BACKGROUND
leaves are the main organs of photosynthesis and play a crucial role in plant development.
leaf rolling is a frequently observed phenotype in fields and rice mutant populations
<cit> .
the appropriate leaf rolling has been regarded as one of the most important elements in rice ideotype breeding as it improves photosynthetic efficiency, accelerates dry-matter accumulation, reduces solar radiation on leaves, decreases leaf transpiration under drought stress and raises grain yield
<cit> .
however, severe leaf rolling could lead to growth retardation, developmental defects and crop yield reduction.
to date, many studies have been performed to characterize the genes controlling leaf rolling by analyses of the rolled leaf mutants in rice.
twelve rolled leaf  mutants in rice are reported and the corresponding genes are mapped genetically, of which, six rl genes are mapped on rice chromosomes by morphological markers and the rest are directly mapped in rice genome by molecular markers
<cit> .
among these  <dig> rl mutants, rl <dig> is the first to be cloned and analyzed in detail.
rolled leaf <dig> /shallot-like <dig>  encodes a kanadi transcription factor, and rl9/sll <dig> mutants display extremely incurved leaves due to the defective development of sclerenchymatous cells on the abaxial side
<cit> .
some other genes are also found to be related to leaf rolling in rice.
loss-of-function of oscsld <dig> gene, which encodes a cellulose synthase-like protein, results in phenotypes of reduced leaf width and semi-rolled leaves, possibly due to the significantly smaller bulliform cells in mutants
<cit> .
loss-of-function of constitutively wilted <dig> /narrow leaf <dig> , encoding a member of the yucca protein, causes rice leaves to roll upward
<cit> .
loss-of-function of curly flag leaf , encoding a ww domain protein, induces rolled leaves which have a wrinkled epidermis and contain papillae with reduced wax content
<cit> .
rolled leaf  <dig> , encoding a 2og-fe  oxygenase family protein, is reported to modulate rice leaf rolling by affecting secondary cell wall formation and bulliform cell development
<cit> .
mutation of semi-rolled leaf  <dig> , encoding a putative gpi-anchored protein, results in leaves rolled adaxially in rice due to the increased number of bulliform cells at the adaxial cell layers
<cit> .
in addition, recent studies show that overexpression of rice gene osago <dig>  which encodes an argonaute  family member, results in the leaf blades curling upward
<cit> .
oshb <dig> is a member of the class iii homeodomain leucine zipper family of genes, overexpression of its microrna166-resistant version could result in adaxially rolled leaves due to the reduced sclerenchyma and formation of bulliform cells on the abaxial side
<cit> .
taken together, the major factors possibly affecting leaf rolling include the alteration of osmotic pressure and/or turgidity in bulliform cells, the impairment of cell polarity establishment and cell differentiation.
however, the mechanisms underlying leaf rolling in rice and, particularly, the involved transcription factors remain largely unknown
<cit> .
myb proteins belong to a super-family of transcription factors, which prevail in all eukaryotes and share a highly conserved dna-binding domain, myb
<cit> .
depending on the number of adjacent repeats in the myb domain, myb proteins can be classified into four subfamilies, including 1r-myb, r2r3-myb, 3r-myb, and 4r-myb
<cit> .
among them, r2r3-myb subfamily is a typical representative one, whose members contain two myb repeats in their dna-binding domains
<cit> .
myb proteins in plants are involved in a variety of plant-specific processes, including primary and secondary metabolism, cell fate and identity, developmental processes, and responses to various stresses
<cit> .
genome-wide analysis shows that there are approximately  <dig> myb genes in rice, of which  <dig> encode r2r3-myb proteins
<cit> .
so far, only a few number of myb proteins, including osgamyb, osmyb <dig>  osmyb <dig>  osmyb3r- <dig>  csa, mybs <dig> and osmyb2p-1
<cit> , have been functionally characterized.
the functions of most of myb proteins are still unknown in rice.
to characterize functions of myb transcription factors in rice, we overexpressed several myb genes in kasalath, an indica cultivar, with the transgene constructs containing the full-length cdnas of rice myb genes, driven by maize ubiquitin promoter.
of the transgenic lines, one line overexpressing the full-length cdna of os08g <dig>  designated as osmyb103l hereafter, displays a rolled leaf phenotype.
osmyb103l encodes an r2r3-myb transcription factor.
our study shows that it localizes in the nucleus and possesses transcriptional activity.
we detail the phenotypes of osmyb103l overexpressing  and rna interference  knockdown plants, including the altered leaf shape, the changed cellulose content, and the impaired mechanical strength.
the roles of osmyb103l in leaf shape formation and cellulose synthesis are discussed.
we propose the potential application of osmyb103l in molecular breeding of rice.
