BACKGROUND
bellringer , also known as vaamana, pennywise, larson and replumless, is a member of the bell family of homeodomain transcription factors and functions in diverse processes in the development of arabidopsis <cit> .
blr was identified independently as a suppresser of the floral homeotic gene agamous
<cit> , as a factor required in fruit development for specification of the replum
<cit>  and as a gene necessary for normal phyllotactic patterning
<cit> .
bell transcription factors were shown to enhance dna binding of maize knotted <dig>
<cit> , the founding member of the knox <dig> gene family
<cit> , and blr was subsequently shown to act synergistically with the knox <dig> homeodomain transcription factor brevipedicellus
<cit> .
blr and knox <dig> proteins heterodimerize
<cit>  and, in nicotiana epidermal cell transient assays, blr translocates to the nucleus on binding to knox <dig> proteins
<cit> .
this event is likely to have evolved early in eukaryotic development as it is required for the haploid-diploid transition in the green algae chlamydomonas <cit> .
although the expression patterns of blr and knox <dig> overlap, they are not identical
<cit>  and blr and knox <dig> mutants show distinctive phenotypes, suggesting that blr function is unlikely to be confined to acting with knox <dig> proteins alone.
nevertheless, similarities are apparent, such as the short stature of both blr and bp plants
<cit>  and the ability of blr and bp to enhance weak alleles of shoot meristemless
<cit> , a knox <dig> gene required for shoot apical meristem maintenance
<cit> , which points to a redundant role for blr and bp in stem cell fate establishment
<cit> .
reports of genetic interactions between blr and bp mutants have, however, been conflicting, with the short stature phenotype of the bp blr double mutation being variously interpreted as additive
<cit> , synergistic
<cit> , or novel - because inflorescence elongation is observed in the single mutants but not in the double
<cit> .
phenotypic differences between blr and bp mutants are unlikely to result from redundancy between family members because multiple knockout lines within the knox <dig> and bell families exhibit diverse phenotypes.
for example, vegetative to reproductive transition defects are observed when blr mutants are combined with mutations in poundfoolish , the gene with which blr shares most similarity in arabidopsis <cit> , which are not observed in multiple knox <dig> mutants
<cit> .
similarly, multiple knox <dig> mutants do not resemble blr plants as one role of bp is repression of knat <dig> and knat <dig> <cit> .
blr is thus likely to have functions both dependent on and independent of knox <dig>
one knox <dig> independent function of blr may include control of phyllotaxy as similar defects are not present in knox <dig> mutants.
normal phyllotaxy requires correct patterning of cell divisions
<cit>  which depend in turn on strictly regulated cell-wall remodelling events
<cit>  themselves driven by mechanical constraints
<cit> .
primordia outgrowth is accompanied by the de-methyl-esterification of pectic polysaccharides in the cell wall, and perturbation of the methyl-esterification status of pectins within the meristem by altering expression levels of pectin methyl-esterase  <dig> has been shown to alter phyllotactic pattern
<cit> .
pme <dig> has been demonstrated to be negatively regulated by blr in the meristem dome and this interaction is essential for maintenance of phyllotaxy, as incorrect positioning of organ initiation in blr mutants is suppressed in blr pme <dig> double mutants.
the relationship between blr and pme <dig> appears to be context specific as blr promotes pme <dig> expression in internodes
<cit> .
the functions of bp and blr do overlap in vascular development.
typically in arabidopsis stems, vascular tissue is radially arranged in collateral bundles with xylem present towards the centre of the stem and phloem positioned towards the outside, such that the xylem, vascular meristem  and phloem are positioned along the mediolateral axis
<cit> .
xylem is characterised by the presence of large vessels for water transport and smaller xylary fibres, both of which have secondary cell walls with the adjacent procambium forming an arc of dividing cells.
within the stem, the vascular tissue represents a developmental series with most recently derived tissue at the top and the oldest at the base
<cit> .
a continuous vascular ring has been described in blr lines, a phenotype enhanced in bp blr double mutants
<cit>  by the appearance of undeveloped vascular bundles
<cit>  containing cells previously only observed occurring in small, isolated strips overlying some vascular bundles of bp plants
<cit> .
here we have exploited transgenic plants over-expressing blr as a tool to identify putative downstream targets of this transcription factor.
in addition to the previously described target of blr, pme <dig> <cit> , our microarray analysis has identified misregulation of many other genes encoding cell-wall-associated proteins following blr over-expression.
defects in cell wall metabolism can affect cell size
<cit>  and we therefore examined blr mutant vascular tissue, where cell size defects have previously been described
<cit> .
we uncovered a genetic interaction between blr, bp and revoluta , a homeodomain transcription factor of the hd-zipiii family that has previously been shown to specify adaxial identity, radial patterning in the stem
<cit>  and xylem differentiation
<cit> .
our results add further support to the notion
<cit>  that in addition to regulating genes at the shoot apex, blr plays a wider role in development through its control of the extracellular matrix.
