BACKGROUND
lim proteins have mainly been described in animals in which they play key roles in a variety of important developmental processes  <cit> .
these proteins contain one or several  double zinc finger motifs, known as lim domains, which function by mediating protein-protein interactions  <cit> .
the three transcription factors, lin <dig> from caenorhabditis elegans <cit> , isl- <dig> from rat  <cit>  and mec- <dig> from c. elegans <cit> , from which the acronym lim is derived, operate during transcription in association with a dna-binding homeodomain.
lim domains have since been found in a wide variety of eukaryotic proteins of diverse functions.
lim domain-containing transcription factors without the homeodomain have also been described.
specifically, the lim-only protein lmo <dig> was found to act as a bridging molecule in assembly of the erythroid dna-binding complex  <cit> , while other lim-domain-containing proteins such as lim kinases are known to participate in regulation of actin dynamics through phosphorylation of cofilin  <cit> .
in contrast, plants possess two distinct sets of lim proteins, one that is plant–specific and has been partially functional characterized  <cit> .
another, cysteine rich protein -like that comprises crps exhibiting the same overall structure found in animals   <cit> .
plants have evolved mechanisms to exploit, survive, or minimize the negative impact of a diverse range of environmental factors including potential pathogens, and in many cases the plant cytoskeleton is instrumental in mediating the plant’s response.
changes in organization of the plant cytoskeleton during plant interactions with these factors are complex and varied, and much still remains to be elucidated, especially in terms of the molecules that signal and induce the dramatic reorganizations that are often observed.
physical and chemical barriers resulting from actin-dependent cytoplasmic aggregation, secretion, and papilla formation appear to constitute an important and probably ancient form of basal resistance to pathogen attack  <cit> .
reorganization of cytoskeletal elements may also be a mechanism responsible for abiotic stress responses.
for example, cytoskeletal elements translocate chloroplasts under high light conditions  <cit> , facilitate gravity sensing  <cit> , and direct cellular response to wounding  <cit> .
a full understanding of the cytoskeletal basis of cytoplasmic aggregation will require identification and characterization of proteins involved in the signaling pathways those induce cytoskeletal rearrangement and responsible proteins for bringing about cytoskeletal reorganization and their function.
this mechanism utilizes regulatory proteins that generate intracellular motility, such as myosin  <cit>  and thick microfilament bundles focusing on the site of infection, such as the actin-bundling proteins villin and fimbrin  <cit> .
in the cytoplasm, actin monomers polymerize into actin filaments , which constitute the core elements of the actin cytoskeleton, providing mechanical support to the cytoplasm and serving as tracks for myosin-dependent intracellular transport  <cit> .
to fulfill its various roles, the actin cytoskeleton requires a sophisticated regulatory system to control its organization and dynamics at both spatial and temporal levels.
primary components of this system are the actin binding proteins  that directly interact with monomeric and/or polymerized actin to promote af nucleation, polymerization, depolymerization, stabilization, severing, capping, and cross-linking  <cit> .
recently, a number of vertebrate lim domain proteins belonging to the cys-rich protein  family and several structurally related plant proteins  have been shown to function as abps .
in arabidopsis, two distinct lim gene subfamilies have been reported.
the first subfamily contains six genes, which encode proteins with two lim domains that share similarity with animal crps  <cit> .
these lim domain-containing proteins bind actin filaments and influence actin cytoskeleton organization  <cit> .
in vitro, chicken crp <dig> and tobacco  wlim <dig> bind directly to afs to trigger the formation of thick actin bundles.
importantly, over-expression of crp <dig> and wlim <dig> proteins has been shown to be sufficient to significantly increase the bundling of afs in rat fibroblasts and tobacco cells, respectively  <cit> .
the genes in the second subfamily encode da <dig> and da <dig> related  proteins with a single lim domain and are specific to plants  <cit> .
da <dig> encodes a putative ubiquitin receptor with two ubiquitin interacting motifs  and a single lim domain, while e.g.
dar <dig> contains a single lim domain and a putative zinc-binding domain but lacks uims suggesting a possible functional divergence with da <dig>  <cit> .
the members of this family are involved in diverse functions e.g.
seed and organ size control, root meristem size control, resistance signaling and cold response  <cit> .
in this study, we retrieved  <dig> brassica rapa lim genes from the brassica database, analyzed the sequences and studied their homology with lim genes of other species.
we also analyzed their phylogenetic classification and discussed their correlation with organ specific expression.
expression of these genes was also analyzed following application of different biotic and abiotic stresses and their association was discussed with biotic and abiotic stress resistance in brassica.
RESULTS
sequence analysis and phylogenetic classification
we identified  <dig> lim proteins, designated as b. rapa lim , after which the sequences were analyzed .
the predicted size of the  <dig> brlims ranged from  <dig> to  <dig> amino acids , and the predicted isoelectric points varied from  <dig>  to  <dig> .
analysis of the protein domain organization showed that proteins contained the characteristic lim domain in the conserved region.
additionally, genomic dna sequence of the  <dig> brlim genes was determined from the b. rapa chromosome sequences and the introns and exons were identified by sequence analysis.
structural information pertaining to the  <dig> brlim genes is presented in additional file 1: figure s <dig>  this analysis confirmed that the identified genes code for lim domain containing proteins.
a blast search of the ncbi database conducted for comparison of these brlims with lim genes of other species revealed that the deduced amino acid sequences of  <dig> brlims shared high homology, primarily with lims of a. thaliana and some other homologous species .