RESULTS
osmyb103l encodes an r2r3-myb transcription factor
to discover transcription factors controlling leaf development, we screened the rice lines ectopically expressing rice myb genes under the control of maize ubiquitin promoter.
one line overexpressing loc_os08g <dig> was selected for further study due to its particular leaf shape, such as upward curling of the leaf blade.
according to the rice genome annotation database , loc_os08g <dig> encodes a putative r2r3-myb family transcription factor with a length of  <dig> amino acids and a molecular mass of approximately  <dig> kd.
the pfam database  shows that the deduced protein has two myb dna-binding domains  at the n-terminus .
as revealed by phylogenetic analysis of the related myb transcription factors in arabidopsis thaliana and rice, os08g <dig> is closely related to at1g <dig>
<cit>  .
protein sequence alignment showed that they are highly conserved in the predicted r2- and r3-myb dna-binding domains .
we hereby designated os08g <dig> as osmyb103l .
nuclear localization is one of the significant features of transcription factors.
to determine its subcellular localization, we fused green fluorescent protein  to the c-terminus of osmyb103l to produce an osmyb103l-gfp fusion protein.
we monitored the fluorescence of the transiently expressed fusion protein in rice protoplasts and onion  epidermis cells.
in both cases, the fluorescence signals of the fusion protein were observed predominantly in nuclei .
while in the gfp alone control, fluorescence signals were observed in nuclei and cytoplasm .
these results indicate that osmyb103l is a nuclear-localized protein.
transcriptional activation is a characteristic feature of many transcription factors.
the full-length, n-terminal myb dna-binding domain and c-terminal putative activation domain were fused in-frame with the gal <dig> dna binding domain, respectively.
we conducted a transcriptional activation analysis in yeast by using the fusion proteins.
the analysis showed that the full-length and c-terminal region of osmyb103l activated the expression of aur1-c and mel <dig> reporter genes in yeast , indicating that osmyb103l has the capability of transcriptional activation.
expression of osmyb103l varies in different organs
we determined the expression level of osmyb103l in various rice organs, including root, culm, leave, and panicle using quantitative real-time pcr .
the expression level of osmyb103l was highest in culm, lower in root, leaf and mature panicle, and lowest in young panicle .
in addition, the expression pattern of osmyb103l was examined by the β-glucuronidase  reporter gene driven by osmyb103l putative promoter in rice plants.
examination of gus activity in three independent transgenic lines revealed that the gus signals were stronger in culm and weaker in vascular bundles of coleoptile, root and leaf .
gus staining was stronger in mature panicle than young panicle .
further examination showed that gus activity was detectable in pedicel, lemma and palea while no gus activity was detected in sterile lemma, lodicule, pistil, and stamen in mature panicle .
these results indicate osmyb103l has diverse expressions in various tissues and organs in rice.
overexpression of osmyb103l results in a leaf rolling phenotype
twelve independent transgenic lines overexpressing osmyb103l  were obtained.
the expression level of osmyb103l was determined by qrt-pcr .
three independent transgenic lines  with the highest expression levels were selected for further study .the most significant phenotype of oe lines was upward curling of the leaf blades .
this phenotype appeared first at the seedling stage and then maintained through the rest of the plant growth .
the measurement of leaf rolling index  was performed at the time of flowering.
the lris of the flag leaf, the second leaf from top, and the third leaf from top were approximately 57%, 52%, and 50%, respectively .
the leaf blade cross-sections did not show significant differences between wild type  and oe in terms of organization of the sclerenchyma and vascular tissues.
it suggests that there is no obvious alteration of leaf polarity in transgenic plants.
however, in contrast to wt, the leaves of transgenic plants displayed smaller bulliform cells in the rolled regions .
overexpression of osmyb103l affects the expression levels of several genes encoding cellulose synthase
to explore the possible molecular mechanism of osmyb103l’s effects on rice leaf development, we examined the expression profile in oe- <dig> and wt leaf blades using digital gene expression profiling analysis .
in all,  <dig>  annotated genes showed expressions with more than twofold difference between oe- <dig> and wt, of which  <dig>  genes were up-regulated and  <dig>  genes were down-regulated .
to obtain an ontological profile of the up- or down-regulated genes, ontological terms were assigned, and the enrichment significance was analyzed by agrigo
<cit> .
twenty-one gene ontology  terms were significantly enriched in the up-regulated gene set, showing that up-regulated genes were involved in multiple biological processes, such as metabolism, biogenesis, localization, and cellular processes .
meanwhile, we noticed that two go terms, cellulose metabolic process  and cellulose biosynthetic process , were possibly related to the leaf rolling trait as reported in rl <dig> mutant
<cit>  .
therefore, we compared the expression levels of the rice cesa genes
<cit> .
our dge data showed that oscesa <dig>  oscesa <dig>  oscesa <dig>  oscesa <dig>  oscesa <dig>  and oscesa <dig> were up-regulated while oscesa <dig> and oscesa <dig> were down-regulated .
we verified the expressions by qrt-pcr for these cesa genes in three independent lines .
four cesa genes, oscesa <dig>  oscesa <dig>  oscesa <dig>  and oscesa <dig>  showed significantly increased transcript levels; while one gene, oscesa <dig>  was down-regulated in the oe plants .
