 h, the wt rice plants had wilting that was more severe than the osgras23-overexpressing plants .
physiological analysis showed that under the mv treatment, the transgenic rice plants accumulated less h2o <dig> and possessed higher superoxide dismutase  and peroxidase  activities compared with the wt plants .
similarly, after 3 μm mv treatment for four days, the wt plants showed a much more severe etiolating phenotype than the osgras23-overexpressing plants , and the chlorophyll content in the leaves of the wt plants was significantly lower than that of the transgenic plants .
these results demonstrated that the overexpression of osgras <dig> enhanced ros scavenging ability and tolerance to oxidative stress of the transgenic rice plants.fig.
<dig> overexpression of osgras <dig> improved tolerance to oxidative stress.
a overexpression and wt rice plants were grown with mv.
total h2o <dig> content , relative sod activity , and relative pod activity  in the leaves of the transgenic and wt rice plants were measured before mv treatment and after the plants were treated with 30 μm mv for 24 h. overexpression and wt rice seeds were grown with 3 μm mv  and the total chlorophyll contents were measured in the leaves .
the data represent the mean ± sd , *p ≤  <dig> , ** p ≤  <dig> , t-test, fw: fresh weight
osgras <dig>  which had transcription activation activity in yeast cells, localized in cell nucleus of tobacco epidermal cells
liscl was reported to be localized in the cell nucleus and had transcription activation activity in yeast and plant cells  <cit> .
to determine the sub-cellular localization of the osgras <dig> protein, a chimeric gene comprised of osgras <dig> coding region and green fluorescent protein  under the control of the camv35s promoter was constructed.
agrobacterium tumefaciens carrying the osgras23-gfp or gfp constructs were infiltrated into tobacco leaves, and the gfp was observed at two days after agroinfiltration.
the tobacco epidermal cells transformed with an empty gfp vector alone displayed fluorescence throughout the cell.
however, in cells expressing the osgras23–gfp construct, fluorescence was found to be localized in the nucleus .
the result indicated that the osgras <dig> protein is a nuclear-localized protein.fig.
<dig> trans-activation assay and sub-cellular localization of osgras <dig>  a trans-activation activities of different portions of osgras <dig> were checked in yeast mav <dig>  bd: gal <dig> dna binding domain; fl: full length; cdl: c-terminus deleted domain; and ndl: n-terminus deleted domain.
b sub-cellular localization of osgras <dig>  gfp and osgras23-gfp fusion gene under the control of the camv 35s promoter separately expressed transiently in the tobacco epidermal cells
to assess the function of the osgras <dig> protein as a transcription factor, the full length of osgras <dig> was fused to the dna binding domain of gal <dig> and introduced into yeast mav <dig> cells.
as fig.
6a shows, the full length of osgras <dig> could strongly induce the expression of the reporter genes, which indicated that the osgras <dig> protein had transcription activation activity.
motifs of osgras <dig>  which are responsible for the transcription activation, were further characterized through checking the transcription activation activities of different partial fragments of osgras <dig> in yeast.
consistent with the previous proposition  <cit> , the relative conserved motif  in the n terminal of osgras <dig> had strong trans-activation activity; whereas, the c-terminal gras domain showed no obvious trans-activation activity .
the results revealed that the osgras <dig> protein is a putative transcription factor and that the n-terminal region of osgras <dig> is required for transcription activation.
osgras <dig> regulated stress-response genes and binding to promoters of several putative target genes
to search the downstream genes of osgras <dig>  the gene expression profiles of osgras23-overexpressing plants and wt plants were analyzed using the affymetrix rice genome genechip.
it was found that  <dig> genes were up-regulated  and  <dig> genes were down-regulated  in the osgras23-overexpressing plants compared with the wt plants under normal condition .
among the up-regulated genes,  <dig> genes were also induced by drought stress in the wt rice plants, which suggests that these up-regulated genes by osgras <dig> may participate in the drought response .
these up-regulated genes encode proteins with diverse functions such as transcription factors, ja induced proteins, protease inhibitors and antioxidant enzymes .fig.