pairwise amino acid sequence comparisons among these  <dig> brlim proteins were also calculated using blast of the ncbi database and shown in additional file 2: table s <dig>  the similarity within these brlim protein sequences ranged from  <dig> to 99% and  <dig> brlims showed above 90% similarity within the species indicating their probable duplication.table  <dig>
in silico
analysis of
lim
genes collected from the
brassica
database
a
brlim1
brlim2
brlim3
brlim4
brlim5
brlim6
brlim7
bra014721c
brlim8
brlim9
bra000154c
brlim10
brlim11
brlim12
brlim13
brlim14
brlim15
d
brlim16
d
brlim17
brlim18
brlim19
e
brlim20
brlim21
brlim22
a
brassica database , bgenbank accession number, c
brassica database accession number, dadditional  <dig> uim domain and eadditional  <dig> uim domain.
homology analysis of
lim
and da1-related  genes of
brassica rapa
a
brlim1
brplim2a
arabidopsis thaliana
nicotiana tabacum
brlim2
a. thaliana
populus tremula x populus alba
brlim3
arabidopsis lyrata subsp.
lyrata
p. tremula x populus alba
brlim4
a. thaliana
a. lyrata subsp.
lyrata
brlim5
brwlim2a
a. lyrata subsp.
lyrata
a. thaliana
brlim6
brwlim2b
a. lyrata subsp.
lyrata
n. tabacum
brlim7
brwlim2c
a. lyrata subsp.
lyrata
p. tremula x populus alba
brlim8
brwlim2d
a. thaliana
hevea brasiliensis
brlim9
brwlim2e
a. lyrata subsp.
lyrata
p. tremula x populus alba
brlim10
brwlim1a
a. thaliana
brassica napus
brlim11
brwlim1b
b. napus
a. thaliana
brlim12
brdar1
a. thaliana
a. thaliana
brlim13
brdar2
a. thaliana
medicago truncatula
brlim14
brdar3
a. thaliana
theobroma cacao
brlim15
brdar4
a. thaliana
t. cacao
brlim16
brdar5
a. thaliana
m. truncatula
brlim17
brdar6
a. thaliana
m. truncatula
brlim18
brdar7
a. thaliana
m. truncatula
brlim19
brdar8
a. thaliana
a. thaliana
brlim20
brplim2e
a. thaliana
n. tabacum
brlim21
brplim2f
a. lyrata subsp.
lyrata
a. thaliana
brlim22
brplim2g
a. thaliana
p. tremula x populus alba
aanalyzed using blast from ncbi, http://www.ncbi.nlm.nih.gov/blast/.
we also retrieved all lim family protein sequences of a. thaliana, populus trichocarpa and oryza sativa from ncbi and constructed a phylogenetic tree with  <dig> deduced amino acid sequences of brlim using the nj method .
in the phylogenetic tree, previously identified lim <dig> and lim <dig> groups  <cit>  were clearly separated and the plant lim family was further divided into four groups, αlim <dig>  βlim <dig>  γlim <dig>  and δlim <dig>  resulting from division of the lim <dig> and lim <dig> groups.
this phylogenetic analysis confirms the existence of the plim <dig>  wlim <dig>  plim <dig>  and wlim <dig> subgroups as previously described  <cit> .
the αlim <dig> group includes the plim <dig> and wlim <dig> subgroups, while βlim <dig> is a newly identified group  <cit> .
the wlim <dig> and plim <dig> subgroups belong to the γlim <dig> and δlim <dig> groups, respectively.
among the  <dig> brlims, brlim <dig> and  <dig> were clustered with wlim <dig> subgroup under the αlim <dig> group.
brlim <dig>   <dig>   <dig>   <dig> and  <dig> were separated with the wlim <dig> subgroup under the γlim <dig> group.
moreover, brlim <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> separated with the plim <dig> subgroup under the δlim <dig> group, showing close relationships with the lim sequences of other species.
multiple alignments among members of the wlim <dig>  wlim <dig>  plim <dig> and dar groups also showed clearly distinct amino acid residues conserved in the domains and other regions of their protein sequences .
in arabidopsis, lim proteins are encoded by genes from two distinct subfamilies, one encoding proteins with two lim domains that are homologous to animal crp proteins  <cit> .
consequently, highly homologous wlim <dig>  plim <dig>  and wlim <dig> proteins of b. rapa of this subfamily also contain two lim domains except brlim <dig>  interestingly, with two lim domains n-terminal extensions were observed in brlim <dig> and  <dig> protein sequences and brlim <dig> had additional  <dig> transmembrane domains in the n-terminal extension .
these n-terminal extensions suggest probable misannotation or divergent functions of these two genes which require further experimental evidence.figure  <dig>
phylogenetic tree showing the relatedness of the deduced full-length amino acid sequences of  <dig> brlim proteins and all lim family proteins of
a. thaliana
,
populus trichocarpa
and
oryza sativa
.
brlim proteins are indicated by square bullets.
the scale represents the frequency of amino acid substitutions between sequences as determined by the poisson evolutionary distance method.
a species acronym is added before each lim protein name: at, a. thaliana; pt, populus trichocarpa and os, oryza sativa.
the old nomenclature of lim genes are indicated under bracket.
details of each gene are given in additional file 3: table s <dig>
alignment of deduced amino acid sequences of a) lim and b) dar protein groups using pir.
solid boxes indicate uim and lim domains.
underline indicates highly conserved c-terminus.
asterisks show conserved cysteine and histidine residues  in the highly conserved lim domains and c-terminus.
numbers in the right margin indicate the positions of amino acid residues.