the qrt-pcr analysis was largely consistent with the dge analysis except three genes, oscesa <dig> and oscesa <dig> of which, due to their relatively low log2ratio in dge data.
cesas are proteins responsible for cellulose synthesis in plants
<cit> , thus alteration of cesa expression may change the cellulose content.
therefore, we measured cellulose content in leaf blades of wt and oe plants.
the results showed that oe leaf blades had 13% higher cellulose content compared to wt .
these results suggest that osmyb103l regulates cesa gene expression and may consequently influence cellulose synthesis in rice.
knockdown of osmyb103l decreases the mechanical strength and cellulose content of leaves
to explore the intrinsic function of osmyb103l, we analyzed the phenotypes of mutants with decreased expression levels.
since the loss-of-function mutants were not available, we created rnai-knockdown plants for osmyb103l.
six independent rnai transgenic lines were obtained.
quantitative rt-pcr analyses showed that the expression level of osmyb103l was significantly decreased in leaves of the rnai lines compared with that of wt .
there were no obvious morphological changes in rnai lines.
however, in the heading stage of the rnai plants, we noticed that the leaves were brittle.
when the leaf blade was bent, wt leaves were flexible while the leaves of rnai lines were broken easily, especially in the midribs .
the force required to break the leaf blades was 50% less for the rnai lines than that for wt .
the weakened mechanical strength may relate to the altered cellulose content in leaf blades.
we measured the cellulose content in leaf blades of wt and rnai plants.
compared with wt, the levels of cellulose content in rnai lines were significantly decreased .
therefore, the decreased mechanical strength of rnai plants might result from the decreased cellulose content.
we determined the expression levels of ten cesa genes by qrt-pcr, and found that three cesa genes, oscesa <dig>  oscesa <dig>  and oscesa <dig> were significantly down-regulated in the rnai lines .
these results suggest that the down-regulated cesa genes in osmyb103l rnai plants may lead to a decreased level of cellulose content and the weakened mechanical strength of transgenic leaf blades.
discussion
by screening phenotypes of a transgenic rice population overexpressing rice genes in myb family, we identified a rice leaf rolling-related r2r3-myb gene, osmyb103l.
we showed its activity of transcriptional activation and described its influence on leaf rolling.
we further showed that the altered expression of osmyb103l resulted in changes in cesa gene expression, cellulose content, and mechanical strength in rice leaf.
these results suggest that osmyb103l may have important biological functions in rice leaf development.
leaf rolling is one of the frequently observed phenotypes in rice and other cereals.
it is one of the important agronomic traits for high yield rice varieties
<cit> .
although many cultivars and mutants in rice show several types of leaf rolling phenotypes and a number of leaf rolling related genes have been identified
<cit> , the mechanisms underlying the rolling phenomena remain to be elucidated.
it is generally realized that two major factors can influence leaf rolling.
the first one is the establishment of cell polarity and differentiation, and the second is physiological factors, such as osmotic pressure or turgidity in bulliform cells
<cit> .
previous studies on mutants defective in leaf development in arabidopsis thaliana, maize, and rice have shown that leaf rolling is related to the defective development of the leaf polarity along the adaxial–abaxial axis
<cit> .
however, we did not observe obvious alterations in leaf polarity in the osmyb103l overexpressing plants by histological analysis  and sem observations .
instead, we found that the bulliform cells in the rolled regions of oe leaves were smaller compared with that of wt leaves .
several investigations have shown that bulliform cells are involved in leaf rolling
<cit> .
loss of turgor pressure in bulliform cells could lead to leaf “roll up” during water stress
<cit> .
here the observed abnormal bulliform cells in the leaves of osmyb103l overexpressing plants are consistent with those described in a number of known rolled leaf mutants
<cit> .
for instance, cow <dig> and rl <dig> mutants showed rolled leaves with serious shrinkage and smaller bulliform cells, but with no obvious defects in leaf polarity
<cit> .
therefore, our results suggest that the abnormal bulliform cells, rather than defects in leaf polarity, may account for the rolled leaf phenotype in osmyb103l overexpressing plants to some extent.
secondary cell walls constitute skeletal frameworks and furnish the plant body with mechanical strength
<cit> .
molecular and genetic studies in the model plant, arabidopsis thaliana, revealed that a group of secondary wall-associated nac domain  transcription factors are master switches regulating a cascade of down-stream transcription factors, such as myb transcription factors that leads to the activation of the secondary wall biosynthetic program
<cit> .
it has been reported that atmyb <dig>  is directly activated by the master switches, swns
<cit> .
in arabidopsis thaliana, overexpression of r2r3-myb gene atmyb <dig> led to a noticeable increase in the secondary wall thickness, while its dominant repression caused reduction in secondary wall thickness of both xylary fibers and interfascicular fibers
<cit> .
it has been proposed that atmyb <dig> has the potential to induce the expression of genes involved in cellulose biosynthesis
<cit> .
the transcriptional network regulating secondary cell wall biosynthesis may be evolutionarily conserved in plants, however, little is known about the detailed regulatory programs in grass species, such as rice
<cit> .