RESULTS
over-expression of bellringer reduces organ size
over-expression studies have been informative in functional analysis of knox <dig> <cit> .
using a similar strategy, blr cdna was used to generate 35s::blr constructs from which blr over-expression  transgenic lines were generated.
plants were observed with short hypocotyls, small rosette leaves, narrow cauline leaves, and short siliques .
blr-ox plants also had shorter, thinner stems than wild type with more axiliary branches .
northern blot analysis showed eight lines to exhibit increased blr levels; blr transcript levels in two of these lines, blr-ox <dig> and blr-ox <dig>  are shown in figure
2a.
the organs of blr-ox lines were smaller than wild type, so differences in cell size and number were investigated in 7 day epidermal hypocotyl cells from blr-ox and wild type plants .
blr-ox epidermal cells were significantly shorter than controls ; similar reductions were observed in longitudinal sections of inflorescence stem epidermis  and transverse sections of pith parenchyma .
this reduction in cell size is thus likely to contribute to the smaller organ size of blr-ox plants.
other histological features, including those in vascular tissue were indistinguishable from wild type.
expression changes in blr-ox plants
to explore the changes in transcription underlying the blr-ox phenotype, transcripts from hypocotyls of blr-ox and control plants were subject to microarray analysis at stage  <dig>
<cit>  of development.
using the ‘significance analysis of microarrays’ strategy
<cit> ,  <dig> genes demonstrated consistent expression changes and acceptable spot confidence of which  <dig> had appropriate q-values .
<dig> genes demonstrated > <dig>  fold expression change .
three lines of evidence point to the microarray data reflecting gene expression changes in planta; first, blr was up-regulated  <dig>  fold in blr-ox compared to col- <dig> .
second, the expression of pme <dig>  previously shown to be negatively regulated by blr is reduced  <dig>  fold  and third, the promoters of  <dig> of the  <dig> genes showing the greatest fold change carried predicted blr consensus dna binding sites .
in contrast, the dna binding sequence for athb <dig>  which in common with blr has a  <dig> base pair consensus sequence, occurs in only  <dig> of the  <dig> promoter regions.
the array data was validated by testing expression of meri- <dig> , a xyloglucan endotransglucosylase which acts to hydrolyze glycosyl bonds, hprg , a hydroxyproline-rich plasma membrane localised glycoprotein, and lpt <dig>  and ltpv.
<dig> , both of which encode endomembrane localised lipid binding/transport proteins, by northern blot .
in all instances expression reflected the array data.
blr-ox
blr-ox
genes with putative roles in extracellular matrix regulation , altered expression in bp mutants
<cit>  or having a blr consensus binding site in the promoter  are shown.
1role, or putative role in modification of the extracellular matrix.
2reduced expression in bp compared to wild-type plants .
3increased expression in bp compared to wild-type plants .
4blr dna binding consensus sequence present in promoter.
expression values are presented as the mean ± standard error for three biological replicates.
to understand better the significance of transcriptional differences between blr-ox and control plants, a gene ontogeny  analysis was carried out.
strikingly, the only significant change proved to be in cell wall associated gene expression, which was enriched from  <dig> % in controls to  <dig> % in blr-ox lines  pointing to a role for blr in regulating cell wall metabolism or organisation.
changes in cell size are regularly accompanied by expression changes in cell wall associated genes and the go analysis thus draws together the gain-of-function phenotypes  and the array data .
lipid transfer proteins
<cit>  and peroxidases
<cit>  loosen cell walls and induce cell expansion; thus a  <dig> fold decrease in expression of ltp and a  <dig>  fold reduction in peroxidise expression in blr-ox plants  is consistent with the reduction in cell size observed in blr-ox lines.
pme <dig>  which is negatively regulated by blr , alters the gelling properties of cell walls
<cit> , while other genes with altered expression are implicated by homology to have roles in cell expansion.
for example members of the thaumatin family  have potential endo-β- <dig>  glucanase activity
<cit> .
extreme blr gain-of-function plants lack organ initiation
organised phyllotaxy, lacking in blr mutants
<cit>  requires accurate cell expansion and division during organogenesis
<cit> , processes reliant on close regulation of cell-wall formation and expansion
<cit> .
we therefore anticipated that phenotypes observed in blr-ox lines could be strengthened to include disruption of organ initiation due to altered cell wall metabolism, particularly as blr-ox lines analysed above exhibited only a relatively small expression increase .
furthermore, in some contexts, blr is localised in the cytoplasm, and its nuclear localisation is dependent on an interacting partner
<cit> .
plants were therefore generated expressing a steroid-inducible version of blr in the form of a blr-gr fusion regulated by the 35s camv promoter which would result in nuclear localisation of blr in a steroid inducible manner, showing independence of an interacting partner.