identical amino acids are indicated by a dark background, while > 50% similarities are indicated by a light background.
to investigate the existence of n-terminal extensions of brlim <dig> and  <dig>  we analyzed expression of both genes over different organs specifying forward and reverse primers at several locations of the sequences .
both genes showed expression, when forward  and reverse  primers were selected at the conserved parts.
conversely, no expression was found, when the forward primers  were selected at the extended parts and the reverse primers  at several locations of the conserved parts.
subsequently, 3’-race pcr using primer # <dig> of brlim <dig> and primer # <dig> of brlim <dig> also showed discontinuation of the transcription towards conserved parts and found to show polya tails indicating the end of the transcript.
alongside, genomic dna amplification specifying these transition portions of both genes revealed no sequence distortion.
thus, it is clear that the extended portions are not part of brlim <dig> and  <dig>  it was a wrong annotation of the splice sites in the original prediction.
the correct exons of brlim <dig> and  <dig> were then drawn in the postulated gene structures  and the primer combinations, # <dig> and # <dig> of brlim <dig> and # <dig> and # <dig> of brlim <dig> located at the corrected exons, were used in latter expression studies.
these corrected sequences were deposited in the genbank under the accession numbers, kj <dig>  and kj <dig> .
these corrected sequences are the basis for all further analysis and all figures and tables were corrected accordingly.
the genes in the second subfamily encode da <dig> and da1-related  proteins specific to plants contain a single conserved lim domain  <cit> .
interestingly, brlim <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were clustered into a new group, showing close relationships with eight da <dig> and dar sequences of a. thaliana and four dar sequences of o. sativa.
additionally, all members of this new group contain only one lim domain, while some also contain one or more uim.
therefore, brlim proteins were renamed according to their phylogenetic relationship as b. rapa dar  .
alignment of the motif sequences of the da <dig> and dar group showed very low similarity with wlim <dig>  wlim <dig> and plim <dig> sequences, indicating their distinctness from these groups .
however, the brdars were highly similar to da <dig> and dar sequences of a. thaliana and o. sativa, and these all contained similar patterns of motifs in their conserved regions.
da <dig> of a. thaliana contains two uims, a single lim domain and the highly conserved c-terminal region  <cit> .
in contrast, dar <dig> contains a single lim domain and a putative zinc-binding domain but lacks uims compared with da <dig> and dar <dig>  <cit> .
similarly, brdar <dig> and  <dig> contain two uims and a single lim domain, while brdar <dig> contains four uim and a single lim domain with highly conserved c-terminal region in their protein sequences.
other brdars contain only a single lim domain and highly conserved c-terminal regions .
thus, the brlim genes showed high similarity with corresponding groups of lim genes of different species, indicating their close evolutionary relationship.
organ specific expression analysis under unstressed condition
expression analysis was performed using specific primers with equal amounts of cdna templates prepared from the mrna of roots, stems, leaves and flower buds of healthy chinese cabbage plants without applying any stresses by rt-pcr and organ specific expression was observed.
specifically, brplim2d, brwlim2a, b, c, d, e, brwlim1a and b showed expression in all the organs tested.
brplim2a, b, c, brplim2e, f and g were only expressed in flower buds .
expression of the genes that were only expressed in flower buds was further analyzed during six developmental stages , and all genes except brplim2e showed higher expression in young buds .
this expression pattern and phylogenetic classification revealed two clear groups of brlim genes, with one group  only being expressed in flower buds and another group  being expressed in all vegetative parts of the chinese cabbage plants tested in this study.
thus, organ specific expressions of brlims showed clear correlations with phylogenetic grouping as well.figure  <dig>
expression analysis of 14
brlim
and 8
brdar
genes, a) over different organs, b) at six growth stages  of flower buds of
brassica rapa
‘sun-3061’ plants and c) microarray expressions of
da1
and seven
dar
genes of
arabidopsis thaliana
at  <dig> developmental stages.
lanes 1– <dig>  pcr products of roots , stems , leaves  and flower buds .
a-f, young to mature stages of flower buds.
dar subfamily genes of b. rapa showed a different expression pattern compared to other lim domain containing genes.
specifically, brdar <dig>   <dig> and  <dig> showed expression in all the organs tested.
brdar <dig>   <dig> and  <dig> showed expression in all organs except roots, while brdar <dig> was only expressed in flower buds.
noticeably, all brdar genes expressed highly in flower buds compared to other organs .
expression of brdar <dig> that was only expressed in flower buds was further analyzed during six developmental stages , and found higher expressions in young and middle aged buds .
microarray expression of da <dig> and dar genes of a. thaliana were also lower at different vegetative parts compared to floral organs and comparatively higher at young flower bud stages .
the plim- <dig> protein of sunflower appeared in the microspore stage in a limited number of cytoplasmic bodies, became undetectable in bicellular pollen, and reappeared in tricellular pollen grains in cortical patches, being particularly concentrated in the f-actin-enriched germination cones of the vegetative cell.
taken together, these findings suggest a dual function of plim- <dig> gene during pollen development  <cit> .
conversely, expression of ntplim <dig> of tobacco in germinating pollen suggests a possible role in pollen germination and/or pollen tube growth  <cit> .
lilim <dig>  an effective actin bundling protein, also plays an important role in pollen tube growth of lilium longiflorum <cit> .
likewise, the brplims expressed in young flower bud stage might play a role in the development of pollen tube.
earlier studies have also suggested the existence of two main lim gene subfamilies that differ in their expression patterns  <cit> .
again, based on phylogenetic analysis of  <dig> lims and comparison of the available expression data, a complex classification of lim genes has been proposed by arnaud et al.,  <cit> .
according to this classification, the arabidopsis lim gene family comprises three vegetative  and three reproductive  isoforms, of which, all plims have been shown to be expressed in pollen  <cit> .
ye et al.