based on our analyses on the osmyb103l and its encoded protein, we deduced that osmyb103l might be an ortholog of atmyb <dig> in rice.
we wondered whether it has the conserved functions in rice, a model plant in monocotyledon, and addressed this question by reverse genetics, including overexpression and rnai-knockdown approaches.
our study showed that overexpression of osmyb103l resulted in rolled leaves and knockdown of osmyb103l led to brittle leaves .
further analyses disclosed that the expression of four rice cesa genes, oscesa <dig>  oscesa <dig>  oscesa <dig>  and oscesa <dig>  was increased significantly in transgenic overexpression rice plants  whereas three of these cesa genes, oscesa <dig>  oscesa <dig>  and oscesa <dig>  were significantly down-regulated in the rnai lines .
osmyb103l is a putative r2r <dig> myb transcription factor, which is expected to regulate gene expression by binding to the promoters of its downstream genes.
putative myb binding sequences  can be identified by using the tools described in place
<cit> .
we did find two and even more cngttr sites, the putative mbss, in the respective promoter regions of these cesa genes .
therefore, we hypothesized that these cesa genes would be the targets of osmyb103l.
further investigation, such as chromatin immunoprecipitation  and electrophoretic mobility shift assay , could be carried out to prove it.
in higher plants, cesas were characterized as catalytic subunits of cellulose synthase complexes
<cit> .
in this study, our results showed that the cellulose content of mature leaves was higher in overexpression lines than that in wt  while the osmyb103l rnai lines had decreased cellulose content in the leaf blades .
the results indicate that osmyb103l may regulate the expression levels of cesa genes, which could subsequently alter the cellulose content.
however, we also noticed that the contents of cellulose were not positively correlated with the expression levels of osmyb103l in transgenic plants, especially in the overexpression lines.
plant cesa genes are members of multigene families
<cit> .
arabidopsis has  <dig> cesa isoforms which can be classified into six orthologous groups having non-redundant functions in cellulose synthesis as revealed by mutational analysis
<cit> .
rice also has  <dig> cesa isoforms, of which oscesa <dig>  oscesa <dig> and oscesa <dig> were showed non-redundant functions in cellulose synthesis specifically in secondary walls
<cit> .
based on these, we speculate that most rice cesas might also play distinctive roles in cellulose synthesis.
some particular cesa gene expression induction may not lead to significant change of cellulose content.
therefore, changed expressions of several cesas in osmyb103l transgenic lines may not induce obviously changes in cellulose content of the transgenic plants.
in addition, our study demonstrated that osmyb103l rnai-knockdown plants possessed brittle leaves with obviously decreased mechanical strength and reduced cellulose content in the leaf blades .
of note, reduced cellulose content and inferior mechanical strength are the common phenotypes of cesa mutants, such as irx <dig> to irx <dig> in arabidopsis and brittle culm <dig>  in rice
<cit> .
thus, we wondered whether mechanical strength was elevated due to the increased cellulose in osmyb103l overexpression plants.
therefore, we also measured the mechanical strength of the overexpression lines of osmyb103l.
nevertheless, the leaves of overexpression plants did not show statistically significant changes in mechanical strength compared to wild type .
mechanical strength is a complex trait, which can be affected by many factors
<cit> .
perhaps, in our case, the increase of cellulose contents was not enough to cause the change of mechanical strength in osmyb103l overexpression plants.
moderate leaf rolling has been regarded as one of the important agronomic traits in high yield rice cultivars
<cit> , and selection of this trait has been applied for rice breeding directly or indirectly in china
<cit> .
one of the successful examples is the breeding of super-hybrid rice, liang-you-pei-jiu
<cit> .
lyp <dig> has moderate xrolling leaves and is widely cultivated throughout southern china due to its high yield and seed quality.
the successful cultivation of lyp <dig> is therefore taken as an example for improving crop yield and quality using the rolled leaf trait
<cit> .
therefore, identification of genes that regulate leaf rolling would be beneficial to breeding.
in this study, we report that overexpression of osmyb103l in rice results in leaf rolling, which suggests that osmyb103l could be a potential target for genetic manipulation of leaf shape and plant architecture in rice.
in addition, leaf brittleness is an important agronomic trait for rice and other crops since it affects not only grain production but also the utilization of cereal straw as animal forage
<cit> .
here, we found that down-regulation of the expression level of osmyb103l decreases mechanical strength in rice plants, suggesting that osmyb103l could be engineered for favorable leaf brittleness in rice, as well as in other cereal and forage crops.
CONCLUSIONS
in this study, an r2r <dig> myb transcription factor, osmyb103l, has been identified in rice.
we show that overexpression of osmyb103l in rice results in leaf rolling, increased cellulose content, and elevated expression of genes encoding cellulose synthase .
besides, the rna interference -knockdown of osmyb103l leads to a weakened mechanical strength and decreased cellulose content in leaves.
moreover, the expression levels of several cesa genes are up-regulated in osmyb103l overexpression lines while down-regulated in osmyb103l-knockdown lines.
collectively, this work demonstrates that osmyb103l impacts leaf shape, cellulose synthesis, and mechanical strength in rice.