<dig> gene expression profile analysis of osgras23-overexpressing transgenic rice plants.
a drought responsive expression pattern of all differently expressed genes in transgenic plants.
oen: genes differently expressed between osgras23-overexpressing transgenic rice plants and wt plants under normal condition.
oed: genes differently expressed between osgras23-overexpressing transgenic rice plants and wt plants under dehydration treatment.
drought: genes differently expressed in the wt plants between dehydration treatment and normal condition.
b classification of up-regulated genes in the transgenic plants compared with the wt plants.
c relative expression levels of some up-regulated genes in transgenic rice plants.
qrt-pcr was used to analyze the expression levels.
the data represent the mean ± se
to further confirm the microarray results, the transcription levels of several up-regulated genes were analyzed using qrt-pcr.
the results were consistent with the microarray results.
the expression of the obviously up-regulated genes  was highly induced in osgras23-overexpressing lines.
the expression of the other genes related to anti-oxidation and defense responses ; os03g <dig> encoding leucoanthocyanidin dioxygenase; and os12g <dig> and os01g <dig> encoding proteinase inhibitors) in osgras23-overexpressing lines was also significantly higher than that of the wt plants .
this suggested that the transcription of these genes may be positively regulated by osgras <dig>
based on the microarray analysis, eight up-regulated genes were selected to confirm whether the osgras <dig> protein could bind to the promoter region of these genes via the yeast one hybrid assay.
the pgad-osgras <dig> plasmid  and the reporter construct phis-cis  were co-transformed into yeast strain y <dig>  as indicated by the activation of the reporter genes, osgras <dig> can bind to the promoters of several genes  .
these results implied that osgras <dig> has dna binding activity and may directly regulate the expressions of these target genes.fig.
<dig> identification of putative target genes regulated by osgras <dig> with yeast one hybrid assay.
a schematic structure of yeast expression construct pgad-osgras <dig> and reporter construct phis <dig> -gtp .
b growth performance of transformants on sd/-leu-/trp/-his medium containing 100 mm or 30 mm 3-at.
gtp1-gtp <dig> indicates the pgad-osgras <dig> plus phis <dig> -cis .
ck-: negative control
discussion
osgras <dig> encodes drought-responsive gras protein
the transcriptional regulation of stress related genes is one of the crucial steps during plant stress responses, and transcription factors play important roles in these processes  <cit> .
for example, osmotic stress activates several transcription factors, including nac proteins, which activate an early response to dehydration <dig>   <cit> .
several bzip proteins have been characterized to be aba -responsive transcription factors  that bind to the abres and have a pivotal role in aba-dependent gene activation .
there remain a number of transcription factors, especially novel type transcription factors, that have not been studied in detail.
in this study, the osgras <dig> protein was characterized to be a novel gras transcription factor that is involved in rice abiotic stress responses.
gras proteins are plant specific proteins, for which a large number of reports have shown that they play important roles in plant growth, development and phytohormone signal transduction.
however, reports referring to gras proteins being involved in abiotic stress were relatively few.
in this study, osgras <dig> was isolated from rice and shown to belong to the liscl subfamily, and it is an ortholog to ntgras <dig>  scl <dig>  and scl <dig> .
previous research showed that the expression of these genes was induced by abiotic stresses  <cit> .
scl <dig> has been found to play an important role in plant stress response  <cit> .
the close genetic relationship between osgras <dig> and these proteins suggests that osgras <dig> might also play a role in plant abiotic stress responses.
the expression of osgras <dig> was induced by drought stress .
the hormones ja and ga but not aba, can also induce the expression of osgras <dig>  furthermore, the promoter of osgras <dig> contained the heat stress responsive element, cis-acting regulatory element involved in meja-responsiveness, ga-responsive element, and other cis-elements involved in stress and phytohormone responsiveness, which were predicted using plantcare .
these results implied that osgras <dig> is a stress-responsive gras protein that may be involved in plant responses to abiotic stresses  and phytohormone signaling .
osgras <dig> protein functioned as a gras transcription factor
some of the previously characterized gras proteins functioned through interactions with other proteins in the signal transduction pathway, and they were found to participate in transcription regulation.
the regulations could be divided into two types: transcription co-regulator or transcription factor  <cit> .
when functioning as transcription factors, several gras proteins, such as liscl and nsp, have both trans-activation activities and the ability to directly binding to dna which was indicated by assays in yeast or plant cells  <cit> .
in the current work, the genetic relationship of the osgras <dig> protein and liscl protein was close , which suggests that the osgras <dig> protein may also act as a transcription factor.
further characterization indicated that the osgras <dig> protein was mainly localized in the cell nucleus, and that it showed obvious trans-activation activity and dna binding ability in yeast cells .
moreover, several genes were up-regulated in osgras23-overexpressing rice plants, which supports that osgras <dig> could positively modulate the expressions of down-stream genes , these results confirmed the hypothesis that osgras <dig> functions as a transcription factor.