<cit>  also suggested the critical roles of arabidopsis plims in actin configuration during pollen germination and tube growth.
specific control of plim actin regulatory activities by ph is particularly relevant with regard to the potential biological functions of these proteins in growing tip region of pollen tubes  <cit> .
expression analysis under stress conditions
biotic stress
biotic stress responses are a crucial issue for the brassica crops and functions of lim genes in biotic stress responses are not well studied yet.
we investigated the responses of the  <dig> brlim genes identified in this study to infection by f. oxysporum f.sp.
conglutinans in chinese cabbage after various times.
brwlim2c and brdar <dig> were found to be up-regulated with the time course of infection.
specifically, brdar <dig> showed very high responses in the late stages of fungal infection, while brwlim2c responded during earlier stages .
atwlim <dig>  atwlim2b, atdar <dig>  atdar <dig> and atdar <dig> genes also showed responses during microarray expression analysis after infection with f. oxysporum in a. thaliana, which were collected from public database .
we also analyzed the expression of all brlims after infection with the soft rot disease causing necrotroph bacteria, pectobacterium carotovorum subsp.
carotovorum; however, no differential expression was observed in chinese cabbage plants .
these results suggest probable involvement of brwlim2c and brdar <dig> with f. oxysporum f.sp.
conglutinans resistance and other brlim and brdar genes might also have association with resistance against other biotic stress agents in brassica crops.figure  <dig>
real-time quantitative pcr expression analysis of a)
brwlim2c
and b)
brdar4
after infection of chinese cabbage  with
fusarium oxysporum
f. sp.
conglutinans.
relative gene expressions in treated samples are presented as fold change compared to mock treated samples.
at each time point gene expression values in mock treated samples are set to  <dig>
plants encounter and must deal with a range of other organisms that may be potential partners or pathogens, and the plant cytoskeleton plays a key role in their response to such organisms.
cell wall appositions, or papillae, are important barriers formed by plants in defense against attempted penetration by fungal and oomycete pathogens  <cit> .
prior to the development of papillae, plant cytosol and subcellular components are rapidly translocated to the site of pathogen penetration via a mechanism known as cytoplasmic aggregation that is dependent on the action of the actin component of the cytoskeleton  <cit> .
this cytoplasmic aggregation has been observed in many plant-microbe interactions  <cit>  and is a common resistance response to pathogens by both dicotyledonous and monocotyledonous plants to invading filamentous pathogens.
microfilaments are the main cytoskeletal element responsible for penetration resistance based on cytoplasmic aggregation and papilla formation at the site of infection.
for example, focusing of actin microfilaments at the penetration site is more extensive in resistant plants than susceptible hosts in flax -melampsora and barley -erysiphe interactions  <cit> , suggesting that accumulation of material at the site of infection is related to the degree of host resistance.
some other previous observations suggest that inhibition of penetration through cytoplasmic aggregation and papilla formation is an early, if not the first, tactic in plant resistance, and that it may be backed up by the hypersensitive response  <cit> .
thus, the physical and chemical barrier resulting from actin-dependent cytoplasmic aggregation, secretion, and papilla formation appears to constitute an important and probably ancient form of basal resistance to pathogen attack  <cit> .
for a full understanding of the cytoskeletal basis of cytoplasmic aggregation, it is very essential to elucidate the role of responsible regulatory proteins that participate in other forms of cytoplasmic streaming, as well as additional factors specific for the localized defense response.
proteins those generate intracellular motility  are likely to be important components of such regulations  <cit> .
in addition, several crp-like plant lim proteins have been shown to function as abps , and lilim <dig> has been proposed as an effective abp, that plays an important role in pollen tube growth of l. longiflorum <cit> .
in vitro, tobacco  wlim <dig> binds directly to afs and triggers the formation of thick actin bundles.
importantly, overexpression of wlim <dig> proteins was sufficient to significantly increase the bundling of afs in tobacco cells  <cit> .
thus, actin binding and bundling function of lim genes could also be an important factor for actin-dependent resistance mechanism against pathogen infection.
in this study, high expression of brwlim2c gene against pathogen infection is tempting to speculate that brlim protein may also be implicated in the perception of pathogen resistance mechanism.
genes in the second subfamily encode da <dig> proteins, which are specific to plants.
the arabidopsis lim domain protein da <dig> was characterized to function as an ubiquitin receptor  <cit> .
ubiquitin is a highly conserved and wide-spread small protein modifier that is engaged in a wide range of cellular  <cit>  and biological processes, such as resistance to disease and abiotic stresses .
the consequences of ubiquitination are accusing the target protein to proteolysis or relocalization or endocytosis  <cit> .
da <dig> is inevitably involved in ubiquitination resulting extension of the cellular proliferation period, thus increasing cell numbers and ultimately plant organ size  <cit> .
our highly expressed brdar <dig> in response to pathogen infection might be involved in biotic stress resistance of b. rapa following aforesaid mechanism.
another member of this family chs3/atdar <dig> containing tir-nbs-lrr domain is associated with resistance signaling and cold response  <cit>  and atdar <dig> with controlling of root meristem size  <cit> .
alongside, no brdar protein contained tir-nbs-lrr domain like as chs3/atdar <dig>  however, reports on the responses of lim genes to biotic stresses are still lacking, and this is the first report of such possible role of lim domain containing genes against biotic stress caused by f. oxysporum f.sp.