 leaves are the main organs of photosynthesis and play a crucial role in plant development. leaf rolling is a frequently observed phenotype in fields and rice mutant populations. the appropriate leaf rolling has been regarded as one of the most important elements in rice ideotype breeding as it improves photosynthetic efficiency, accelerates dry-matter accumulation, reduces solar radiation on leaves, decreases leaf transpiration under drought stress and raises grain yield. however, severe leaf rolling could lead to growth retardation, developmental defects and crop yield reduction. to date, many studies have been performed to characterize the genes controlling leaf rolling by analyses of the rolled leaf mutants in rice. twelve rolled leaf mutants in rice are reported and the corresponding genes are mapped genetically, of which, six rl genes are mapped on rice chromosomes by morphological markers and the rest are directly mapped in rice genome by molecular markers. among these rl mutants, rl is the first to be cloned and analyzed in detail. rolled leaf /shallot-like encodes a kanadi transcription factor, and rl9/sll mutants display extremely incurved leaves due to the defective development of sclerenchymatous cells on the abaxial side. some other genes are also found to be related to leaf rolling in rice. loss-of-function of oscsld gene, which encodes a cellulose synthase-like protein, results in phenotypes of reduced leaf width and semi-rolled leaves, possibly due to the significantly smaller bulliform cells in mutants. loss-of-function of constitutively wilted /narrow leaf, encoding a member of the yucca protein, causes rice leaves to roll upward. loss-of-function of curly flag leaf, encoding a ww domain protein, induces rolled leaves which have a wrinkled epidermis and contain papillae with reduced wax content. rolled leaf, encoding a 2og-fe oxygenase family protein, is reported to modulate rice leaf rolling by affecting secondary cell wall formation and bulliform cell development. mutation of semi-rolled leaf, encoding a putative gpi-anchored protein, results in leaves rolled adaxially in rice due to the increased number of bulliform cells at the adaxial cell layers. in addition, recent studies show that overexpression of rice gene osago which encodes an argonaute family member, results in the leaf blades curling upward. oshb is a member of the class iii homeodomain leucine zipper family of genes, overexpression of its microrna166-resistant version could result in adaxially rolled leaves due to the reduced sclerenchyma and formation of bulliform cells on the abaxial side. taken together, the major factors possibly affecting leaf rolling include the alteration of osmotic pressure and/or turgidity in bulliform cells, the impairment of cell polarity establishment and cell differentiation. however, the mechanisms underlying leaf rolling in rice and, particularly, the involved transcription factors remain largely unknown. myb proteins belong to a super-family of transcription factors, which prevail in all eukaryotes and share a highly conserved dna-binding domain, myb. depending on the number of adjacent repeats in the myb domain, myb proteins can be classified into four subfamilies, including 1r-myb, r2r3-myb, 3r-myb, and 4r-myb. among them, r2r3-myb subfamily is a typical representative one, whose members contain two myb repeats in their dna-binding domains. myb proteins in plants are involved in a variety of plant-specific processes, including primary and secondary metabolism, cell fate and identity, developmental processes, and responses to various stresses. genome-wide analysis shows that there are approximately myb genes in rice, of which encode r2r3-myb proteins. so far, only a few number of myb proteins, including osgamyb, osmyb osmyb osmyb3r- csa, mybs and osmyb2p-1, have been functionally characterized. the functions of most of myb proteins are still unknown in rice. to characterize functions of myb transcription factors in rice, we overexpressed several myb genes in kasalath, an indica cultivar, with the transgene constructs containing the full-length cdnas of rice myb genes, driven by maize ubiquitin promoter. of the transgenic lines, one line overexpressing the full-length cdna of os08g designated as osmyb103l hereafter, displays a rolled leaf phenotype. osmyb103l encodes an r2r3-myb transcription factor. our study shows that it localizes in the nucleus and possesses transcriptional activity. we detail the phenotypes of osmyb103l overexpressing and rna interference knockdown plants, including the altered leaf shape, the changed cellulose content, and the impaired mechanical strength. the roles of osmyb103l in leaf shape formation and cellulose synthesis are discussed. we propose the potential application of osmyb103l in molecular breeding of rice.  osmyb103l encodes an r2r3-myb transcription factor to discover transcription factors controlling leaf development, we screened the rice lines ectopically expressing rice myb genes under the control of maize ubiquitin promoter. one line overexpressing loc_os08g was selected for further study due to its particular leaf shape, such as upward curling of the leaf blade. according to the rice genome annotation database, loc_os08g encodes a putative r2r3-myb family transcription factor with a length of amino acids and a molecular mass of approximately kd. the pfam database shows that the deduced protein has two myb dna-binding domains at the n-terminus. as revealed by phylogenetic analysis of the related myb transcription factors in arabidopsis thaliana and rice, os08g is closely related to at1g. protein sequence alignment showed that they are highly conserved in the predicted r2- and r3-myb dna-binding domains. we hereby designated os08g as osmyb103l. nuclear localization is one of the significant features of transcription factors. to determine its subcellular localization, we fused green fluorescent protein to the c-terminus of osmyb103l to produce an osmyb103l-gfp fusion