the expression of several genes in transgenic rice plants was obviously higher than that of the wt rice , and this strongly suggested these genes may be directly regulated by osgras <dig>  the yeast one hybrid assay further confirmed the osgras <dig> protein could directly bind to the promoters of several up-regulated genes, such as os03g <dig> which encodes leucoanthocyanidin dioxygenase .
however, it was found that osgras <dig> could not bind to the promoters of the genes encoding ros scavenging enzymes , which implies that these genes related to anti-oxidation activity were indirectly regulated by osgras <dig>
the gras protein generally contains the conserved gras domain in the c-terminus, whereas the n-terminus was relatively disordered.
previous bioinformatics research proposed that the motif richness in acidic residues flanking the repeated hydrophobic/aromatic residues in the n-terminus might be associated with transcription activation  <cit> .
while alternative previous reports referred to the n terminus of nsp1/nsp <dig> and liscl as being the main trans-activation domain  <cit> .
in this study, the n-terminus domain of osgras <dig> was also responsible for the trans-activation activity.
further analysis found that the main trans-activation motif was the first conserved motif  but not the second motif  , which is similar to the liscl protein.
osgras <dig> positively regulated rice drought tolerance through upregulating genes related to stress responses
a few studies have found that the gras proteins are involved in abiotic stress responses.
pescl <dig> was recently isolated from populus euphratica oliv, and its overexpression in arabidopsis showed improved drought and salt tolerance  <cit> .
ntgras <dig> was cloned from tobacco and shown to belong to the liscl subfamily, and its expression was induced by drought, salt, and h2o <dig> treatments  <cit> .
della proteins are also involved in the ros reaction  <cit>  and development coordination during abiotic stress  <cit> .
here, overexpression of osgras <dig> conferred enhanced resistance to drought stress and oxidative stress on transgenic rice , which supplies novel evidence for gras proteins functioning in rice abiotic stress responses.
as a putative transcription factor, osgras <dig> may participate in plant responses to stress through regulating the transcription of downstream genes.
microarray analysis showed that a number of drought-induced genes were indeed up-regulated in the osgras23-overexpressing rice plants .
the up-regulated genes encode both regulatory and functional proteins, such as transcription factors, protein kinases, anti-oxidants, proteinase inhibitors, and enzymes related to metabolism .
the homologues of these genes were reported to play roles in plant stress tolerance.
for instance, ros scavenging enzymes, including peroxiredoxin, peroxidase, and glutathione-s-transferase, have been verified to be responsible for alleviating oxidative damage and enhancing plant stress tolerance .
the activities of ros scavenging enzymes  were increased and accumulated h2o <dig> was reduced in the osgras23-overexpressing plants under oxidative stress , which further suggests that the enhanced ros scavenging ability in the transgenic plants might partly contribute to the enhanced drought tolerance of the transgenic plants.
proteinase inhibitors, such as bowman birk trypsin inhibitors, were also revealed to confer plant stress tolerance probably through inhibiting the degradation of the stress-mitigating protein  <cit> .
leucoanthocyanidin dioxygenase is involved in the biosynthesis of anthocyanin which is one class of flavonoids  <cit> ; previous studies have shown that flavonoids are associated with plant stress adaptation  <cit> .
it was also noticed that some genes that were up-regulated in the transgenic plants encoded lectin precursors, protease inhibitors, and ja induced proteins, which suggests that osgras <dig> might also be involved in the defense responses mediated by ja.
taken together, osgras <dig> increases transcription of genes related to the stress responses  and positively regulates rice drought tolerance.
CONCLUSIONS
we isolated a rice gras gene, osgras <dig>  from a rice drought resistance qtl interval and characterized its function.
drought, nacl, ja, and ga treatments induced the expression of osgras <dig>  the osgras <dig> protein was localized in the nucleus and possessed a strong transcriptional activation activity.
furthermore, the osgras <dig> protein could bind to the promoters of several target genes and modulated the expressions of a series of stress-related genes.
overexpression of osgras <dig> conferred transgenic rice plants with improved drought resistance.
we can therefore conclude that osgras <dig> encodes a novel stress-responsive gras transcription factor and positively regulates the rice drought stress response.

