BACKGROUND
for multicellular organisms, organ size is a fundamental attribute of body morphology  <cit> .
in animals, the final size of an organ is primarily determined by cell growth, proliferation, and apoptosis.
these processes are primarily mediated by two major pathways: the target of the rapamycin  pathway that regulates cell growth and the hippo pathway that coordinately controls cell growth, proliferation, and apoptosis  <cit> .
in plants, because cell apoptosis does not generally contribute to morphogenesis in most organs  <cit> , the development of an organ to its characteristic size depends mainly on cell proliferation and expansion  <cit> .
recent studies in arabidopsis have identified a number of genes involved in the regulation of either cell proliferation or cell expansion that are now known to affect final organ size  <cit> .
however, many such factors appear to be involved in multiple pathways to affect cell number or size  <cit> , suggesting that the mechanisms of organ-size control in plants are more complicated than those in animals  <cit> .
this might be partly because plants are immobile and have to be highly responsive to the ever-changing environments  <cit> .
during the development of plant determinate organs such as leaves and floral organs, cell proliferation and expansion occur at two distinct but overlapping phases  <cit> .
in a leaf, for example, at the early stages of development the cells within leaf primordia undergo a period of cell proliferation and differentiation; cell expansion subsequently begins at the leaf tip and proceeds progressively in a basipetal direction, a process during which the transition from cell proliferation to expansion is established  <cit> .
finally, the leaf grows by post-mitotic cell expansion and reaches its characteristic final size.
recent studies in arabidopsis have suggested strongly that the timing of the transition from the cell proliferation to the cell expansion phase appears to be a critical determinant of overall organ size  <cit> .
this notion is supported by the large number of identified factors involved in organ-size control that are known to alter the duration of cell proliferation, and thereby alter total cell number and final organ size  <cit> .
for instance, some positive regulators that participate in organ-size control such as aintegumenta , auxin-regulated gene involved in organ size , and kluh/cyp78a <dig>  can prolong the cell proliferation phase and thus increase cell numbers  <cit> .
there are some negative regulators such as da <dig> and enhance of da <dig> /big brother , had a role in restricting the period of cell proliferation and thus the organ size  <cit> .
furthermore, factors involved in the regulation of cell expansion during organ growth have been identified in arabidopsis, including the regulatory particle aaa-atpase 2a , expansin <dig> , argos-like , and target of rapamycin   <cit> .
these factors were shown to affect either the duration or the rate of cell expansion, and are thus known to alter the final size of cells and of organs.
interestingly, there is a “compensatory mechanism” that coordinates cell proliferation and expansion during organ growth  <cit> .
for instance, the an <dig> mutant had fewer but larger cells than did wild-type plants, and further analysis indicated that the an <dig> cells seemed to generate and transmit an intercellular signal that could enhance post-mitotic cell expansion  <cit> .
it appears that when the cell number within an organ was decreased below a threshold level, the plants often triggered further post-mitotic cell expansion to compensate for the reduction in cell number  <cit> .
the mechanisms responsible for the coordination of cell proliferation and expansion during plant organogenesis are far from being understood.
the arabidopsis organ size related  family has been implicated in the regulation of aerial organ size  <cit> .
argos, the founding member of the osr homologues, controlled the growth of aerial organs, mainly through prolonging the duration of cell proliferation by promoting the continued expression of ant and cycd3; <dig>  <cit> .
however, argos-like , a close homolog of argos, was found to regulate the organ-size by affecting cell expansion  <cit> .
our recent work demonstrated that organ size related <dig>  promoted organ growth by affecting both cell proliferation and expansion  <cit> .
osr <dig>  argos, and arl all share a conserved osr domain that is sufficient to promote organ growth  <cit> .
moreover, these three osr genes are differentially regulated by various plant hormones, suggesting that they may mediate different signals to affect the cell proliferation and/or expansion process.
indeed, a recent study showed that zea mays argos <dig>  had a function similar to that of argos.
overexpression of zar <dig> in maize enhanced maize organ growth, increased yield, and improved tolerance to drought stress  <cit> .
osr genes have only been identified in plants, and it is very interesting that different osr members with a similar osr domain could mediate cell proliferation and/or cell expansion, the two different cellular events that have been widely considered to be separately controlled  <cit> .
there are four osr homologues in the arabidopsis genome, and these were likely generated by gene duplication events  <cit> .
to gain further insight into the evolutionary function of the osr genes, we investigated the role of the fourth osr gene, osr <dig>  during organ growth.
here, we report that osr <dig> participates in the regulation of organ growth by primarily affecting cell expansion in a manner redundant with arl.
further analysis of osr <dig> argos- <dig> arli triple and argos- <dig> osr <dig> arli osr2i quadruple mutants revealed that these four osr genes have redundant and cooperative roles in the regulation of cell proliferation and/or expansion.
our characterization of functional divergence among these four osr genes implies a novel mechanism underlying the coordinative regulation of cell proliferation and expansion during plant organogenesis.