protein. we monitored the fluorescence of the transiently expressed fusion protein in rice protoplasts and onion epidermis cells. in both cases, the fluorescence signals of the fusion protein were observed predominantly in nuclei. while in the gfp alone control, fluorescence signals were observed in nuclei and cytoplasm. these results indicate that osmyb103l is a nuclear-localized protein. transcriptional activation is a characteristic feature of many transcription factors. the full-length, n-terminal myb dna-binding domain and c-terminal putative activation domain were fused in-frame with the gal dna binding domain, respectively. we conducted a transcriptional activation analysis in yeast by using the fusion proteins. the analysis showed that the full-length and c-terminal region of osmyb103l activated the expression of aur1-c and mel reporter genes in yeast, indicating that osmyb103l has the capability of transcriptional activation. expression of osmyb103l varies in different organs we determined the expression level of osmyb103l in various rice organs, including root, culm, leave, and panicle using quantitative real-time pcr. the expression level of osmyb103l was highest in culm, lower in root, leaf and mature panicle, and lowest in young panicle. in addition, the expression pattern of osmyb103l was examined by the β-glucuronidase reporter gene driven by osmyb103l putative promoter in rice plants. examination of gus activity in three independent transgenic lines revealed that the gus signals were stronger in culm and weaker in vascular bundles of coleoptile, root and leaf. gus staining was stronger in mature panicle than young panicle. further examination showed that gus activity was detectable in pedicel, lemma and palea while no gus activity was detected in sterile lemma, lodicule, pistil, and stamen in mature panicle. these results indicate osmyb103l has diverse expressions in various tissues and organs in rice. overexpression of osmyb103l results in a leaf rolling phenotype twelve independent transgenic lines overexpressing osmyb103l were obtained. the expression level of osmyb103l was determined by qrt-pcr. three independent transgenic lines with the highest expression levels were selected for further study.the most significant phenotype of oe lines was upward curling of the leaf blades. this phenotype appeared first at the seedling stage and then maintained through the rest of the plant growth. the measurement of leaf rolling index was performed at the time of flowering. the lris of the flag leaf, the second leaf from top, and the third leaf from top were approximately 57%, 52%, and 50%, respectively. the leaf blade cross-sections did not show significant differences between wild type and oe in terms of organization of the sclerenchyma and vascular tissues. it suggests that there is no obvious alteration of leaf polarity in transgenic plants. however, in contrast to wt, the leaves of transgenic plants displayed smaller bulliform cells in the rolled regions. overexpression of osmyb103l affects the expression levels of several genes encoding cellulose synthase to explore the possible molecular mechanism of osmyb103l’s effects on rice leaf development, we examined the expression profile in oe- and wt leaf blades using digital gene expression profiling analysis. in all, annotated genes showed expressions with more than twofold difference between oe- and wt, of which genes were up-regulated and genes were down-regulated. to obtain an ontological profile of the up- or down-regulated genes, ontological terms were assigned, and the enrichment significance was analyzed by agrigo. twenty-one gene ontology terms were significantly enriched in the up-regulated gene set, showing that up-regulated genes were involved in multiple biological processes, such as metabolism, biogenesis, localization, and cellular processes. meanwhile, we noticed that two go terms, cellulose metabolic process and cellulose biosynthetic process, were possibly related to the leaf rolling trait as reported in rl mutant. therefore, we compared the expression levels of the rice cesa genes. our dge data showed that oscesa oscesa oscesa oscesa oscesa and oscesa were up-regulated while oscesa and oscesa were down-regulated. we verified the expressions by qrt-pcr for these cesa genes in three independent lines. four cesa genes, oscesa oscesa oscesa and oscesa showed significantly increased transcript levels; while one gene, oscesa was down-regulated in the oe plants. the qrt-pcr analysis was largely consistent with the dge analysis except three genes, oscesa and oscesa of which, due to their relatively low log2ratio in dge data. cesas are proteins responsible for cellulose synthesis in plants, thus alteration of cesa expression may change the cellulose content. therefore, we measured cellulose content in leaf blades of wt and oe plants. the results showed that oe leaf blades had 13% higher cellulose content compared to wt. these results suggest that osmyb103l regulates cesa gene expression and may consequently influence cellulose synthesis in rice. knockdown of osmyb103l decreases the mechanical strength and cellulose content of leaves to explore the intrinsic function of osmyb103l, we analyzed the phenotypes of mutants with decreased expression levels. since the loss-of-function mutants were not available, we created rnai-knockdown plants for osmyb103l. six independent rnai transgenic lines were obtained. quantitative rt-pcr analyses showed that the expression level of osmyb103l was significantly decreased in leaves of the rnai lines compared with that of wt. there were no obvious morphological changes in rnai lines. however, in the heading stage of the rnai plants, we noticed that the leaves were brittle. when the leaf blade was bent, wt leaves were flexible while the leaves of rnai lines were broken easily, especially in the midribs. the force required to break the leaf blades was 50% less for the rnai lines than that for wt. the weakened mechanical strength