to confirm that expression changes of cell wall modifying enzymes following blr-gr induction were consistent with those observed in blr-ox lines, plants were subjected to dexamethasone  induction for 72 h, as maximum activity of direct transcriptional targets in this system has been demonstrated as occurring between  <dig> and 128 hours
<cit> .
following dex induction, expression of both meri- <dig> and exp <dig> was elevated , as in blr-ox lines  providing further evidence for blr regulation of the extracellular matrix.
when blr-gr seeds were germinated on 10 μm dex, only cotyledons were visible after 14 days , suggesting that high levels of blr in the nucleus lead to extremely limited growth.
this observation may explain the relatively low increases in blr expression in blr-ox as plants with higher expression levels may not have been recovered in the t <dig> generation.
seedlings germinated for 3 days in the absence of steroid and subsequently transferred to 10 μm dex , produced leaves with severely reduced expansion .
visible leaves no longer emerged after 21 days and the apex became enlarged , suggesting that growth continued in the absence of organ initiation.
sections cut through these enlarging blr–gri apices revealed an absence of lateral organ primordia or any other clearly differentiated cell types , suggesting that high levels of nuclear blr results in anisotropic growth.
normal organ initiation and phyllotaxy in arabidopsis requires interplay between blr repression of pme <dig> in the shoot apical meristem
<cit> , and loss of this repression during formation of organ primordia
<cit> .
loss of pme <dig> repression has been proposed to allow other cell wall modifying components to access the wall.
our results support this hypothesis for, in our blr-gri plants, blr would continue to negatively regulate pme <dig> with the consequent inhibition of organ initiation we observed.
organ initiation requires both the correct patterning of cell divisions
<cit>  and strictly regulated cell wall remodelling
<cit> .
the organisation of cell divisions in the apex has been shown to depend on mechanical pressures
<cit> , which are likely to be influenced by the cell wall
<cit> .
the current model for cell-wall remodelling during organ initiation at the apex holds that the breakage and annealing of cell-wall components is controlled by connections with microtubules, possibly through regulation of cellulose-microfibril length
<cit> .
treatment of arabidopsis apices with oryzalin, which depolymerises microtubules and prevents ordered division, results in a bulbous apex and loss of lateral-organ initiation when combined with disrupted auxin signalling
<cit> .
the cell wall plays a central part in the transmission of chemical and mechanical signals between cells, and the positioning of developmental cues at the apex of sunflowers is reported to be sensitive to mechanical interventions
<cit> .
our data suggest that blr modulates the expression of genes encoding cell wall associated proteins, and our observation that plants strongly over-expressing blr-gri fusions generate bulbous apices incapable of forming lateral organs is consistent with the view that closely-regulated cell wall development is essential for ordered initiation of lateral organs at the shoot apex.
rev enhances the bp blr phenotype
the array experiments identified genes involved in cell expansion to be misregulated in blr-ox plants.
expression changes in genes involved in cell expansion are also a feature of ectopic knox <dig>  expression in poplar stem vascular tissue
<cit> , and several putative blr targets identified in blr-ox arrays are reciprocally regulated in bp mutants
<cit>  .
bp is required for patterning the inflorescence stem as sectors of differentiation-defective epidermal, cortical and vascular cells are present in bp mutant stems
<cit> .
furthermore, bp negatively regulates biosynthesis of lignin
<cit> , a cell wall polymer, that is present in cells that have secondary walls including those of the vascular tissue.
despite exhaustive analysis, blr and wild type hypocotyls proved indistinguishable ; however we saw clear differences in the vascular tissue of the inflorescence stem where blr is expressed
<cit> .
loss of function blr phenotypes have been described in this region
<cit>  in the relatively newly formed vascular tissue at the top of the stem .
here blr mutants were found to form a continuous ring of vascular tissue, while bp blr double mutants demonstrated an enhanced blr phenotype as cells in the continuous vascular ring failed to differentiate and contained small, densely stained cells where xylem cells with secondary cell walls formed in wild type plants
<cit> .
we analysed vascular tissue at a later stage in development, adjacent to the first cauline leaf on the primary inflorescence stem.
blr vascular bundles were patterned differently from those of wild type, as the arc of procambial cells present in wild type appeared shallower in blr .
blr mutants with the strongest phenotypes also had vascular bundles lacking large xylem vessels and containing apparently only xylary fibres, suggesting that there was either a failure of vessel specification, or a defect in vessel cell size .
‘stripes’ of differentiation-defective cells overlying some vascular bundles in bp mutants gave rise to two distinct vascular morphologies ; within the stripes xylem differentiation was altered because as with blr mutants, xylem lacked large vessels, however, in contrast to blr mutants, many of the smaller cells had not generated secondary cell walls
<cit>  .
in areas outside the differentiation defective stripe, vascular patterning was indistinguishable from wild type .
bp blr vascular tissue has been described as reminiscent of that present in differentiation defective areas of bp mutants
<cit> .
in bp blr bundles outside the differentiation-defective stripe, small darkly staining cells as described by smith et al.