RESULTS
osr <dig> is expressed primarily in organs undergoing growth by cell expansion
we previously reported that arabidopsis osr <dig> was involved in the regulation of organ growth and that it contributes to final organ size primarily through enhancement of cell proliferation  <cit> .
we also showed that three identified osr proteins shared a conserved osr domain with an identical lpplpppp motif and two putative transmembrane helices, which was sufficient to promote organ growth  <cit> .
the arabidopsis thaliana genome also has another gene, at2g <dig>  which encodes a protein of only  <dig> amino acids that phylogenetically belongs to the osr member   <cit> .
careful alignment of its amino acid sequence with those of osr <dig>  argos, and arl showed that at2g <dig> had the conserved lpplpppp motif and the c terminal transmembrane helix of the osr domain.
however, it lacked the n terminal transmembrane helix but instead had a plasma membrane-localized signal peptide predicted by phobius and ipsort  .
moreover, at2g <dig> is located alongside osr <dig> in the genome, suggesting that these two genes may originate from a gene duplication event  <cit> .
therefore, we designated at2g <dig> as organ size-related  <dig> .
to explore whether or not osr <dig> is functional during organogenesis, we initially monitored osr <dig> expression in various organs with qrt-pcr analysis.
as shown in figure 1a, osr <dig> was expressed primarily in the expanding organs, including leaf, inflorescence, and flower.
we then examined the tissue-specific expression of osr <dig> using the t <dig> independent transgenic plants carrying an osr <dig> promoter:β-glucuronidase  construct.
in two-week-old seedlings, high gus expression was observed in the organs or organ regions where active cell expansion and elongation were occurring, such as the upper parts of leaves, and the elongating regions of lateral roots ; abundant expression was also detected in the cotyledons .
however, weak or nearly absent gus signals were observed in juvenile leaves, shoot, and root tips, where cells were predominantly undergoing the cell proliferation process .
in the inflorescence and developing floral organs, gus staining was mainly detected in buds, sepals, and filaments .
careful examination of gus expression in developing first leaves showed that weak gus signal was detected at the juvenile stage, while abundant gus staining was observed when a leaf was undergoing expanding growth.
subsequently, the gus signal was almost disappeared in fully-expanded leaves .
these observations indicate that osr <dig> is expressed predominantly in organs that are undergoing cell expansion growth.figure  <dig>
expression of
osr2
.
expression of osr <dig> in various organs assayed by qrt-pcr.
the data were from three biological replicates and are presented as mean values ± se.
rt, root; st, stem; dl, dividing leaf; el, expanding leaf; ol, old leaf; in, inflorescence; fl, flower.
expression patterns of osr <dig> assayed by gus staining of transgenic plants carrying a proosr2:gus construct.
gus staining was shown in seedling , shoot , lateral root , and the floral organs .
bars, 2 mm.
since the three previously-identified osr members were known to be er-localized  <cit> , we further examined the cellular localization of osr <dig> in leaf epidermal cells of proosr2:osr2-gfp transgenic plants.
unlike other osr proteins, the osr2-gfp fusion protein signal was detected in the plasma membrane , indicating that osr <dig> is plasma membrane-localized.
ectopic expression of osr <dig> enlarges organs primarily by enhancing cell expansion
to investigate the role of osr <dig> in organ development, we generated transgenic arabidopsis thaliana  plants harboring a pro35s:osr <dig> construct.
all  <dig> of the t <dig> transgenic lines  overexpressing osr <dig> exhibited, to varying extents, enlarged organs as compared with the empty vector control plants.
careful examination of three of the independent t <dig> homozygous lines  showed that the ectopic expression of osr <dig> resulted in an obviously increased size of various organs such as leaves, cotyledons, roots, floral organs, and siliques .
detailed quantification of fully expanded third leaves revealed that the average blade areas of the pro35s:osr <dig> l <dig>  l <dig>  and l <dig> plants were increased by  <dig> ,  <dig> , and  <dig> %, respectively, as compared with those of control plants .
qrt-pcr analysis verified that the increased sizes of organs were indeed closely associated with the elevated osr <dig> expression in these transgenic lines .
the transgenic plants overexpressing osr <dig> also displayed longer roots, hypocotyls, siliques, and petioles, and the plant heights were also increased in these plants as compared with the controls .
additionally, the flowering time in osr <dig> transgenic plants was delayed by about four days .figure  <dig>
morphological and cytological characterization of
pro35s:osr2
transgenic plants.
morphology of 28-day-old control and three independent lines of pro35s:osr <dig> transgenic plants .
bar, 1 cm.
blade areas of fully-expanded third leaves of control and three pro35s:osr <dig> transgenic lines.
at least five leaves for each genotype were examined; data are shown as mean values ± se; student’s t-test: **p <  <dig> .
qrt-pcr analysis of osr <dig> expression in control and transgenic plants presented in .
the data were from three biological replicates and are presented as mean values ± se; student’s t-test: **p <  <dig> .
the cell area and estimated cell number of epidermal cells in fully-expanded third leaves of control and three pro35s:osr <dig> transgenic plants described in .
at least five leaves were examined for each line, and data are shown as mean values ± se; student’s t-test: **p <  <dig> .
bar, 50 μm.
the cell area and estimated cell number of mesophyll cells in fully-expanded third leaves of control and pro35s:osr <dig>  transgenic plants.
bar, 50 μm.
data are shown as mean values ± se; student’s t-test: **p <  <dig> .
we then used leaves and cotyledons as representative organs to investigate the contribution of cell proliferation and cell expansion to the enlarged organs of the p35s:osr <dig> plants.
as shown in figure 2d and e, as compared with those of the control leaves, the average size of the epidermal cells in fully-expanded third leaves of l <dig>  l <dig>  and l <dig> plants was increased by  <dig> ,  <dig> , and  <dig> %, respectively.
the estimated numbers of epidermal cells per leaf were not obviously altered in the transgenic plants .
similarly, mesophyll cells in p35s:osr <dig> leaves were enlarged, whereas the estimated cell numbers did not differ from the control leaves .
consistently, ectopic expression of osr <dig> resulted in larger mesophyll cells in cotyledons, and again the mesophyll cell number in cotyledons did not differ from the control .
these observations demonstrate that the enlarged organs in p35s:osr <dig> resulted primarily from enhanced cell expansion rather than cell proliferation.
osr <dig> affects the cell expansion rate during organ growth
to define the exact role of osr <dig> in cell expansion rate and duration, we further compared the expansion kinetics of epidermal cells in both the tips and the basal regions of the third leaves in control and p35s:osr <dig> l <dig> plants.