may relate to the altered cellulose content in leaf blades. we measured the cellulose content in leaf blades of wt and rnai plants. compared with wt, the levels of cellulose content in rnai lines were significantly decreased. therefore, the decreased mechanical strength of rnai plants might result from the decreased cellulose content. we determined the expression levels of ten cesa genes by qrt-pcr, and found that three cesa genes, oscesa oscesa and oscesa were significantly down-regulated in the rnai lines. these results suggest that the down-regulated cesa genes in osmyb103l rnai plants may lead to a decreased level of cellulose content and the weakened mechanical strength of transgenic leaf blades. discussion by screening phenotypes of a transgenic rice population overexpressing rice genes in myb family, we identified a rice leaf rolling-related r2r3-myb gene, osmyb103l. we showed its activity of transcriptional activation and described its influence on leaf rolling. we further showed that the altered expression of osmyb103l resulted in changes in cesa gene expression, cellulose content, and mechanical strength in rice leaf. these results suggest that osmyb103l may have important biological functions in rice leaf development. leaf rolling is one of the frequently observed phenotypes in rice and other cereals. it is one of the important agronomic traits for high yield rice varieties. although many cultivars and mutants in rice show several types of leaf rolling phenotypes and a number of leaf rolling related genes have been identified, the mechanisms underlying the rolling phenomena remain to be elucidated. it is generally realized that two major factors can influence leaf rolling. the first one is the establishment of cell polarity and differentiation, and the second is physiological factors, such as osmotic pressure or turgidity in bulliform cells. previous studies on mutants defective in leaf development in arabidopsis thaliana, maize, and rice have shown that leaf rolling is related to the defective development of the leaf polarity along the adaxial–abaxial axis. however, we did not observe obvious alterations in leaf polarity in the osmyb103l overexpressing plants by histological analysis and sem observations. instead, we found that the bulliform cells in the rolled regions of oe leaves were smaller compared with that of wt leaves. several investigations have shown that bulliform cells are involved in leaf rolling. loss of turgor pressure in bulliform cells could lead to leaf “roll up” during water stress. here the observed abnormal bulliform cells in the leaves of osmyb103l overexpressing plants are consistent with those described in a number of known rolled leaf mutants. for instance, cow and rl mutants showed rolled leaves with serious shrinkage and smaller bulliform cells, but with no obvious defects in leaf polarity. therefore, our results suggest that the abnormal bulliform cells, rather than defects in leaf polarity, may account for the rolled leaf phenotype in osmyb103l overexpressing plants to some extent. secondary cell walls constitute skeletal frameworks and furnish the plant body with mechanical strength. molecular and genetic studies in the model plant, arabidopsis thaliana, revealed that a group of secondary wall-associated nac domain transcription factors are master switches regulating a cascade of down-stream transcription factors, such as myb transcription factors that leads to the activation of the secondary wall biosynthetic program. it has been reported that atmyb is directly activated by the master switches, swns. in arabidopsis thaliana, overexpression of r2r3-myb gene atmyb led to a noticeable increase in the secondary wall thickness, while its dominant repression caused reduction in secondary wall thickness of both xylary fibers and interfascicular fibers. it has been proposed that atmyb has the potential to induce the expression of genes involved in cellulose biosynthesis. the transcriptional network regulating secondary cell wall biosynthesis may be evolutionarily conserved in plants, however, little is known about the detailed regulatory programs in grass species, such as rice. based on our analyses on the osmyb103l and its encoded protein, we deduced that osmyb103l might be an ortholog of atmyb in rice. we wondered whether it has the conserved functions in rice, a model plant in monocotyledon, and addressed this question by reverse genetics, including overexpression and rnai-knockdown approaches. our study showed that overexpression of osmyb103l resulted in rolled leaves and knockdown of osmyb103l led to brittle leaves. further analyses disclosed that the expression of four rice cesa genes, oscesa oscesa oscesa and oscesa was increased significantly in transgenic overexpression rice plants whereas three of these cesa genes, oscesa oscesa and oscesa were significantly down-regulated in the rnai lines. osmyb103l is a putative r2r myb transcription factor, which is expected to regulate gene expression by binding to the promoters of its downstream genes. putative myb binding sequences can be identified by using the tools described in place. we did find two and even more cngttr sites, the putative mbss, in the respective promoter regions of these cesa genes. therefore, we hypothesized that these cesa genes would be the targets of osmyb103l. further investigation, such as chromatin immunoprecipitation and electrophoretic mobility shift assay, could be carried out to prove it. in higher plants, cesas were characterized as catalytic subunits of cellulose synthase complexes. in this study, our results showed that the cellulose content of mature leaves was higher in overexpression lines than that in wt while the osmyb103l rnai lines had decreased cellulose content in the leaf blades. the results indicate that osmyb103l may regulate the expression levels of cesa genes, which could subsequently alter the cellulose content. however, we also noticed that the contents of cellulose were not positively