 drought is a major environmental stress factor that reduces agricultural productivity. rice is one of the most important crops worldwide, and it consumes a large amount of fresh water resources, e.g. about 50 % in china. developing water-saving and drought resistant rice varieties is an effective strategy to achieve food security and prevent the detrimental effects of drought and water deficit. elucidating the hereditary basis and molecular mechanism that underlies the drought resistance in rice is indispensable and vital for the development of new rice varieties with improved drought resistance. drought and water deficit can decrease photosynthetic capacity, result in oxidative damage to chloroplasts, limit metabolic reactions, and reduce dry matter accumulation and partitioning. to cope with drought stress, plants have developed various strategies, which include developing larger and deeper root systems to increase water absorption from the deep soil, regulating stomata closure to reduce water loss, accumulation of compatible solutes and protective proteins, and increasing the level of antioxidants. on exposure of plants to drought stresses, a series of genes are induced, the products of which would then participate in the stress responses. transcription of these stress-response genes is largely controlled by transcription factors. a number of transcription factors have been identified in the past few years that have been demonstrated to play an essential role in regulating plant responses to stresses. for instance, ap transcription factors including dreb and cbf proteins bind to the dehydration response element and control expression of stress-responsive genes. overexpression of dreb1b and dreb1a in arabidopsis enhanced freezing tolerance and dehydration/salt tolerance, respectively. in rice, ap transcription activators such as osdreb1a have been isolated. osdreb1a was induced by dehydration and high salinity stress, and overexpression of osdreb1a in transgenic arabidopsis improved stress tolerance. recently, several other types of transcription factors in rice including snca, dst, myb, and zip have been identified to play important roles in drought resistance through regulating stomata closure, reactive oxygen species scavenging, or other physiological processes. although the transcription factor genes have been extensively studied, further studies are still needed to identify other novel transcription factors that are involved in stress responses. gras proteins are plant specific proteins, and homologues have been found in many higher plants such as arabidopsis, tomato, petunia, rice, and barley. the name is derived from the three initially identified members, gibberellin-acid insensitive, repressor of ga and scarecrow. gras proteins are typically composed of 400– amino acid residues and exhibit considerable sequence homology to each other in their respective c-terminal domains, whereas the n-terminal amino acid sequences are highly divergent. gras family proteins are divided into several sub-families such as della, shr, scr, pat, liscl, and scl. in the past few years, studies have demonstrated that gras proteins play diverse roles in gibberellin signal transduction, root development, meristem development, light signaling, biotic stress, and abiotic stress responses. della proteins are one of the most extensively studied gras subfamilies, and they function as repressors of gibberellin responsive plant growth and are key regulatory targets in the ga signaling pathway. dellas have also been revealed to participate in the regulation of plant responses to jasmonic acid signaling and light signaling through interactions with the jaz protein and the light-responsive transcription factor pifs, respectively. in general, gras proteins have been hypothesized to be transcription factors; however, there are only a few reports, such as those about liscl and nsp1/nsp, that show gras proteins acting as classic transcription factors, which have transcription activation activity and can directly bind to dna. there are at least gras genes in rice, among which, several genes, such as moc slr scr, dlt, and osgras, have been well characterized, whereas the functions of other gras genes in rice are unknown. in this work, osgras a gras gene that is localized in a rice drought resistant qtl interval, was isolated. its expression pattern and function in rice drought resistance were investigated. it was found that osgras was induced by osmotic stress, and overexpression of this gene enhanced the drought resistance of transgenic rice plants. the protein possessed trans-activation activity, and it could bind to the promoter of the putative target genes. these results reveal that the osgras protein acts as a transcription factor and is involved in the drought stress response.  