as shown in figure 3a and b, the cell expansion rates of both the tips and the bases of p35s:osr <dig> leaves were indeed much higher than those of controls from day  <dig> to day  <dig>  whereas the cell expansion rates in the two genotypes were almost the same after day 21; cell expansion ceased after day  <dig>  these observations strongly suggest that ectopic expression of osr <dig> mainly affects the rate of cell expansion rather than the duration of cell expansion phase.figure  <dig>
osr2
increases the rate of cell expansion in developing leaves.
relative cell expansion rate of the epidermal cells at the tip  and base  of the third leaves of control and pro35s:osr <dig> transgenic plants .
at least five leaves from each genotype were examined.
nuclear polyploidization analysis of the leaf cells in the control and l <dig> pro35s:osr <dig> transgenic lines.
fully-expanded blades of the third leaves were used for cell nuclear ploidy analysis with a flow cytometer.
the percentages of cells with different nuclear polyploidy levels were observed from three independent biological duplicates, and are shown as mean values ± se.
as cell endoreduplication is known to contribute to final cell size  <cit> , we investigated whether the enlarged cells in the p35s:osr <dig> leaves were attributable to alteration of endoreduplication.
flow cytometry analysis of the nuclei of fully expanded leaves of 27-day-old plants showed that, although the percentages of cells from 4c and 8c were slightly different between the two genotypes, the overall distribution of cells from 2c to 16c remained comparable between two genotypes .
this result indicates that osr2-mediated cell expansion is not related to the nuclear dna endoreduplication of leaf cells.
osr <dig> functions redundantly with arl to mediate cell expansion
to further investigate the role of osr <dig> in organ growth, we obtained the t-dna insertion mutant osr <dig>  from the arabidopsis biological resource center , in which a t-dna fragment was inserted into the exon of osr <dig> .
rt-pcr analysis showed that osr <dig> was an osr <dig> knock-out mutant .
however, osr <dig> plants didn’t show an obvious reduction in organ size when compared with wild-type  plants .
since the tissue-specific expression pattern of osr <dig> was quite similar to that of arl , we speculated that osr <dig> may have functional redundancy with arl.
to test this, we introduced an arl specific rna-interference construct  into both wt and osr <dig> plants to generate arli and osr <dig> arli plants.
qrt-pcr analysis validated that the expression of arl was obviously knocked down in the independent t <dig> lines .
the final size of the third leaf of the arli transgenic plants was reduced by about 9% as compared with that of either the wt or osr <dig>  by contrast, leaf size in osr <dig> arli plants l <dig> and l <dig> was decreased by about  <dig>  and  <dig> %, respectively .
consistently, careful examination of the number and size of epidermal cells in the third leaf confirmed that the knockout of osr <dig> led to size reduction of epidermal cells in arli plants but had no obvious effect on cell number .
consistently, transgenic plants harboring a 35s promoter-driven specific osr <dig> rnai construct  did not exhibit any organ-size phenotype.
however, introduction of a pro35s:osr <dig> rnai construct into arli plants enhanced the size reduction of leaves , confirming that osr <dig> acts redundantly with arl in the regulation of cell expansion during organ growth.figure  <dig>
osr2
acts redundantly with
arl
.
morphology and the size of third leave of 26-day-old wt, osr <dig>  arli, and osr <dig> arli  plants.
at least five fully-expanded leaves of each genotype were used for determination of the leaf size; the data are shown as mean values ± se; student’s t-test: *p <  <dig> , ***p <  <dig> .
bars, 2 mm.
the epidermal cell area and estimated cell number of the third leave described in .
data were from at least five leaves of each genotype and are shown as mean values ± se; student’s t-test: *p <  <dig> , **p <  <dig> .
as the three previously-identified osr genes are known to be differentially responsive to various hormones  <cit> , we also investigated the transcriptional regulation of osr <dig> by plant hormones.
interestingly, the qrt-pcr analysis of seedlings treated with various hormones revealed that, similar to arl, osr <dig> was induced by epi-brassinolide  , and this induction was disrupted in the br signaling mutants bri1- <dig> and bin <dig> .
moreover, introduction of a p35s:osr <dig> construct into bri1- <dig> and bin <dig> could partially restore leaf and petiole growth, and the expansion defect in bri1- <dig> and bin <dig> leaf cells was partially restored by overexpression of osr <dig> .
these findings suggest that osr <dig> may also be involved in br-related cell expansion, further supporting the likely functional redundancy between osr <dig> and arl during organ growth.
osr genes cooperatively regulate cell proliferation and expansion during organogenesis
our previous work revealed that argos and osr <dig> regulated organ growth by primarily affecting cell proliferation in a redundant manner  <cit> .
to examine the cooperative role of the osr family members in organ growth, we generated an osr <dig> argos- <dig> arli triple mutant and an argos- <dig> osr <dig> arli osr2i quadruple mutant by introducing a pro35s:osr <dig> rnai construct into argos- <dig> osr <dig> arli plants .
qrt-pcr analysis confirmed that the expression of arl was knocked down in the osr <dig> argos- <dig> arli triple mutant, and the expression of both arl and osr <dig> transcripts was decreased in the argos- <dig> osr <dig> arli osr2i quadruple mutant .
as expected, the introduction of argos mutation to osr <dig> arli plants further reduced the final size of their organs, and this reduction was enhanced in the quadruple mutant argos- <dig> osr <dig> arli osr2i plants .
cytological analysis of epidermal cells of the third leaves in these plants indicated that this reduction was primarily caused by a decrease in cell numbers , confirming the role of argos and osr <dig> in cell proliferation.
these differential roles of osr genes in cell proliferation or expansion were further verified by examining the kinetics of cell proliferation and expansion during organ growth in these genotypes .
notably, we observed that epidermal cells of leaves in the osr quadruple mutant were slightly smaller than those of the osr <dig> arli plants .