<cit> , were observed towards the centre of the stem  suggesting that these cells were derived early in vascular development.
however, xylem cells generated later in development  underwent significant differentiation in bp blr double mutants because secondary cell walls were present, but as with extreme blr mutants, all xylem cells were reminiscent of xylary fibres .
in contrast, we observed very few xylem cells with secondary walls in bp blr vascular bundles within the differentiation-defective tissue associated with the bp mutation, even at this late stage of development .
consequently, and consistent with other reports
<cit> , blr enhances bp in the context of the vascular tissue and this interaction is particularly evident in mature bp blr vascular tissue within differentiation-defective regions of the stem .
we reasoned that analysis of bp and blr mutant combinations in a background of altered xylem identity may reveal redundant functions of bp and blr.
rev has been associated with radial patterning in the stem
<cit>  and has previously been shown to be required for development of interfascicular fibres
<cit> , for xylem differentiation and maintenance of xylem cell number
<cit> .
the role of rev in the xylem is likely to be partially redundant as phenotypes appear enhanced in rev phb/+ or rev phv mutants
<cit> .
furthermore in the shoot apex, as with bp <cit>  and blr <cit> , rev enhances the phenotype of weak alleles of stm <cit>  suggesting that rev may have overlapping functions with blr.
we therefore examined inflorescence stem vascular tissue in bp, blr and rev combinatorial mutants.
within rev vascular bundles xylem tissue resembled wild type in that both vessels and parenchyma were present, however the arc of procambium was shallower as in blr plants , a phenotype also occurring in bp rev mutant vascular tissue, outside of differentiation defective tissue .
within differentiation defective tissue, the xylem cells were generally similar to those of bp single mutants except that the relatively few cells that developed secondary walls were extremely small compared with those of bp .
blr rev and blr vascular bundles were similar but, strikingly, the xylem in vascular bundles of blr bp rev triple mutants comprised small and darkly stained xylem cells, whether or not the bundles lay within the stripe of differentiation-defective cells .
in extreme cases, no cells with secondary walls were seen in vascular bundles .
genetic interactions between blr, rev and bp in the stem vary depending on tissue type.
vascular cell division between vascular bundles is one of the hallmarks of secondary growth .
bp and blr repress this process as bp blr mutants have a continuous ring of vascular tissue
<cit> .
however, rev appears to be required for the large increase in vascular tissue present in bp blr because bp blr rev lines demonstrate a reduction in vascular tissue compared to bp blr .
this observation supports a role for rev  in promoting secondary growth, a function that has previously been suggested for its poplar orthologue
<cit> .
blr, rev and bp do not have contrasting roles in regulating xylem differentiation.
the presence of xylem cells with secondary walls was reduced in double and triple mutant combinations  suggesting that blr, rev and bp act together in this process.
consequently, blr, rev and bp interactions are context dependent.
although the primary defect observed in blr-ox was one of reduction in cell size, similar to that observed in vascular tissue of blr mutant combinations, the relationship between cell size and expression of particular cell wall modifying genes is not always straightforward
<cit> .
for example, over-expression of an e2ff transcription factor causes a reduction in cell size, accompanied by a decrease in expression of expansins , a udp-glucose-glucosyl transferase  and meri- <dig> <cit>  .
however, over-expression of meri- <dig> is reported elsewhere as promoting precocious elongation
<cit> .
blr control of the extracellular matrix is thus likely to be similarly complex.
blr-ox plants have smaller cells than those of wild-type, but blr mutants, when combined with bp and rev also resulted in vascular tissue with smaller cells.
CONCLUSIONS
control of cell expansion is essential for normal plant development.
our results demonstrate that blr controls cell expansion and differentiation in vascular development in a process also involving bp and rev transcription factors.
microarray analysis of plants over-expressing blr indicates that blr targets many genes involved in cell wall regulation.
as dynamic control of the cell wall is essential for cell expansion, we suggest that the striking phenotypes of plants over- and under-expressing blr may result from the disruption of this process.
closely regulated cell wall dynamics are also required for a normal pattern of organ initiation at the shoot apex and this process has previously been shown to be regulated by blr repression of pme <dig> <cit> .
our experiments with blr-gr suggest that further transcriptional targets of blr may also play important parts in this process.