osgras encodes a gras protein that belongs to the liscl subfamily in our previous work, rice drought resistance qtls were mapped using a ril population derived from the cross between upland rice irat and paddy rice zhenshan97b. a qtl interval on chromosome which contains several drought resistance qtls, was selected for further study. some key candidate genes in this qtl interval were chosen through bioinformatics and gene expression profile analysis; among which, one candidate gene coding a gras transcription factor was isolated from the upland rice and identified as osgras. the amino acid sequence alignment analysis showed that osgras possesses a typical gras domain that included the leucine heptad, vhiid, pfyre, and saw motifs in its c-terminus. phylogenetic analysis indicated that osgras groups with the liscl branch contained arabidopsis homologs atscl and atscl.fig. molecular phylogenetic tree of representative members of gras family and osgras proteins are mainly from arabidopsis and oryza sativa, among others. the sequence alignment and phylogenetic tree construction were performed using the mega software. the della and liscl clades are indicated by gray boxes. the protein accession numbers in the arabidopsis information resource and genbank database are as follows: atscl at1g214520; atscl at1g50420; atscl at1g50600; atscl at4g00150; atscl at3g50650; atscl at5g52510; atscl at2g37650; atscl at5g59450; atscl at4g17230; atscl at1g07530; atscl at2g04890; atshr, at4g37450; atscr, at3g54220; atlas, at1g55580; atpat at5g48150; atrga, at2g01570; atrgl at1g66350; atrgl at3g03450; atrgl at5g17490; osshr a2yn; osshr a2xia; osslr ac; osscr a2zax; osscr a2zhl; osgras np_001053706; liscl, bac77269; and ntgras abe expression pattern of osgras23 to investigate and predict the function of osgras its expression patterns under various stresses and phytohormone treatments were investigated by real-time quantitative rt-pcr. as shown in fig.  the expression of osgras was significantly induced by polyethylene glycol, dehydration, salt, ga, and ja treatment in the rice leaves.fig. expression patterns of osgras a relative expression level of osgras under hormone treatment including aba, ga, and ja. b relative expression level of osgras under stresses include peg, dehydration, and nacl. c relative expression of osgras in different tissues of rice plants under normal condition. all the expression levels of osgars- were examined by quantitative real-time pcr. the data represent the mean ± se. d osgras promoter: gus expression pattern in transgenic rice plants. gus staining in the leaves, sheath, spike, stem, root, and root tip the tissue expression pattern of osgras was further investigated by transforming rice with a β-glucuronidase construct driven by the osgras promoter. after staining the transgenic rice plants, strong gus signals were detected in the root tips and spikes. there were also gus signals in the stem and leaves, but the signals were relatively weak. real-time pcr further confirmed that osgras expression was higher in rice panicles and roots than in the stems and leaves. molecular characterization of osgras23-overexpressing transgenic rice plants to investigate the biological function of osgras transgenic rice plants over-expressing osgras under the control of the constitutive camv35s promoter were produced. real-time pcr analysis showed that the expression levels of osgras in these transgenic rice plants were significantly higher than in the wild type zhonghua plants. no significant differences in morphological and agronomic traits between the wt and transgenic plants in the adult plant stage were observed.fig. molecular identification of osgras23-overexpressing transgenic lines. a schematic diagram of overexpression construct. lb, left border; hpt, hygromycin phosphotransferase; pcamv35s, cauliflower mosaic virus 35s promoter; tnos, nopaline synthase gene terminator; and rb, right border. b relative expression levels of osgras in transgenic rice lines. wt: wild type, oe1-oe58: transgenic lines. the gene expression level was analyzed by quantitative real-time pcr. the data represent the mean ± se. c phenotypes of transgenic rice plants and wt plants grown in pvc pipes overexpression of osgras improved tolerance of transgenic rice plants to drought and oxidative stresses to further determine the function of osgras on the plant stress responses, various stress treatments on the transgenic rice and wt plants were performed. under the dehydration stress condition, osgras23-overexpressing rice plants showed a lower degree of wilting and better recovery compared with wt plants, and 37-45 % of osgras23-overexpressing plants recovered, which was significantly more than that of the wt plants. when grown in a paddy field under drought stress during the later tillering stage, the seed setting rate and yield of osgras23-overexpressing plants were significantly higher than those of the wt plants. these results indicated that overexpression of osgras could improve the drought resistance of the transgenic rice plants.fig. drought resistance assay of osgras23-overexpressing transgenic rice. a three-week-old transgenic rice plants and wt plants grown in the 96-well plates and cultivated with culture solution, and exposed to dehydration stress for one day before being transferred to the culture solution. b survival rate of wt and transgenic lines after dehydration stress. the data represent the mean ± sd,*p ≤ . c, d seed setting rate and yield of transgenic rice plants under drought stress at the reproductive stage. the data represent the mean ± sd,*p ≤ , ** p ≤ , t-test the seedlings of the wt and osgras23-overexpressing plants were also treated with 30 μm methyl viologen, which is an oxidative stress inducer, to determine the tolerance to oxidative stress. after treatment for 24 h, the wt rice plants had wilting that was more severe than the osgras23-overexpressing plants. physiological analysis showed that under the mv treatment, the transgenic rice plants accumulated less h2o and possessed higher superoxide dismutase and peroxidase activities compared with the wt plants. similarly, after 3 μm mv treatment for four days, the wt plants showed a much more severe etiolating phenotype than the osgras23-overexpressing plants, and the chlorophyll content in the leaves of the wt plants was significantly lower than that of the transgenic