conglutinans in chinese cabbage.
thus, brlim and brdar genes might be involved in biotic stress resistance related functions in b. rapa.
abiotic stresses
in addition to biotic stresses, expression of brlims was also analyzed in response to abiotic stress conditions such as cold, aba and ph stresses.
commonly ten brlims among them, showed differential expressions in response to the abiotic stresses applied in this study.
specifically, brplim2d, brwlim2a, b, c, d, e, brwlim1a, b, brdar <dig> and  <dig> showed differential expression after cold, aba and ph stress treatments compared to mock treated samples  of chinese cabbage .
brplim2d, brwlim2a, b and brdar <dig> were down-regulated in response to cold stress throughout the treatment period, while brwlim2c, d, e, brwlim1a, b and brdar <dig> were up-regulated during the first 8 h of treatment, after which they were down-regulated.
following aba stress treatments, brplim2d, brwlim2b, e and brdar <dig> were also down-regulated throughout the treatment period, while brwlim2a, c, d, brwlim1a, b and brdar <dig> were up-regulated during the first 8 h of treatment, after which they were down-regulated.
moreover, brplim2d, brwlim2a and brdar <dig> were down-regulated and brwlim1a, b brwlim2b and c were up-regulated throughout the treatment period in response to ph  <dig>   <dig> and  <dig> stress treatments compared to mock treated samples  which were maintained after transferring into a fresh medium with ph  <dig> .
brwlim2d and brwlim2e were down-regulated in response to ph  <dig> and  <dig>  while was up-regulated in response to ph  <dig>  brdar <dig> was down-regulated in response to ph  <dig> and up-regulated in response to ph  <dig> and  <dig>  interestingly, brplim2d and brdar <dig> were down-regulated and brwlim1a, b and brwlim2c were up-regulated in response to all abiotic stress treatments applied in this study.
atwlim <dig>  atwlim2a, atwlim2b, dar <dig>  dar <dig>  dar <dig> and dar <dig> genes also showed responses after cold stress treatments in a. thaliana during microarray expressions study collected from public database .figure  <dig>
real-time quantitative pcr expression analysis of
brlim
and
brdar
genes after a) cold, b) aba and c) ph stress treatments in chinese cabbage
.
relative gene expressions in treated samples are presented as fold change compared to mock treated samples.
at each time point gene expression values in mock treated samples are set to  <dig>
plants actin cytoskeleton is a major signaling target and in response to numerous abiotic and biotic stimuli, it changes dramatically in various ways, ranging from filament bundling, to massive actin depolymerization, to assembly of new filament arrays .
plant cells can detect mechanical stress on the epidermal cell surface and respond by reorganizing subcellular components like, actin microfilaments, endoplasmic reticulum  and peroxisomes in a manner similar to that induced during attack by potential fungal or oomycete pathogens  <cit> .
expression analysis of poplar lims in tension wood also suggested a connection between plant lims and mechanical stress  <cit> .
thus, rearrangement of cytoskeletal elements through actin binding and bundling may also be the mechanism of abiotic stress responses of the crp-like lim genes.
alongside, aforementioned ubiquitination mechanism of da <dig> and related proteins might also be implicated in abiotic stress resistance function of brdar proteins.
in a very recent study, expression of gmada <dig> gene of soybeans  showed responses to salt, drought, acid and alkali stresses and aba treatment  <cit> .
atda <dig> expression occurs in response to aba trteatment  <cit>  and chs3/atdar <dig> is associated with resistance signaling and cold response in a. thaliana <cit> .
supporting this report bi et al.
<cit>  hypothesized that disease resistance and environmental stress response pathways are intricately tied in plants, although the degree of overlap and the precise signaling events required for each pathway remain unclear.
thus, the results of this study indicate the likely involvement of the analyzed genes towards abiotic stress tolerance in brassica.
CONCLUSIONS
this present study identified  <dig> lim domain containing genes in b. rapa and classified into two distinct functional groups.
one group is plant–specific and designated as brdar, and another is crp-like designated as brlim.
these sequences were analyzed, compared them with lim domain containing genes of other plants and analyzed their expression after applying biotic and abiotic stresses in chinese cabbage.
overall, brplim2d, brwlim2a, b, c, d, e, brwlim1a, b, brdar <dig> and  <dig> responded after cold, aba and ph stress treatments in b. rapa, of which brwlim2c and brdar <dig> showed responses to f. oxysporum f.sp.
conglutinans infection as well.
thus, these eight brlims and two brdars might be promising candidates for multiple abiotic stresses resistance, while brwlim2c and brdar <dig> might be the most promising among them for both biotic and abiotic stresses resistance in brassica crops.
the remaining brlims and brdars might also be useful resources for induction of resistance to other biotic and abiotic stresses and/or various developmental processes of brassica.