 bellringer, also known as vaamana, pennywise, larson and replumless, is a member of the bell family of homeodomain transcription factors and functions in diverse processes in the development of arabidopsis. blr was identified independently as a suppresser of the floral homeotic gene agamous, as a factor required in fruit development for specification of the replum and as a gene necessary for normal phyllotactic patterning. bell transcription factors were shown to enhance dna binding of maize knotted, the founding member of the knox gene family, and blr was subsequently shown to act synergistically with the knox homeodomain transcription factor brevipedicellus. blr and knox proteins heterodimerize and, in nicotiana epidermal cell transient assays, blr translocates to the nucleus on binding to knox proteins. this event is likely to have evolved early in eukaryotic development as it is required for the haploid-diploid transition in the green algae chlamydomonas. although the expression patterns of blr and knox overlap, they are not identical and blr and knox mutants show distinctive phenotypes, suggesting that blr function is unlikely to be confined to acting with knox proteins alone. nevertheless, similarities are apparent, such as the short stature of both blr and bp plants and the ability of blr and bp to enhance weak alleles of shoot meristemless, a knox gene required for shoot apical meristem maintenance, which points to a redundant role for blr and bp in stem cell fate establishment. reports of genetic interactions between blr and bp mutants have, however, been conflicting, with the short stature phenotype of the bp blr double mutation being variously interpreted as additive, synergistic, or novel - because inflorescence elongation is observed in the single mutants but not in the double. phenotypic differences between blr and bp mutants are unlikely to result from redundancy between family members because multiple knockout lines within the knox and bell families exhibit diverse phenotypes. for example, vegetative to reproductive transition defects are observed when blr mutants are combined with mutations in poundfoolish, the gene with which blr shares most similarity in arabidopsis, which are not observed in multiple knox mutants. similarly, multiple knox mutants do not resemble blr plants as one role of bp is repression of knat and knat. blr is thus likely to have functions both dependent on and independent of knox one knox independent function of blr may include control of phyllotaxy as similar defects are not present in knox mutants. normal phyllotaxy requires correct patterning of cell divisions which depend in turn on strictly regulated cell-wall remodelling events themselves driven by mechanical constraints. primordia outgrowth is accompanied by the de-methyl-esterification of pectic polysaccharides in the cell wall, and perturbation of the methyl-esterification status of pectins within the meristem by altering expression levels of pectin methyl-esterase has been shown to alter phyllotactic pattern. pme has been demonstrated to be negatively regulated by blr in the meristem dome and this interaction is essential for maintenance of phyllotaxy, as incorrect positioning of organ initiation in blr mutants is suppressed in blr pme double mutants. the relationship between blr and pme appears to be context specific as blr promotes pme expression in internodes. the functions of bp and blr do overlap in vascular development. typically in arabidopsis stems, vascular tissue is radially arranged in collateral bundles with xylem present towards the centre of the stem and phloem positioned towards the outside, such that the xylem, vascular meristem and phloem are positioned along the mediolateral axis. xylem is characterised by the presence of large vessels for water transport and smaller xylary fibres, both of which have secondary cell walls with the adjacent procambium forming an arc of dividing cells. within the stem, the vascular tissue represents a developmental series with most recently derived tissue at the top and the oldest at the base. a continuous vascular ring has been described in blr lines, a phenotype enhanced in bp blr double mutants by the appearance of undeveloped vascular bundles containing cells previously only observed occurring in small, isolated strips overlying some vascular bundles of bp plants. here we have exploited transgenic plants over-expressing blr as a tool to identify putative downstream targets of this transcription factor. in addition to the previously described target of blr, pme, our microarray analysis has identified misregulation of many other genes encoding cell-wall-associated proteins following blr over-expression. defects in cell wall metabolism can affect cell size and we therefore examined blr mutant vascular tissue, where cell size defects have previously been described. we uncovered a genetic interaction between blr, bp and revoluta, a homeodomain transcription factor of the hd-zipiii family that has previously been shown to specify adaxial identity, radial patterning in the stem and xylem differentiation. our results add further support to the notion that in addition to regulating genes at the shoot apex, blr plays a wider role in development through its control of the extracellular matrix.  over-expression of bellringer reduces organ size over-expression studies have been informative in functional analysis of knox. using a similar strategy, blr cdna was used to generate 35s::blr constructs from which blr over-expression transgenic lines were generated. plants were observed with short hypocotyls, small rosette leaves, narrow cauline leaves, and short siliques. blr-ox plants also had shorter, thinner stems than wild type with more axiliary branches. northern blot analysis showed eight lines to exhibit increased blr levels; blr transcript levels in two of these lines, blr-ox and blr-ox are shown in figure 2a. the organs of blr-ox lines were smaller than wild type, so differences in cell size and number were investigated in 7 day epidermal hypocotyl cells from blr-ox and wild type plants. blr-ox epidermal cells were significantly shorter than controls; similar reductions were observed in longitudinal sections of inflorescence stem epidermis