plants. these results demonstrated that the overexpression of osgras enhanced ros scavenging ability and tolerance to oxidative stress of the transgenic rice plants.fig. overexpression of osgras improved tolerance to oxidative stress. a overexpression and wt rice plants were grown with mv. total h2o content, relative sod activity, and relative pod activity in the leaves of the transgenic and wt rice plants were measured before mv treatment and after the plants were treated with 30 μm mv for 24 h. overexpression and wt rice seeds were grown with 3 μm mv and the total chlorophyll contents were measured in the leaves. the data represent the mean ± sd, *p ≤ , ** p ≤ , t-test, fw: fresh weight osgras which had transcription activation activity in yeast cells, localized in cell nucleus of tobacco epidermal cells liscl was reported to be localized in the cell nucleus and had transcription activation activity in yeast and plant cells. to determine the sub-cellular localization of the osgras protein, a chimeric gene comprised of osgras coding region and green fluorescent protein under the control of the camv35s promoter was constructed. agrobacterium tumefaciens carrying the osgras23-gfp or gfp constructs were infiltrated into tobacco leaves, and the gfp was observed at two days after agroinfiltration. the tobacco epidermal cells transformed with an empty gfp vector alone displayed fluorescence throughout the cell. however, in cells expressing the osgras23–gfp construct, fluorescence was found to be localized in the nucleus. the result indicated that the osgras protein is a nuclear-localized protein.fig. trans-activation assay and sub-cellular localization of osgras a trans-activation activities of different portions of osgras were checked in yeast mav bd: gal dna binding domain; fl: full length; cdl: c-terminus deleted domain; and ndl: n-terminus deleted domain. b sub-cellular localization of osgras gfp and osgras23-gfp fusion gene under the control of the camv 35s promoter separately expressed transiently in the tobacco epidermal cells to assess the function of the osgras protein as a transcription factor, the full length of osgras was fused to the dna binding domain of gal and introduced into yeast mav cells. as fig. 6a shows, the full length of osgras could strongly induce the expression of the reporter genes, which indicated that the osgras protein had transcription activation activity. motifs of osgras which are responsible for the transcription activation, were further characterized through checking the transcription activation activities of different partial fragments of osgras in yeast. consistent with the previous proposition, the relative conserved motif in the n terminal of osgras had strong trans-activation activity; whereas, the c-terminal gras domain showed no obvious trans-activation activity. the results revealed that the osgras protein is a putative transcription factor and that the n-terminal region of osgras is required for transcription activation. osgras regulated stress-response genes and binding to promoters of several putative target genes to search the downstream genes of osgras the gene expression profiles of osgras23-overexpressing plants and wt plants were analyzed using the affymetrix rice genome genechip. it was found that genes were up-regulated and genes were down-regulated in the osgras23-overexpressing plants compared with the wt plants under normal condition. among the up-regulated genes, genes were also induced by drought stress in the wt rice plants, which suggests that these up-regulated genes by osgras may participate in the drought response. these up-regulated genes encode proteins with diverse functions such as transcription factors, ja induced proteins, protease inhibitors and antioxidant enzymes.fig. gene expression profile analysis of osgras23-overexpressing transgenic rice plants. a drought responsive expression pattern of all differently expressed genes in transgenic plants. oen: genes differently expressed between osgras23-overexpressing transgenic rice plants and wt plants under normal condition. oed: genes differently expressed between osgras23-overexpressing transgenic rice plants and wt plants under dehydration treatment. drought: genes differently expressed in the wt plants between dehydration treatment and normal condition. b classification of up-regulated genes in the transgenic plants compared with the wt plants. c relative expression levels of some up-regulated genes in transgenic rice plants. qrt-pcr was used to analyze the expression levels. the data represent the mean ± se to further confirm the microarray results, the transcription levels of several up-regulated genes were analyzed using qrt-pcr. the results were consistent with the microarray results. the expression of the obviously up-regulated genes was highly induced in osgras23-overexpressing lines. the expression of the other genes related to anti-oxidation and defense responses; os03g encoding leucoanthocyanidin dioxygenase; and os12g and os01g encoding proteinase inhibitors) in osgras23-overexpressing lines was also significantly higher than that of the wt plants. this suggested that the transcription of these genes may be positively regulated by osgras based on the microarray analysis, eight up-regulated genes were selected to confirm whether the osgras protein could bind to the promoter region of these genes via the yeast one hybrid assay. the pgad-osgras plasmid and the reporter construct phis-cis were co-transformed into yeast strain y as indicated by the activation of the reporter genes, osgras can bind to the promoters