this is consistent with the previous finding that osr <dig> had some effects on cell expansion during organogenesis  <cit> .figure  <dig>
cooperative roles of
osr2
and other
osr
genes in cell proliferation and expansion.
morphology of 28-day-old wt, osr <dig> arli, osr <dig> argos- <dig> arli, and argos- <dig> osr <dig> arli osr2i plants.
bar, 1 cm.
the leaf area , epidermal cell area , and estimated epidermal cell number  of the third leaves of the 30-day-old plants described in .
at least five leaves were examined for each genotype; the data are shown as mean values ± se; the letters  indicate statistical significance  among the genotypes according to one-way anova testing .
the cellular kinetics of developing third leaves of wt and osr mutant plants.
the epidermal cell size  and estimated epidermal cell number  are shown as the ratio of mutant/wt from 9– <dig> day-old plants.
at least five leaves of each genotype were examined.
discussion
characterization of osr <dig> defines a new osr member involved in the regulation of organ growth
the development of an organ relies on the coordination of cell proliferation and expansion; these two different cellular events determine the overall organ size.
cell proliferation, which sets the cell number of an organ, plays an important role in organ growth and final size control  <cit> .
post-mitotic cell expansion, which determines the final cell size, is also critical for organ development and size regulation  <cit> .
recently, several genes that participate in the regulation of cell expansion and thus cell size have been also reported to affect final organ size, such as exp <dig>  rpt2a, tor, arl, and erbb- <dig> epidermal growth factor receptor binding protein  <dig>   <cit> .
here, we showed that osr <dig> participates in the regulation of cell expansion during organ growth.
osr <dig> was expressed in the organs or tissues undergoing cell expansion and elongation, and ectopic expression of osr <dig> increased overall organ size by enhancing the cell expansion rate.
although the loss-of-function mutant of osr <dig> had no obvious phenotype, our detailed examination revealed that osr <dig> acted redundantly with arl, another osr member that had been shown previously to affect cell expansion and thus final organ size  <cit> .
consistently, the expression pattern of osr <dig> in various organs and the response of osr <dig> expression to hormone treatment were quite similar to those of arl, supporting the supposition that these genes have functional redundancy in organ growth.
therefore, our work identified osr <dig> as a new regulator of plant organ growth and final organ size, thereby providing an alternative to use osr <dig> in efforts to manipulate biomass production in plants.
osr-mediated organ growth may represent an evolutionary mechanism of the cooperative regulation of cell proliferation and expansion
there are four osr genes in the arabidopsis genome, and phylogenetic analysis showed that these four genes likely originated from gene duplication events.
argos and arl were clustered into a group and osr <dig> and osr <dig> were clustered into a separate group  <cit> .
osr <dig> and osr <dig> are located adjacent in the arabidopsis genome.
surprisingly, argos and osr <dig> are expressed predominately in organs or tissues undergoing growth by cell proliferation, and they are known to act upstream of ant and redundantly regulate the duration of cell proliferation and thus the final organ size  <cit> .
osr <dig> and arl are expressed primarily in organs and tissues in which cell expansion/elongation is occurring, and our work here demonstrates that these genes mediate the cell expansion process and thus organ size.
it is interesting that the different members of a co-evolved family function differentially in the regulation of cell proliferation or expansion, two fundamental cellular events that have long been considered to be separately controlled in multicellular organisms  <cit> .
therefore, osr-mediated organ growth might represent an evolved mechanism of cooperative control of cell division and expansion at the organ level.
since osrs were identified only in plants, and osr mutations only resulted in organ growth phenotypes, it is likely that the osr regulatory pathway represents a mechanism with which plants respond to growth signals or environmental cues to modify their growth and final organ size.
indeed, the osr genes were transcriptionally regulated by treatment with different hormones.
argos expression is known to be induced by auxin and cytokinin; osr <dig> expression is induced by ethylene, but repressed by aba and br; the expression of arl and osr <dig> is induced by br  <cit> ; zea mays argos <dig>  is known to regulate the final size of maize organs and thus contributes to increased yields, particularly in drought stress conditions  <cit> .
the molecular roles of osr family members during plant organogenesis
our characterization of osr members raises a critical question: how do the different members of the osr family participate in the regulation of two different cellular events?
more importantly, although cell division and cell expansion are strictly coordinated at both the cellular and organ levels, the two processes are generally considered to be controlled by the different mechanisms  <cit> .
there has been little evidence so far that these two events share the same molecular machinery.
moreover, our previous work demonstrated that the osr domain was sufficient to promote the organ growth, and that osr <dig> also had some effect on the cell expansion process  <cit> .
obviously, the involvement of different osr genes in cell proliferation and/or expansion relies on their temporal-spatial expression in developing organs; argos and osr <dig> are expressed primarily in the cell proliferation phase while arl and osr <dig> are expressed primarily in the cell expansion phase  <cit> .
it still remains unclear how they facilitate the regulatory role in cell division or expansion with a similar osr domain.
our previous work showed that three osr proteins were likely localized to the er, and here we showed that osr <dig> is localized to plasma membrane.
we still do not know whether the er-localized osr proteins or the plasma membrane-localized osr <dig> are functional or processing forms.
because osrs are small proteins, we also cannot exclude the possibility that osrs may be functional as peptide signals.
finally, it is more likely that different osr members may differentially interact with the variety of proteins, which would in turn diverge the roles of osr in cell proliferation or expansion.
therefore, it will be critical to identify proteins that interact with osr proteins and to dissect and characterize their downstream targets.
such efforts will be necessary for elucidating the molecular mechanism underlying osr-mediated cell proliferation and expansion at the organ level.
CONCLUSIONS
in conclusion, we revealed that the arabidopsis osr <dig> participates in regulation of cell expansion during organogenesis and thus the organ size.
our findings of the differential and cooperative roles of osr genes in regulation of cell proliferation or expansion strongly suggest that osr-mediated organ growth may represent an evolutionary mechanism of the cooperative regulation of cell proliferation and expansion during plant organogenesis.

 for multicellular organisms, organ size is a fundamental attribute of body morphology. in animals, the final size of an organ is primarily determined by cell growth, proliferation, and apoptosis. these processes are primarily mediated by two major pathways: the target of the rapamycin pathway that regulates cell growth and the hippo pathway that coordinately controls cell growth, proliferation, and apoptosis. in plants, because cell apoptosis does not generally contribute to morphogenesis in most organs, the development of an organ to its characteristic size depends mainly on cell proliferation and expansion. recent studies in arabidopsis have identified a number of genes involved in the regulation of either cell proliferation or cell expansion that are now known to affect final organ size. however, many such factors appear to be involved in multiple pathways to affect cell number or size, suggesting that the mechanisms of organ-size control in plants are more complicated than those in animals. this might be partly because plants are immobile and have to be highly responsive to the ever-changing environments. during the development of plant determinate organs such as leaves and floral organs, cell proliferation and expansion occur at two distinct but overlapping phases. in a leaf, for example, at the early stages of development the cells within leaf primordia undergo a period of cell proliferation and differentiation; cell expansion subsequently begins at the leaf tip and proceeds progressively in a basipetal direction, a process during which the transition from cell proliferation to expansion is established. finally, the leaf grows by post-mitotic cell expansion and reaches its characteristic final size. recent studies in arabidopsis have suggested strongly that the timing of the transition from the cell proliferation to the cell expansion phase appears to be a critical determinant of overall organ size. this notion is supported by the large number of identified factors involved in organ-size control that are known to alter the duration of cell proliferation, and thereby alter total cell number and final organ size. for instance, some positive regulators that participate in organ-size control such as aintegumenta, auxin-regulated gene involved in organ size, and kluh/cyp78a can prolong the cell proliferation phase and thus increase cell numbers. there are some negative regulators such as da and enhance of da /big brother, had a role in restricting the period of cell proliferation and thus the organ size. furthermore, factors involved in the regulation of cell expansion during organ growth have been identified in arabidopsis, including the regulatory particle aaa-atpase 2a, expansin, argos-like, and target of rapamycin. these factors were shown to affect either the duration or the rate of cell expansion, and are thus known to alter the final size of cells and of organs. interestingly, there is a “compensatory mechanism” that coordinates cell proliferation and expansion during organ growth. for instance, the an mutant had fewer but larger cells than did wild-type plants, and further analysis indicated that the an cells seemed to generate and transmit an intercellular signal that could enhance post-mitotic cell expansion. it appears that when the cell number within an organ was decreased below a threshold level, the plants often triggered further post-mitotic cell expansion to compensate for the reduction in cell number. the mechanisms responsible for the coordination of cell proliferation and expansion during plant organogenesis are far from being understood. the arabidopsis organ size related family has been implicated in the regulation of aerial organ size. argos, the founding member of the osr homologues, controlled the growth of aerial organs, mainly through prolonging the duration of cell proliferation by promoting the continued expression of ant and cycd3;. however, argos-like, a close homolog of argos, was found to regulate the organ-size by affecting cell expansion. our recent work demonstrated that organ size related promoted organ growth by affecting both cell proliferation and expansion. osr argos, and arl all share a conserved osr domain that is sufficient to promote organ growth. moreover, these three osr genes are differentially regulated by various plant hormones, suggesting that they may mediate different signals to affect the cell proliferation and/or expansion process. indeed, a recent study showed that zea mays argos had a function similar to that of argos. overexpression of zar in maize enhanced maize organ growth, increased yield, and improved tolerance to drought stress. osr genes have only been identified in plants, and it is very interesting that different osr members with a similar osr domain could mediate cell proliferation and/or cell expansion, the two different cellular events that have been widely considered to be separately controlled. there are four osr homologues in the arabidopsis genome, and these were likely generated by gene duplication events. to gain further insight into the evolutionary function of the osr genes, we investigated the role of the fourth osr gene, osr during organ growth. here, we report that osr participates in the regulation of organ growth by primarily affecting cell expansion in a manner redundant with arl. further analysis of osr argos- arli triple and argos- osr arli osr2i quadruple mutants revealed that these four osr genes have redundant and cooperative roles in the regulation of cell proliferation and/or expansion. our characterization of functional divergence among these four osr genes implies a novel mechanism underlying the coordinative regulation of cell proliferation and expansion during plant organogenesis.  osr is expressed primarily in organs undergoing growth by cell expansion we previously reported that arabidopsis osr was involved in the regulation of organ growth and that it contributes to final organ size primarily through enhancement of cell proliferation. we also showed that three identified osr proteins shared a conserved osr domain with an identical lpplpppp motif and two putative transmembrane helices, which was sufficient to promote organ growth. the arabidopsis thaliana genome also has another gene, at2g which encodes a protein of only amino acids that phylogenetically belongs to the osr member. careful alignment of its amino acid sequence with those of osr argos, and arl showed that at2g had the conserved lpplpppp motif and the c terminal transmembrane helix of the osr domain. however, it lacked the n terminal transmembrane helix but instead had a plasma membrane-localized signal peptide predicted by phobius and ipsort. moreover, at2g is located alongside osr in the genome, suggesting that these two genes may originate from a gene duplication event. therefore, we designated at2g as organ size-related. to explore whether or not osr is functional during organogenesis, we