 lim proteins have mainly been described in animals in which they play key roles in a variety of important developmental processes. these proteins contain one or several double zinc finger motifs, known as lim domains, which function by mediating protein-protein interactions. the three transcription factors, lin from caenorhabditis elegans, isl- from rat and mec- from c. elegans, from which the acronym lim is derived, operate during transcription in association with a dna-binding homeodomain. lim domains have since been found in a wide variety of eukaryotic proteins of diverse functions. lim domain-containing transcription factors without the homeodomain have also been described. specifically, the lim-only protein lmo was found to act as a bridging molecule in assembly of the erythroid dna-binding complex, while other lim-domain-containing proteins such as lim kinases are known to participate in regulation of actin dynamics through phosphorylation of cofilin. in contrast, plants possess two distinct sets of lim proteins, one that is plant–specific and has been partially functional characterized. another, cysteine rich protein -like that comprises crps exhibiting the same overall structure found in animals. plants have evolved mechanisms to exploit, survive, or minimize the negative impact of a diverse range of environmental factors including potential pathogens, and in many cases the plant cytoskeleton is instrumental in mediating the plant’s response. changes in organization of the plant cytoskeleton during plant interactions with these factors are complex and varied, and much still remains to be elucidated, especially in terms of the molecules that signal and induce the dramatic reorganizations that are often observed. physical and chemical barriers resulting from actin-dependent cytoplasmic aggregation, secretion, and papilla formation appear to constitute an important and probably ancient form of basal resistance to pathogen attack. reorganization of cytoskeletal elements may also be a mechanism responsible for abiotic stress responses. for example, cytoskeletal elements translocate chloroplasts under high light conditions, facilitate gravity sensing, and direct cellular response to wounding. a full understanding of the cytoskeletal basis of cytoplasmic aggregation will require identification and characterization of proteins involved in the signaling pathways those induce cytoskeletal rearrangement and responsible proteins for bringing about cytoskeletal reorganization and their function. this mechanism utilizes regulatory proteins that generate intracellular motility, such as myosin and thick microfilament bundles focusing on the site of infection, such as the actin-bundling proteins villin and fimbrin. in the cytoplasm, actin monomers polymerize into actin filaments, which constitute the core elements of the actin cytoskeleton, providing mechanical support to the cytoplasm and serving as tracks for myosin-dependent intracellular transport. to fulfill its various roles, the actin cytoskeleton requires a sophisticated regulatory system to control its organization and dynamics at both spatial and temporal levels. primary components of this system are the actin binding proteins that directly interact with monomeric and/or polymerized actin to promote af nucleation, polymerization, depolymerization, stabilization, severing, capping, and cross-linking. recently, a number of vertebrate lim domain proteins belonging to the cys-rich protein family and several structurally related plant proteins have been shown to function as abps. in arabidopsis, two distinct lim gene subfamilies have been reported. the first subfamily contains six genes, which encode proteins with two lim domains that share similarity with animal crps. these lim domain-containing proteins bind actin filaments and influence actin cytoskeleton organization. in vitro, chicken crp and tobacco wlim bind directly to afs to trigger the formation of thick actin bundles. importantly, over-expression of crp and wlim proteins has been shown to be sufficient to significantly increase the bundling of afs in rat fibroblasts and tobacco cells, respectively. the genes in the second subfamily encode da and da related proteins with a single lim domain and are specific to plants. da encodes a putative ubiquitin receptor with two ubiquitin interacting motifs and a single lim domain, while e.g. dar contains a single lim domain and a putative zinc-binding domain but lacks uims suggesting a possible functional divergence with da. the members of this family are involved in diverse functions e.g. seed and organ size control, root meristem size control, resistance signaling and cold response. in this study, we retrieved brassica rapa lim genes from the brassica database, analyzed the sequences and studied their homology with lim genes of other species. we also analyzed their phylogenetic classification and discussed their correlation with organ specific expression. expression of these genes was also analyzed following application of different biotic and abiotic stresses and their association was discussed with biotic and abiotic stress resistance in brassica.  sequence analysis and phylogenetic classification we identified lim proteins, designated as b. rapa lim, after which the sequences were analyzed. the predicted size of the brlims ranged from to amino acids, and the predicted isoelectric points varied from to. analysis of the protein domain organization showed that proteins contained the characteristic lim domain in the conserved region. additionally, genomic dna sequence of the brlim genes was determined from the b. rapa chromosome sequences and the introns and exons were identified by sequence analysis. structural information pertaining to the brlim genes is presented in additional file 1: figure s this analysis confirmed that the identified genes code for lim domain containing proteins. a blast search of the ncbi database conducted for comparison of these brlims with lim genes of other species revealed that the deduced amino acid sequences of brlims shared high homology, primarily with lims of a. thaliana and some other homologous species. pairwise amino acid sequence comparisons among these brlim proteins were also calculated using blast of the ncbi database and shown in additional file 2: table s the similarity within these brlim protein sequences ranged from to 99% and brlims showed above 90% similarity within the species indicating their probable duplication.table in silico analysis of lim genes collected from the brassica database a brlim1 brlim2 brlim3 brlim4 brlim5 brlim6 brlim7 bra014721c brlim8 brlim9 bra000154c brlim10 brlim11 brlim12 brlim13 brlim14 brlim15 d brlim16 d brlim17 brlim18 brlim19 e brlim20 brlim21 brlim22 a brassica database, bgenbank accession number, c brassica database accession number, dadditional uim domain and eadditional uim domain. homology analysis of lim and da1-related genes of brassica rapa a brlim1 brplim2a arabidopsis thaliana nicotiana