of several genes. these results implied that osgras has dna binding activity and may directly regulate the expressions of these target genes.fig. identification of putative target genes regulated by osgras with yeast one hybrid assay. a schematic structure of yeast expression construct pgad-osgras and reporter construct phis -gtp. b growth performance of transformants on sd/-leu-/trp/-his medium containing 100 mm or 30 mm 3-at. gtp1-gtp indicates the pgad-osgras plus phis -cis. ck-: negative control discussion osgras encodes drought-responsive gras protein the transcriptional regulation of stress related genes is one of the crucial steps during plant stress responses, and transcription factors play important roles in these processes. for example, osmotic stress activates several transcription factors, including nac proteins, which activate an early response to dehydration. several bzip proteins have been characterized to be aba -responsive transcription factors that bind to the abres and have a pivotal role in aba-dependent gene activation. there remain a number of transcription factors, especially novel type transcription factors, that have not been studied in detail. in this study, the osgras protein was characterized to be a novel gras transcription factor that is involved in rice abiotic stress responses. gras proteins are plant specific proteins, for which a large number of reports have shown that they play important roles in plant growth, development and phytohormone signal transduction. however, reports referring to gras proteins being involved in abiotic stress were relatively few. in this study, osgras was isolated from rice and shown to belong to the liscl subfamily, and it is an ortholog to ntgras scl and scl. previous research showed that the expression of these genes was induced by abiotic stresses. scl has been found to play an important role in plant stress response. the close genetic relationship between osgras and these proteins suggests that osgras might also play a role in plant abiotic stress responses. the expression of osgras was induced by drought stress. the hormones ja and ga but not aba, can also induce the expression of osgras furthermore, the promoter of osgras contained the heat stress responsive element, cis-acting regulatory element involved in meja-responsiveness, ga-responsive element, and other cis-elements involved in stress and phytohormone responsiveness, which were predicted using plantcare. these results implied that osgras is a stress-responsive gras protein that may be involved in plant responses to abiotic stresses and phytohormone signaling. osgras protein functioned as a gras transcription factor some of the previously characterized gras proteins functioned through interactions with other proteins in the signal transduction pathway, and they were found to participate in transcription regulation. the regulations could be divided into two types: transcription co-regulator or transcription factor. when functioning as transcription factors, several gras proteins, such as liscl and nsp, have both trans-activation activities and the ability to directly binding to dna which was indicated by assays in yeast or plant cells. in the current work, the genetic relationship of the osgras protein and liscl protein was close, which suggests that the osgras protein may also act as a transcription factor. further characterization indicated that the osgras protein was mainly localized in the cell nucleus, and that it showed obvious trans-activation activity and dna binding ability in yeast cells. moreover, several genes were up-regulated in osgras23-overexpressing rice plants, which supports that osgras could positively modulate the expressions of down-stream genes, these results confirmed the hypothesis that osgras functions as a transcription factor. the expression of several genes in transgenic rice plants was obviously higher than that of the wt rice, and this strongly suggested these genes may be directly regulated by osgras the yeast one hybrid assay further confirmed the osgras protein could directly bind to the promoters of several up-regulated genes, such as os03g which encodes leucoanthocyanidin dioxygenase. however, it was found that osgras could not bind to the promoters of the genes encoding ros scavenging enzymes, which implies that these genes related to anti-oxidation activity were indirectly regulated by osgras the gras protein generally contains the conserved gras domain in the c-terminus, whereas the n-terminus was relatively disordered. previous bioinformatics research proposed that the motif richness in acidic residues flanking the repeated hydrophobic/aromatic residues in the n-terminus might be associated with transcription activation. while alternative previous reports referred to the n terminus of nsp1/nsp and liscl as being the main trans-activation domain. in this study, the n-terminus domain of osgras was also responsible for the trans-activation activity. further analysis found that the main trans-activation motif was the first conserved motif but not the second motif, which is similar to the liscl protein. osgras positively regulated rice drought tolerance through upregulating genes related to stress responses a few studies have found that the gras proteins are involved in abiotic stress responses. pescl was recently isolated from populus euphratica oliv, and its overexpression in arabidopsis showed improved drought and salt tolerance. ntgras was cloned from tobacco and shown to belong to the liscl subfamily, and its expression was induced by drought, salt, and h2o treatments. della