initially monitored osr expression in various organs with qrt-pcr analysis. as shown in figure 1a, osr was expressed primarily in the expanding organs, including leaf, inflorescence, and flower. we then examined the tissue-specific expression of osr using the t independent transgenic plants carrying an osr promoter:β-glucuronidase construct. in two-week-old seedlings, high gus expression was observed in the organs or organ regions where active cell expansion and elongation were occurring, such as the upper parts of leaves, and the elongating regions of lateral roots; abundant expression was also detected in the cotyledons. however, weak or nearly absent gus signals were observed in juvenile leaves, shoot, and root tips, where cells were predominantly undergoing the cell proliferation process. in the inflorescence and developing floral organs, gus staining was mainly detected in buds, sepals, and filaments. careful examination of gus expression in developing first leaves showed that weak gus signal was detected at the juvenile stage, while abundant gus staining was observed when a leaf was undergoing expanding growth. subsequently, the gus signal was almost disappeared in fully-expanded leaves. these observations indicate that osr is expressed predominantly in organs that are undergoing cell expansion growth.figure expression of osr2. expression of osr in various organs assayed by qrt-pcr. the data were from three biological replicates and are presented as mean values ± se. rt, root; st, stem; dl, dividing leaf; el, expanding leaf; ol, old leaf; in, inflorescence; fl, flower. expression patterns of osr assayed by gus staining of transgenic plants carrying a proosr2:gus construct. gus staining was shown in seedling, shoot, lateral root, and the floral organs. bars, 2 mm. since the three previously-identified osr members were known to be er-localized, we further examined the cellular localization of osr in leaf epidermal cells of proosr2:osr2-gfp transgenic plants. unlike other osr proteins, the osr2-gfp fusion protein signal was detected in the plasma membrane, indicating that osr is plasma membrane-localized. ectopic expression of osr enlarges organs primarily by enhancing cell expansion to investigate the role of osr in organ development, we generated transgenic arabidopsis thaliana plants harboring a pro35s:osr construct. all of the t transgenic lines overexpressing osr exhibited, to varying extents, enlarged organs as compared with the empty vector control plants. careful examination of three of the independent t homozygous lines showed that the ectopic expression of osr resulted in an obviously increased size of various organs such as leaves, cotyledons, roots, floral organs, and siliques. detailed quantification of fully expanded third leaves revealed that the average blade areas of the pro35s:osr l l and l plants were increased by,, and %, respectively, as compared with those of control plants. qrt-pcr analysis verified that the increased sizes of organs were indeed closely associated with the elevated osr expression in these transgenic lines. the transgenic plants overexpressing osr also displayed longer roots, hypocotyls, siliques, and petioles, and the plant heights were also increased in these plants as compared with the controls. additionally, the flowering time in osr transgenic plants was delayed by about four days.figure morphological and cytological characterization of pro35s:osr2 transgenic plants. morphology of 28-day-old control and three independent lines of pro35s:osr transgenic plants. bar, 1 cm. blade areas of fully-expanded third leaves of control and three pro35s:osr transgenic lines. at least five leaves for each genotype were examined; data are shown as mean values ± se; student’s t-test: **p < . qrt-pcr analysis of osr expression in control and transgenic plants presented in. the data were from three biological replicates and are presented as mean values ± se; student’s t-test: **p < . the cell area and estimated cell number of epidermal cells in fully-expanded third leaves of control and three pro35s:osr transgenic plants described in. at least five leaves were examined for each line, and data are shown as mean values ± se; student’s t-test: **p < . bar, 50 μm. the cell area and estimated cell number of mesophyll cells in fully-expanded third leaves of control and pro35s:osr transgenic plants. bar, 50 μm. data are shown as mean values ± se; student’s t-test: **p < . we then used leaves and cotyledons as representative organs to investigate the contribution of cell proliferation and cell expansion to the enlarged organs of the p35s:osr plants. as shown in figure 2d and e, as compared with those of the control leaves, the average size of the epidermal cells in fully-expanded third leaves of l l and l plants was increased by,, and %, respectively. the estimated numbers of epidermal cells per leaf were not obviously altered in the transgenic plants. similarly, mesophyll cells in p35s:osr leaves were enlarged, whereas the estimated cell numbers did not differ from the control leaves. consistently, ectopic expression of osr resulted in larger mesophyll cells in cotyledons, and again the mesophyll cell number in cotyledons did not differ from the control. these observations demonstrate that the enlarged organs in p35s:osr resulted primarily from enhanced cell expansion rather than cell proliferation. osr affects the cell expansion rate during organ growth to define the exact role of osr in cell expansion rate and duration, we further compared the expansion kinetics of epidermal cells in both the tips and the basal regions of the third leaves in control and p35s:osr l plants. as shown in figure 3a and b, the cell expansion rates of both the tips and the bases of p35s:osr leaves were indeed much higher than those of controls from day to day whereas the cell expansion rates in the two genotypes were almost the same after day 21; cell expansion ceased after day these observations strongly suggest that ectopic expression of osr mainly affects the rate of cell expansion rather than the duration of cell expansion phase.figure osr2 increases the rate of cell expansion in developing leaves. relative cell expansion rate of the epidermal cells at the tip and base of the third leaves of control and pro35s:osr transgenic plants. at least five leaves from each genotype were examined. nuclear polyploidization analysis of the leaf cells in the control and l pro35s:osr transgenic lines. fully-expanded blades of the third leaves were used for cell nuclear ploidy analysis with a flow cytometer. the percentages of cells with different nuclear polyploidy levels were observed from three independent biological duplicates, and are shown as mean values ± se. as cell endoreduplication is known to contribute to final cell size, we investigated whether the enlarged cells in the p35s:osr leaves were attributable to alteration of endoreduplication. flow cytometry analysis of the nuclei of fully expanded leaves of 27-day-old plants showed that, although the percentages of cells from 4c and 8c were slightly different between the two genotypes, the overall distribution of cells from 2c to 16c remained comparable between two genotypes. this result indicates that osr2-mediated cell expansion is not related to the nuclear dna endoreduplication of leaf cells. osr functions redundantly with arl to mediate cell expansion