correlated with the expression levels of osmyb103l in transgenic plants, especially in the overexpression lines. plant cesa genes are members of multigene families. arabidopsis has cesa isoforms which can be classified into six orthologous groups having non-redundant functions in cellulose synthesis as revealed by mutational analysis. rice also has cesa isoforms, of which oscesa oscesa and oscesa were showed non-redundant functions in cellulose synthesis specifically in secondary walls. based on these, we speculate that most rice cesas might also play distinctive roles in cellulose synthesis. some particular cesa gene expression induction may not lead to significant change of cellulose content. therefore, changed expressions of several cesas in osmyb103l transgenic lines may not induce obviously changes in cellulose content of the transgenic plants. in addition, our study demonstrated that osmyb103l rnai-knockdown plants possessed brittle leaves with obviously decreased mechanical strength and reduced cellulose content in the leaf blades. of note, reduced cellulose content and inferior mechanical strength are the common phenotypes of cesa mutants, such as irx to irx in arabidopsis and brittle culm in rice. thus, we wondered whether mechanical strength was elevated due to the increased cellulose in osmyb103l overexpression plants. therefore, we also measured the mechanical strength of the overexpression lines of osmyb103l. nevertheless, the leaves of overexpression plants did not show statistically significant changes in mechanical strength compared to wild type. mechanical strength is a complex trait, which can be affected by many factors. perhaps, in our case, the increase of cellulose contents was not enough to cause the change of mechanical strength in osmyb103l overexpression plants. moderate leaf rolling has been regarded as one of the important agronomic traits in high yield rice cultivars, and selection of this trait has been applied for rice breeding directly or indirectly in china. one of the successful examples is the breeding of super-hybrid rice, liang-you-pei-jiu. lyp has moderate xrolling leaves and is widely cultivated throughout southern china due to its high yield and seed quality. the successful cultivation of lyp is therefore taken as an example for improving crop yield and quality using the rolled leaf trait. therefore, identification of genes that regulate leaf rolling would be beneficial to breeding. in this study, we report that overexpression of osmyb103l in rice results in leaf rolling, which suggests that osmyb103l could be a potential target for genetic manipulation of leaf shape and plant architecture in rice. in addition, leaf brittleness is an important agronomic trait for rice and other crops since it affects not only grain production but also the utilization of cereal straw as animal forage. here, we found that down-regulation of the expression level of osmyb103l decreases mechanical strength in rice plants, suggesting that osmyb103l could be engineered for favorable leaf brittleness in rice, as well as in other cereal and forage crops.  in this study, an r2r myb transcription factor, osmyb103l, has been identified in rice. we show that overexpression of osmyb103l in rice results in leaf rolling, increased cellulose content, and elevated expression of genes encoding cellulose synthase. besides, the rna interference -knockdown of osmyb103l leads to a weakened mechanical strength and decreased cellulose content in leaves. moreover, the expression levels of several cesa genes are up-regulated in osmyb103l overexpression lines while down-regulated in osmyb103l-knockdown lines. collectively, this work demonstrates that osmyb103l impacts leaf shape, cellulose synthesis, and mechanical strength in rice. 
knockdown of osmyb103l by rna interference led to a decreased level of cellulose content and reduced mechanical strength in leaves. these findings suggest that osmyb103l may target cesa genes for regulation of cellulose synthesis and could potentially be engineered for desirable leaf shape and mechanical strength in rice. further analyses showed that expression levels of several cellulose synthase genes were significantly increased, as was the cellulose content in osmyb103l overexpressing lines. meanwhile, the expression levels of several cesa genes were decreased in these knockdown lines. in this study, we report the functional characterization of a rice gene, osmyb103l, which encodes an r2r3-myb transcription factor. overexpression of osmyb103l in rice resulted in a rolled leaf phenotype. myb transcription factors are one of the largest gene families and have important roles in plant development, metabolism and stress responses.
 the shape of grass leaves possesses great value in both agronomy and developmental biology research. leaf rolling is one of the important traits in rice breeding. myb transcription factors are one of the largest gene families and have important roles in plant development, metabolism and stress responses. however, little is known about their functions in rice.  in this study, we report the functional characterization of a rice gene, osmyb103l, which encodes an r2r3-myb transcription factor. osmyb103l was localized in the nucleus with transactivation activity. overexpression of osmyb103l in rice resulted in a rolled leaf phenotype. further analyses showed that expression levels of several cellulose synthase genes were significantly increased, as was the cellulose content in osmyb103l overexpressing lines. knockdown of osmyb103l by rna interference led to a decreased level of cellulose content and reduced mechanical strength in leaves. meanwhile, the expression levels of several cesa genes were decreased in these knockdown lines.  these findings suggest that osmyb103l may target cesa genes for regulation of cellulose synthesis and could potentially be engineered for desirable leaf shape and mechanical strength in rice. osmyb103lleaf rollingmyb transcription factorcellulose 