and transverse sections of pith parenchyma. this reduction in cell size is thus likely to contribute to the smaller organ size of blr-ox plants. other histological features, including those in vascular tissue were indistinguishable from wild type. expression changes in blr-ox plants to explore the changes in transcription underlying the blr-ox phenotype, transcripts from hypocotyls of blr-ox and control plants were subject to microarray analysis at stage of development. using the ‘significance analysis of microarrays’ strategy, genes demonstrated consistent expression changes and acceptable spot confidence of which had appropriate q-values. genes demonstrated > fold expression change. three lines of evidence point to the microarray data reflecting gene expression changes in planta; first, blr was up-regulated fold in blr-ox compared to col-. second, the expression of pme previously shown to be negatively regulated by blr is reduced fold and third, the promoters of of the genes showing the greatest fold change carried predicted blr consensus dna binding sites. in contrast, the dna binding sequence for athb which in common with blr has a base pair consensus sequence, occurs in only of the promoter regions. the array data was validated by testing expression of meri-, a xyloglucan endotransglucosylase which acts to hydrolyze glycosyl bonds, hprg, a hydroxyproline-rich plasma membrane localised glycoprotein, and lpt and ltpv., both of which encode endomembrane localised lipid binding/transport proteins, by northern blot. in all instances expression reflected the array data. blr-ox blr-ox genes with putative roles in extracellular matrix regulation, altered expression in bp mutants or having a blr consensus binding site in the promoter are shown. 1role, or putative role in modification of the extracellular matrix. 2reduced expression in bp compared to wild-type plants. 3increased expression in bp compared to wild-type plants. 4blr dna binding consensus sequence present in promoter. expression values are presented as the mean ± standard error for three biological replicates. to understand better the significance of transcriptional differences between blr-ox and control plants, a gene ontogeny analysis was carried out. strikingly, the only significant change proved to be in cell wall associated gene expression, which was enriched from % in controls to % in blr-ox lines pointing to a role for blr in regulating cell wall metabolism or organisation. changes in cell size are regularly accompanied by expression changes in cell wall associated genes and the go analysis thus draws together the gain-of-function phenotypes and the array data. lipid transfer proteins and peroxidases loosen cell walls and induce cell expansion; thus a fold decrease in expression of ltp and a fold reduction in peroxidise expression in blr-ox plants is consistent with the reduction in cell size observed in blr-ox lines. pme which is negatively regulated by blr, alters the gelling properties of cell walls, while other genes with altered expression are implicated by homology to have roles in cell expansion. for example members of the thaumatin family have potential endo-β- glucanase activity. extreme blr gain-of-function plants lack organ initiation organised phyllotaxy, lacking in blr mutants requires accurate cell expansion and division during organogenesis, processes reliant on close regulation of cell-wall formation and expansion. we therefore anticipated that phenotypes observed in blr-ox lines could be strengthened to include disruption of organ initiation due to altered cell wall metabolism, particularly as blr-ox lines analysed above exhibited only a relatively small expression increase. furthermore, in some contexts, blr is localised in the cytoplasm, and its nuclear localisation is dependent on an interacting partner. plants were therefore generated expressing a steroid-inducible version of blr in the form of a blr-gr fusion regulated by the 35s camv promoter which would result in nuclear localisation of blr in a steroid inducible manner, showing independence of an interacting partner. to confirm that expression changes of cell wall modifying enzymes following blr-gr induction were consistent with those observed in blr-ox lines, plants were subjected to dexamethasone induction for 72 h, as maximum activity of direct transcriptional targets in this system has been demonstrated as occurring between and 128 hours. following dex induction, expression of both meri- and exp was elevated, as in blr-ox lines providing further evidence for blr regulation of the extracellular matrix. when blr-gr seeds were germinated on 10 μm dex, only cotyledons were visible after 14 days, suggesting that high levels of blr in the nucleus lead to extremely limited growth. this observation may explain the relatively low increases in blr expression in blr-ox as plants with higher expression levels may not have been recovered in the t generation. seedlings germinated for 3 days in the absence of steroid and subsequently transferred to 10 μm dex, produced leaves with severely reduced expansion. visible leaves no longer emerged after 21 days and the apex became enlarged, suggesting that growth continued in the absence of organ initiation. sections cut through these enlarging blr–gri apices revealed an absence of lateral organ primordia or any other clearly differentiated cell types, suggesting that high levels of nuclear blr results in anisotropic growth. normal organ initiation and phyllotaxy in arabidopsis requires interplay between blr repression of pme in the shoot apical meristem, and loss of this repression during formation of organ primordia. loss of pme repression has been proposed to allow other cell wall modifying components to access the wall. our results support this hypothesis for, in our blr-gri plants, blr would continue to negatively regulate pme with the consequent inhibition of organ initiation we observed. organ initiation requires both the correct patterning of cell divisions and strictly regulated cell wall remodelling. the organisation of cell divisions in the apex has been shown to depend on mechanical pressures, which are likely to be influenced by the cell wall. the current model for cell-wall remodelling during organ initiation at the apex holds that the breakage and annealing of cell-wall components is controlled by connections with microtubules, possibly through regulation of cellulose-microfibril length. treatment of arabidopsis apices with oryzalin, which depolymerises microtubules and prevents ordered division, results in a bulbous apex and loss of lateral-organ initiation when combined with disrupted auxin signalling. the cell wall plays a central part in the transmission of chemical and mechanical signals between cells, and the positioning of developmental cues at the apex of sunflowers is reported to be sensitive to mechanical interventions. our data suggest that blr modulates the expression of genes encoding cell wall associated proteins, and our observation that plants strongly over-expressing blr-gri fusions generate bulbous apices incapable of forming lateral organs is consistent with the view that closely-regulated cell wall development is essential for ordered initiation of lateral organs at the shoot apex. rev enhances the bp blr phenotype the array experiments identified genes involved in cell expansion to be misregulated in blr-ox plants. expression changes in genes involved in cell expansion are also a feature of ectopic knox expression in poplar stem vascular tissue, and several putative blr targets identified in blr-ox arrays are reciprocally regulated in bp mutants. bp is required for patterning the inflorescence stem as sectors of differentiation-defective epidermal, cortical and vascular cells are present in bp mutant stems. furthermore, bp negatively regulates biosynthesis of lignin, a cell wall polymer, that is present in cells that have secondary walls including those of the vascular tissue. despite exhaustive analysis, blr and wild type hypocotyls proved indistinguishable; however we saw clear differences in the vascular tissue of the inflorescence stem where blr is expressed. loss of function blr phenotypes have been described in this region in the relatively newly formed vascular tissue at the top of the stem. here blr mutants were found to form a continuous ring of vascular tissue, while bp blr double mutants demonstrated an enhanced blr phenotype as cells in the continuous vascular ring failed to differentiate and contained small, densely stained cells where xylem cells with secondary cell walls formed in wild type plants. we analysed vascular tissue at a later stage in development, adjacent to the first cauline leaf on the primary inflorescence stem. blr vascular bundles were patterned differently from those of wild type, as the arc of procambial cells present in wild type appeared shallower in blr. blr mutants with the strongest phenotypes also had vascular bundles lacking large xylem vessels and containing apparently only xylary fibres, suggesting that there was either a failure of vessel specification, or a defect in vessel cell size. ‘stripes’ of differentiation-defective cells overlying some vascular bundles in bp mutants gave rise to two distinct vascular morphologies; within the stripes xylem differentiation was altered because as with blr mutants, xylem lacked large vessels, however, in contrast to blr mutants, many of the smaller cells had not generated secondary cell walls. in areas outside the differentiation defective stripe, vascular patterning was indistinguishable from wild type. bp blr vascular tissue has been described as reminiscent of that present in differentiation defective areas of bp mutants. in bp blr bundles outside the differentiation-defective stripe, small darkly staining cells as described by smith et al., were observed towards the centre of the stem suggesting that these cells were derived early in vascular development. however, xylem cells generated later in development underwent significant differentiation in bp blr double mutants because secondary cell walls were present, but as with extreme blr mutants, all xylem cells were reminiscent of xylary fibres. in contrast, we observed very few xylem cells with secondary walls in bp blr vascular bundles within the differentiation-defective tissue associated with the bp mutation, even at this late stage of development. consequently, and consistent with other reports, blr enhances bp in the context of the vascular tissue and this interaction is particularly evident in mature bp blr vascular tissue within differentiation-defective regions of the stem. we reasoned that analysis of bp and blr mutant combinations in a background of altered xylem identity may reveal redundant functions of bp and blr. rev has been associated with radial patterning in the stem and has previously been shown to be required for development of interfascicular fibres, for xylem differentiation and maintenance of xylem cell number. the role of rev in the xylem is likely to be partially redundant as phenotypes appear enhanced in rev phb/+ or rev phv mutants. furthermore in the shoot apex, as with bp and blr, rev enhances the phenotype of weak alleles of stm suggesting that rev may have overlapping functions with blr. we therefore examined inflorescence stem vascular tissue in bp, blr and rev combinatorial mutants. within rev vascular bundles xylem tissue resembled wild type in that both vessels and parenchyma were present, however the arc of procambium was shallower as in blr plants, a phenotype also occurring in bp rev mutant vascular tissue, outside of differentiation defective tissue. within differentiation defective tissue, the xylem cells were generally similar to those of bp single mutants except that the relatively few cells that developed secondary walls were extremely small compared with those of bp. blr rev and blr vascular bundles were similar but, strikingly, the xylem in vascular bundles of blr bp rev triple mutants comprised small and darkly stained xylem cells, whether or not the bundles lay within the stripe of differentiation-defective cells. in extreme cases, no cells with secondary walls were seen in vascular bundles. genetic interactions between blr, rev and bp in the stem vary depending on tissue type. vascular cell division between vascular bundles is one of the hallmarks of secondary growth. bp and blr repress this process as bp blr mutants have a continuous ring of vascular tissue. however, rev