tabacum brlim2 a. thaliana populus tremula x populus alba brlim3 arabidopsis lyrata subsp. lyrata p. tremula x populus alba brlim4 a. thaliana a. lyrata subsp. lyrata brlim5 brwlim2a a. lyrata subsp. lyrata a. thaliana brlim6 brwlim2b a. lyrata subsp. lyrata n. tabacum brlim7 brwlim2c a. lyrata subsp. lyrata p. tremula x populus alba brlim8 brwlim2d a. thaliana hevea brasiliensis brlim9 brwlim2e a. lyrata subsp. lyrata p. tremula x populus alba brlim10 brwlim1a a. thaliana brassica napus brlim11 brwlim1b b. napus a. thaliana brlim12 brdar1 a. thaliana a. thaliana brlim13 brdar2 a. thaliana medicago truncatula brlim14 brdar3 a. thaliana theobroma cacao brlim15 brdar4 a. thaliana t. cacao brlim16 brdar5 a. thaliana m. truncatula brlim17 brdar6 a. thaliana m. truncatula brlim18 brdar7 a. thaliana m. truncatula brlim19 brdar8 a. thaliana a. thaliana brlim20 brplim2e a. thaliana n. tabacum brlim21 brplim2f a. lyrata subsp. lyrata a. thaliana brlim22 brplim2g a. thaliana p. tremula x populus alba aanalyzed using blast from ncbi, http://www.ncbi.nlm.nih.gov/blast/. we also retrieved all lim family protein sequences of a. thaliana, populus trichocarpa and oryza sativa from ncbi and constructed a phylogenetic tree with deduced amino acid sequences of brlim using the nj method. in the phylogenetic tree, previously identified lim and lim groups were clearly separated and the plant lim family was further divided into four groups, αlim βlim γlim and δlim resulting from division of the lim and lim groups. this phylogenetic analysis confirms the existence of the plim wlim plim and wlim subgroups as previously described. the αlim group includes the plim and wlim subgroups, while βlim is a newly identified group. the wlim and plim subgroups belong to the γlim and δlim groups, respectively. among the brlims, brlim and were clustered with wlim subgroup under the αlim group. brlim and were separated with the wlim subgroup under the γlim group. moreover, brlim and separated with the plim subgroup under the δlim group, showing close relationships with the lim sequences of other species. multiple alignments among members of the wlim wlim plim and dar groups also showed clearly distinct amino acid residues conserved in the domains and other regions of their protein sequences. in arabidopsis, lim proteins are encoded by genes from two distinct subfamilies, one encoding proteins with two lim domains that are homologous to animal crp proteins. consequently, highly homologous wlim plim and wlim proteins of b. rapa of this subfamily also contain two lim domains except brlim interestingly, with two lim domains n-terminal extensions were observed in brlim and protein sequences and brlim had additional transmembrane domains in the n-terminal extension. these n-terminal extensions suggest probable misannotation or divergent functions of these two genes which require further experimental evidence.figure phylogenetic tree showing the relatedness of the deduced full-length amino acid sequences of brlim proteins and all lim family proteins of a. thaliana, populus trichocarpa and oryza sativa. brlim proteins are indicated by square bullets. the scale represents the frequency of amino acid substitutions between sequences as determined by the poisson evolutionary distance method. a species acronym is added before each lim protein name: at, a. thaliana; pt, populus trichocarpa and os, oryza sativa. the old nomenclature of lim genes are indicated under bracket. details of each gene are given in additional file 3: table s alignment of deduced amino acid sequences of a) lim and b) dar protein groups using pir. solid boxes indicate uim and lim domains. underline indicates highly conserved c-terminus. asterisks show conserved cysteine and histidine residues in the highly conserved lim domains and c-terminus. numbers in the right margin indicate the positions of amino acid residues. identical amino acids are indicated by a dark background, while > 50% similarities are indicated by a light background. to investigate the existence of n-terminal extensions of brlim and we analyzed expression of both genes over different organs specifying forward and reverse primers at several locations of the sequences. both genes showed expression, when forward and reverse primers were selected at the conserved parts. conversely, no expression was found, when the forward primers were selected at the extended parts and the reverse primers at several locations of the conserved parts. subsequently, 3’-race pcr using primer # of brlim and primer # of brlim also showed discontinuation of the transcription towards conserved parts and found to show polya tails indicating the end of the transcript. alongside, genomic dna amplification specifying these transition portions of both genes revealed no sequence distortion. thus, it is clear that the extended portions are not part of brlim and it was a wrong annotation of the splice sites in the original prediction. the correct exons of brlim and were then drawn in the postulated gene structures and the primer combinations, # and # of brlim and # and # of brlim located at the corrected exons, were used in latter expression studies. these corrected sequences were deposited in the genbank under the accession numbers, kj and kj. these corrected sequences are the basis for all further analysis and all figures and tables were corrected accordingly. the genes in the second subfamily encode da and da1-related proteins specific to plants contain a single conserved lim domain. interestingly, brlim and were clustered into a new group, showing close relationships with eight da and dar sequences of a. thaliana and four dar sequences of o. sativa. additionally, all members of this new group contain only one lim domain, while some also contain one or more uim. therefore, brlim proteins were renamed according to their phylogenetic relationship as b. rapa dar. alignment of the motif sequences of the da and dar group showed very low similarity with wlim wlim and plim sequences, indicating their distinctness from these groups. however, the brdars were highly similar to da and dar sequences of a. thaliana and o. sativa, and these all contained similar patterns of motifs in their conserved regions. da of a. thaliana contains two uims, a single lim domain and the highly conserved c-terminal region. in contrast, dar contains a single lim domain and a putative zinc-binding domain but lacks uims compared with da and dar. similarly, brdar and contain two uims and a single lim domain, while brdar contains four uim and a single lim domain with highly conserved c-terminal region in their protein sequences. other brdars contain only a single lim domain and highly conserved c-terminal regions. thus, the brlim genes showed high similarity with corresponding groups of lim genes of different species, indicating their close evolutionary relationship. organ specific expression analysis under unstressed condition expression analysis was performed using specific primers with equal