to further investigate the role of osr in organ growth, we obtained the t-dna insertion mutant osr from the arabidopsis biological resource center, in which a t-dna fragment was inserted into the exon of osr. rt-pcr analysis showed that osr was an osr knock-out mutant. however, osr plants didn’t show an obvious reduction in organ size when compared with wild-type plants. since the tissue-specific expression pattern of osr was quite similar to that of arl, we speculated that osr may have functional redundancy with arl. to test this, we introduced an arl specific rna-interference construct into both wt and osr plants to generate arli and osr arli plants. qrt-pcr analysis validated that the expression of arl was obviously knocked down in the independent t lines. the final size of the third leaf of the arli transgenic plants was reduced by about 9% as compared with that of either the wt or osr by contrast, leaf size in osr arli plants l and l was decreased by about and %, respectively. consistently, careful examination of the number and size of epidermal cells in the third leaf confirmed that the knockout of osr led to size reduction of epidermal cells in arli plants but had no obvious effect on cell number. consistently, transgenic plants harboring a 35s promoter-driven specific osr rnai construct did not exhibit any organ-size phenotype. however, introduction of a pro35s:osr rnai construct into arli plants enhanced the size reduction of leaves, confirming that osr acts redundantly with arl in the regulation of cell expansion during organ growth.figure osr2 acts redundantly with arl. morphology and the size of third leave of 26-day-old wt, osr arli, and osr arli plants. at least five fully-expanded leaves of each genotype were used for determination of the leaf size; the data are shown as mean values ± se; student’s t-test: *p < , ***p < . bars, 2 mm. the epidermal cell area and estimated cell number of the third leave described in. data were from at least five leaves of each genotype and are shown as mean values ± se; student’s t-test: *p < , **p < . as the three previously-identified osr genes are known to be differentially responsive to various hormones, we also investigated the transcriptional regulation of osr by plant hormones. interestingly, the qrt-pcr analysis of seedlings treated with various hormones revealed that, similar to arl, osr was induced by epi-brassinolide, and this induction was disrupted in the br signaling mutants bri1- and bin. moreover, introduction of a p35s:osr construct into bri1- and bin could partially restore leaf and petiole growth, and the expansion defect in bri1- and bin leaf cells was partially restored by overexpression of osr. these findings suggest that osr may also be involved in br-related cell expansion, further supporting the likely functional redundancy between osr and arl during organ growth. osr genes cooperatively regulate cell proliferation and expansion during organogenesis our previous work revealed that argos and osr regulated organ growth by primarily affecting cell proliferation in a redundant manner. to examine the cooperative role of the osr family members in organ growth, we generated an osr argos- arli triple mutant and an argos- osr arli osr2i quadruple mutant by introducing a pro35s:osr rnai construct into argos- osr arli plants. qrt-pcr analysis confirmed that the expression of arl was knocked down in the osr argos- arli triple mutant, and the expression of both arl and osr transcripts was decreased in the argos- osr arli osr2i quadruple mutant. as expected, the introduction of argos mutation to osr arli plants further reduced the final size of their organs, and this reduction was enhanced in the quadruple mutant argos- osr arli osr2i plants. cytological analysis of epidermal cells of the third leaves in these plants indicated that this reduction was primarily caused by a decrease in cell numbers, confirming the role of argos and osr in cell proliferation. these differential roles of osr genes in cell proliferation or expansion were further verified by examining the kinetics of cell proliferation and expansion during organ growth in these genotypes. notably, we observed that epidermal cells of leaves in the osr quadruple mutant were slightly smaller than those of the osr arli plants. this is consistent with the previous finding that osr had some effects on cell expansion during organogenesis.figure cooperative roles of osr2 and other osr genes in cell proliferation and expansion. morphology of 28-day-old wt, osr arli, osr argos- arli, and argos- osr arli osr2i plants. bar, 1 cm. the leaf area, epidermal cell area, and estimated epidermal cell number of the third leaves of the 30-day-old plants described in. at least five leaves were examined for each genotype; the data are shown as mean values ± se; the letters indicate statistical significance among the genotypes according to one-way anova testing. the cellular kinetics of developing third leaves of wt and osr mutant plants. the epidermal cell size and estimated epidermal cell number are shown as the ratio of mutant/wt from 9– day-old plants. at least five leaves of each genotype were examined. discussion characterization of osr defines a new osr member involved in the regulation of organ growth the development of an organ relies on the coordination of cell proliferation and expansion; these two different cellular events determine the overall organ size. cell proliferation, which sets the cell number of an organ, plays an important role in organ growth and final size control. post-mitotic cell expansion, which determines the final cell size, is also critical for organ development and size regulation. recently, several genes that participate in the regulation of cell expansion and thus cell size have been also reported to affect final organ size, such as exp rpt2a, tor, arl, and erbb- epidermal growth factor receptor binding protein. here, we showed that osr participates in the regulation of cell expansion during organ growth. osr was expressed in the organs or tissues undergoing cell expansion and elongation, and ectopic expression of osr increased overall organ size by enhancing the cell expansion rate. although the loss-of-function mutant of osr had no obvious phenotype, our detailed examination revealed that osr acted redundantly with arl, another osr member that had been shown previously to affect cell expansion and thus final organ size. consistently, the expression pattern of osr in various organs and the response of osr expression to hormone treatment were quite similar to those of arl, supporting the supposition that these genes have functional redundancy in organ growth. therefore, our work identified osr as a new regulator of plant organ growth and final organ size, thereby providing an alternative to use osr in efforts to manipulate biomass production in plants. osr-mediated organ growth may represent an evolutionary mechanism of the cooperative regulation of cell proliferation and expansion there are four osr genes in the arabidopsis genome, and phylogenetic analysis showed that these four genes likely originated from gene duplication events. argos and arl were clustered into a group and osr and osr were clustered into a separate group. osr and osr are located adjacent in the arabidopsis genome. surprisingly, argos and osr are expressed predominately in organs or tissues undergoing