appears to be required for the large increase in vascular tissue present in bp blr because bp blr rev lines demonstrate a reduction in vascular tissue compared to bp blr. this observation supports a role for rev in promoting secondary growth, a function that has previously been suggested for its poplar orthologue. blr, rev and bp do not have contrasting roles in regulating xylem differentiation. the presence of xylem cells with secondary walls was reduced in double and triple mutant combinations suggesting that blr, rev and bp act together in this process. consequently, blr, rev and bp interactions are context dependent. although the primary defect observed in blr-ox was one of reduction in cell size, similar to that observed in vascular tissue of blr mutant combinations, the relationship between cell size and expression of particular cell wall modifying genes is not always straightforward. for example, over-expression of an e2ff transcription factor causes a reduction in cell size, accompanied by a decrease in expression of expansins, a udp-glucose-glucosyl transferase and meri-. however, over-expression of meri- is reported elsewhere as promoting precocious elongation. blr control of the extracellular matrix is thus likely to be similarly complex. blr-ox plants have smaller cells than those of wild-type, but blr mutants, when combined with bp and rev also resulted in vascular tissue with smaller cells.  control of cell expansion is essential for normal plant development. our results demonstrate that blr controls cell expansion and differentiation in vascular development in a process also involving bp and rev transcription factors. microarray analysis of plants over-expressing blr indicates that blr targets many genes involved in cell wall regulation. as dynamic control of the cell wall is essential for cell expansion, we suggest that the striking phenotypes of plants over- and under-expressing blr may result from the disruption of this process. closely regulated cell wall dynamics are also required for a normal pattern of organ initiation at the shoot apex and this process has previously been shown to be regulated by blr repression of pme. our experiments with blr-gr suggest that further transcriptional targets of blr may also play important parts in this process. 
bellringer, one such transcription factor, is involved in diverse developmental processes in arabidopsis, acting in vascular differentiation, phyllotaxy, flower and fruit development. some were defective in cell size and demonstrated misregulation of genes predominantly affecting cell wall development. microarray analysis strongly indicates that the striking vascular phenotypes of blr bp rev triple mutants and plants over-expressing blr result from the misregulation of a suite of genes, targets of blr in wild type plants, that determine cell size and structure in the developing vasculature. cell wall dynamics are also affected in blr mutant plants, and blr has previously been shown to regulate vascular development in conjunction with bp. homeodomain transcription factors play critical roles in metazoan development. blr also has a redundant role in meristem maintenance. other lines with more extreme phenotypes failed to generate lateral organs, consistent with blr repressing transcription in the shoot apex. our data support a role for blr in ordering the shoot apex and, in conjunction with bp and rev, playing a part in determining the composition and organisation of the vascular system. cell wall remodelling underpins many of these processes, and blr has recently been shown to regulate expression of pectin methyl-esterase, a cell wall modifying enzyme in control of phyllotaxy. we have further explored the role of blr in plant development by analysing phenotypes and gene expression in a series of plants over-expressing blr, and generating combinatorial mutants with blr, brevipedicellus, a member of the knox family of transcription factors that has previously been shown to interact with blr, and the homeodomain transcription factor revoluta, required for radial patterning of the stem.
 homeodomain transcription factors play critical roles in metazoan development. bellringer, one such transcription factor, is involved in diverse developmental processes in arabidopsis, acting in vascular differentiation, phyllotaxy, flower and fruit development. blr also has a redundant role in meristem maintenance. cell wall remodelling underpins many of these processes, and blr has recently been shown to regulate expression of pectin methyl-esterase, a cell wall modifying enzyme in control of phyllotaxy. we have further explored the role of blr in plant development by analysing phenotypes and gene expression in a series of plants over-expressing blr, and generating combinatorial mutants with blr, brevipedicellus, a member of the knox family of transcription factors that has previously been shown to interact with blr, and the homeodomain transcription factor revoluta, required for radial patterning of the stem.  plants over-expressing blr exhibited a wide range of phenotypes. some were defective in cell size and demonstrated misregulation of genes predominantly affecting cell wall development. other lines with more extreme phenotypes failed to generate lateral organs, consistent with blr repressing transcription in the shoot apex. cell wall dynamics are also affected in blr mutant plants, and blr has previously been shown to regulate vascular development in conjunction with bp. we found that when bp and blr were combined with rev, a set of defects was observed that were distinct from those of bp blr lines. in these triple mutants xylem development was most strikingly affected, resulting in an almost complete lack of vessels and xylem parenchyma with secondary thickening.  our data support a role for blr in ordering the shoot apex and, in conjunction with bp and rev, playing a part in determining the composition and organisation of the vascular system. microarray analysis strongly indicates that the striking vascular phenotypes of blr bp rev triple mutants and plants over-expressing blr result from the misregulation of a suite of genes, targets of blr in wild type plants, that determine cell size and structure in the developing vasculature. 