proteins are also involved in the ros reaction and development coordination during abiotic stress. here, overexpression of osgras conferred enhanced resistance to drought stress and oxidative stress on transgenic rice, which supplies novel evidence for gras proteins functioning in rice abiotic stress responses. as a putative transcription factor, osgras may participate in plant responses to stress through regulating the transcription of downstream genes. microarray analysis showed that a number of drought-induced genes were indeed up-regulated in the osgras23-overexpressing rice plants. the up-regulated genes encode both regulatory and functional proteins, such as transcription factors, protein kinases, anti-oxidants, proteinase inhibitors, and enzymes related to metabolism. the homologues of these genes were reported to play roles in plant stress tolerance. for instance, ros scavenging enzymes, including peroxiredoxin, peroxidase, and glutathione-s-transferase, have been verified to be responsible for alleviating oxidative damage and enhancing plant stress tolerance. the activities of ros scavenging enzymes were increased and accumulated h2o was reduced in the osgras23-overexpressing plants under oxidative stress, which further suggests that the enhanced ros scavenging ability in the transgenic plants might partly contribute to the enhanced drought tolerance of the transgenic plants. proteinase inhibitors, such as bowman birk trypsin inhibitors, were also revealed to confer plant stress tolerance probably through inhibiting the degradation of the stress-mitigating protein. leucoanthocyanidin dioxygenase is involved in the biosynthesis of anthocyanin which is one class of flavonoids; previous studies have shown that flavonoids are associated with plant stress adaptation. it was also noticed that some genes that were up-regulated in the transgenic plants encoded lectin precursors, protease inhibitors, and ja induced proteins, which suggests that osgras might also be involved in the defense responses mediated by ja. taken together, osgras increases transcription of genes related to the stress responses and positively regulates rice drought tolerance.  we isolated a rice gras gene, osgras from a rice drought resistance qtl interval and characterized its function. drought, nacl, ja, and ga treatments induced the expression of osgras the osgras protein was localized in the nucleus and possessed a strong transcriptional activation activity. furthermore, the osgras protein could bind to the promoters of several target genes and modulated the expressions of a series of stress-related genes. overexpression of osgras conferred transgenic rice plants with improved drought resistance. we can therefore conclude that osgras encodes a novel stress-responsive gras transcription factor and positively regulates the rice drought stress response. 
gras transcription factors are plant-specific proteins that play diverse roles in plant development. however, the functions of a number of gras genes identified in rice are unknown, especially the gras genes related to rice drought resistance have not been characterized. osgras23-overexpressing rice plants showed improved drought resistance and oxidative stress tolerance as well as less h2o accumulation compared with the wild-type plants. drought resistancegrasricetranscription factorissue-copyright-statement© the author 2015 a trans-activation assay in yeast cells demonstrated that the osgras protein possessed a strong transcriptional activation activity. the osgras23-gfp fused protein was localized in the nucleus of tobacco epidermal cells. our results demonstrate that osgras encodes a stress-responsive gras transcription factor and positively modulates rice drought tolerance via the induction of a number of stress-responsive genes. furthermore, microarray analysis showed that several anti-oxidation related genes were up-regulated in the osgras23-overexpressing rice plants. drought is a major abiotic stress factors that reduces agricultural productivity.
 drought is a major abiotic stress factors that reduces agricultural productivity. gras transcription factors are plant-specific proteins that play diverse roles in plant development. however, the functions of a number of gras genes identified in rice are unknown, especially the gras genes related to rice drought resistance have not been characterized.  in this study, a novel gras transcription factor gene named osgras which is located in a drought-resistant qtl interval on chromosome of rice, was isolated. the expression of osgras was induced by drought, nacl, and jasmonic acid treatments. the osgras23-gfp fused protein was localized in the nucleus of tobacco epidermal cells. a trans-activation assay in yeast cells demonstrated that the osgras protein possessed a strong transcriptional activation activity. osgras23-overexpressing rice plants showed improved drought resistance and oxidative stress tolerance as well as less h2o accumulation compared with the wild-type plants. furthermore, microarray analysis showed that several anti-oxidation related genes were up-regulated in the osgras23-overexpressing rice plants. the yeast one hybrid test indicated that osgras could bind to the promoters of its potential target genes.  our results demonstrate that osgras encodes a stress-responsive gras transcription factor and positively modulates rice drought tolerance via the induction of a number of stress-responsive genes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords drought resistancegrasricetranscription factorissue-copyright-statement© the author 2015 