growth by cell proliferation, and they are known to act upstream of ant and redundantly regulate the duration of cell proliferation and thus the final organ size. osr and arl are expressed primarily in organs and tissues in which cell expansion/elongation is occurring, and our work here demonstrates that these genes mediate the cell expansion process and thus organ size. it is interesting that the different members of a co-evolved family function differentially in the regulation of cell proliferation or expansion, two fundamental cellular events that have long been considered to be separately controlled in multicellular organisms. therefore, osr-mediated organ growth might represent an evolved mechanism of cooperative control of cell division and expansion at the organ level. since osrs were identified only in plants, and osr mutations only resulted in organ growth phenotypes, it is likely that the osr regulatory pathway represents a mechanism with which plants respond to growth signals or environmental cues to modify their growth and final organ size. indeed, the osr genes were transcriptionally regulated by treatment with different hormones. argos expression is known to be induced by auxin and cytokinin; osr expression is induced by ethylene, but repressed by aba and br; the expression of arl and osr is induced by br; zea mays argos is known to regulate the final size of maize organs and thus contributes to increased yields, particularly in drought stress conditions. the molecular roles of osr family members during plant organogenesis our characterization of osr members raises a critical question: how do the different members of the osr family participate in the regulation of two different cellular events? more importantly, although cell division and cell expansion are strictly coordinated at both the cellular and organ levels, the two processes are generally considered to be controlled by the different mechanisms. there has been little evidence so far that these two events share the same molecular machinery. moreover, our previous work demonstrated that the osr domain was sufficient to promote the organ growth, and that osr also had some effect on the cell expansion process. obviously, the involvement of different osr genes in cell proliferation and/or expansion relies on their temporal-spatial expression in developing organs; argos and osr are expressed primarily in the cell proliferation phase while arl and osr are expressed primarily in the cell expansion phase. it still remains unclear how they facilitate the regulatory role in cell division or expansion with a similar osr domain. our previous work showed that three osr proteins were likely localized to the er, and here we showed that osr is localized to plasma membrane. we still do not know whether the er-localized osr proteins or the plasma membrane-localized osr are functional or processing forms. because osrs are small proteins, we also cannot exclude the possibility that osrs may be functional as peptide signals. finally, it is more likely that different osr members may differentially interact with the variety of proteins, which would in turn diverge the roles of osr in cell proliferation or expansion. therefore, it will be critical to identify proteins that interact with osr proteins and to dissect and characterize their downstream targets. such efforts will be necessary for elucidating the molecular mechanism underlying osr-mediated cell proliferation and expansion at the organ level.  in conclusion, we revealed that the arabidopsis osr participates in regulation of cell expansion during organogenesis and thus the organ size. our findings of the differential and cooperative roles of osr genes in regulation of cell proliferation or expansion strongly suggest that osr-mediated organ growth may represent an evolutionary mechanism of the cooperative regulation of cell proliferation and expansion during plant organogenesis. 
these findings, together with our previous studies, strongly suggest that osr-mediated organ growth may represent an evolutionary mechanism for the cooperative regulation of cell proliferation and expansion during plant organogenesis. moreover, morphological and cytological analysis of triple and quadruple osr mutants verified that the four osr members differentially but cooperatively participate in the regulation of cell proliferation and cell expansion and thus the final organ size. our results reveal that osr is functional in the regulation of cell expansion during organ growth, which further implicates the involvement of osr members in the regulation of both cell proliferation and expansion and thus the final organ size. in plants, the growth of an aerial organ to its characteristic size relies on the coordination of cell proliferation and expansion. however, the mechanism underlying the cooperative and coordinative regulation of cell proliferation and expansion during organ growth remains poorly understood. arabidopsiscell expansioncell proliferationosrorgan sizeissue-copyright-statement© the author 2014 this study characterized a new arabidopsis organ size related gene, osr which participates in the regulation of cell expansion process during organ growth. these two different processes occur successively during organ development, with a period of overlap. osr was expressed primarily in tissues or organs undergoing growth by cell expansion, and the ectopic expression of osr resulted in enlarged organs, primarily through enhancement of cell expansion. we further show that osr functions redundantly with argos-like, another osr gene that regulates cell expansion in organ growth.
 in plants, the growth of an aerial organ to its characteristic size relies on the coordination of cell proliferation and expansion. these two different processes occur successively during organ development, with a period of overlap. however, the mechanism underlying the cooperative and coordinative regulation of cell proliferation and expansion during organ growth remains poorly understood.  this study characterized a new arabidopsis organ size related gene, osr which participates in the regulation of cell expansion process during organ growth. osr was expressed primarily in tissues or organs undergoing growth by cell expansion, and the ectopic expression of osr resulted in enlarged organs, primarily through enhancement of cell expansion. we further show that osr functions redundantly with argos-like, another osr gene that regulates cell expansion in organ growth. moreover, morphological and cytological analysis of triple and quadruple osr mutants verified that the four osr members differentially but cooperatively participate in the regulation of cell proliferation and cell expansion and thus the final organ size.  our results reveal that osr is functional in the regulation of cell expansion during organ growth, which further implicates the involvement of osr members in the regulation of both cell proliferation and expansion and thus the final organ size. these findings, together with our previous studies, strongly suggest that osr-mediated organ growth may represent an evolutionary mechanism for the cooperative regulation of cell proliferation and expansion during plant organogenesis. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords arabidopsiscell expansioncell proliferationosrorgan sizeissue-copyright-statement© the author 2014 