amounts of cdna templates prepared from the mrna of roots, stems, leaves and flower buds of healthy chinese cabbage plants without applying any stresses by rt-pcr and organ specific expression was observed. specifically, brplim2d, brwlim2a, b, c, d, e, brwlim1a and b showed expression in all the organs tested. brplim2a, b, c, brplim2e, f and g were only expressed in flower buds. expression of the genes that were only expressed in flower buds was further analyzed during six developmental stages, and all genes except brplim2e showed higher expression in young buds. this expression pattern and phylogenetic classification revealed two clear groups of brlim genes, with one group only being expressed in flower buds and another group being expressed in all vegetative parts of the chinese cabbage plants tested in this study. thus, organ specific expressions of brlims showed clear correlations with phylogenetic grouping as well.figure expression analysis of 14 brlim and 8 brdar genes, a) over different organs, b) at six growth stages of flower buds of brassica rapa ‘sun-3061’ plants and c) microarray expressions of da1 and seven dar genes of arabidopsis thaliana at developmental stages. lanes 1– pcr products of roots, stems, leaves and flower buds. a-f, young to mature stages of flower buds. dar subfamily genes of b. rapa showed a different expression pattern compared to other lim domain containing genes. specifically, brdar and showed expression in all the organs tested. brdar and showed expression in all organs except roots, while brdar was only expressed in flower buds. noticeably, all brdar genes expressed highly in flower buds compared to other organs. expression of brdar that was only expressed in flower buds was further analyzed during six developmental stages, and found higher expressions in young and middle aged buds. microarray expression of da and dar genes of a. thaliana were also lower at different vegetative parts compared to floral organs and comparatively higher at young flower bud stages. the plim- protein of sunflower appeared in the microspore stage in a limited number of cytoplasmic bodies, became undetectable in bicellular pollen, and reappeared in tricellular pollen grains in cortical patches, being particularly concentrated in the f-actin-enriched germination cones of the vegetative cell. taken together, these findings suggest a dual function of plim- gene during pollen development. conversely, expression of ntplim of tobacco in germinating pollen suggests a possible role in pollen germination and/or pollen tube growth. lilim an effective actin bundling protein, also plays an important role in pollen tube growth of lilium longiflorum. likewise, the brplims expressed in young flower bud stage might play a role in the development of pollen tube. earlier studies have also suggested the existence of two main lim gene subfamilies that differ in their expression patterns. again, based on phylogenetic analysis of lims and comparison of the available expression data, a complex classification of lim genes has been proposed by arnaud et al.,. according to this classification, the arabidopsis lim gene family comprises three vegetative and three reproductive isoforms, of which, all plims have been shown to be expressed in pollen. ye et al. also suggested the critical roles of arabidopsis plims in actin configuration during pollen germination and tube growth. specific control of plim actin regulatory activities by ph is particularly relevant with regard to the potential biological functions of these proteins in growing tip region of pollen tubes. expression analysis under stress conditions biotic stress biotic stress responses are a crucial issue for the brassica crops and functions of lim genes in biotic stress responses are not well studied yet. we investigated the responses of the brlim genes identified in this study to infection by f. oxysporum f.sp. conglutinans in chinese cabbage after various times. brwlim2c and brdar were found to be up-regulated with the time course of infection. specifically, brdar showed very high responses in the late stages of fungal infection, while brwlim2c responded during earlier stages. atwlim atwlim2b, atdar atdar and atdar genes also showed responses during microarray expression analysis after infection with f. oxysporum in a. thaliana, which were collected from public database. we also analyzed the expression of all brlims after infection with the soft rot disease causing necrotroph bacteria, pectobacterium carotovorum subsp. carotovorum; however, no differential expression was observed in chinese cabbage plants. these results suggest probable involvement of brwlim2c and brdar with f. oxysporum f.sp. conglutinans resistance and other brlim and brdar genes might also have association with resistance against other biotic stress agents in brassica crops.figure real-time quantitative pcr expression analysis of a) brwlim2c and b) brdar4 after infection of chinese cabbage with fusarium oxysporum f. sp. conglutinans. relative gene expressions in treated samples are presented as fold change compared to mock treated samples. at each time point gene expression values in mock treated samples are set to plants encounter and must deal with a range of other organisms that may be potential partners or pathogens, and the plant cytoskeleton plays a key role in their response to such organisms. cell wall appositions, or papillae, are important barriers formed by plants in defense against attempted penetration by fungal and oomycete pathogens. prior to the development of papillae, plant cytosol and subcellular components are rapidly translocated to the site of pathogen penetration via a mechanism known as cytoplasmic aggregation that is dependent on the action of the actin component of the cytoskeleton. this cytoplasmic aggregation has been observed in many plant-microbe interactions and is a common resistance response to pathogens by both dicotyledonous and monocotyledonous plants to invading filamentous pathogens. microfilaments are the main cytoskeletal element responsible for penetration resistance based on cytoplasmic aggregation and papilla formation at the site of infection. for example, focusing of actin microfilaments at the penetration site is more extensive in resistant plants than susceptible hosts in flax -melampsora and barley -erysiphe interactions, suggesting that accumulation of material at the site of infection is related to the degree of host resistance. some other previous observations suggest that inhibition of penetration through cytoplasmic aggregation and papilla formation is an early, if not the first, tactic in plant resistance, and that it may be backed up by the hypersensitive response. thus, the physical and chemical barrier resulting from actin-dependent cytoplasmic aggregation, secretion, and papilla formation appears to constitute an important and probably ancient form of basal resistance to pathogen attack. for a full understanding of the cytoskeletal basis of cytoplasmic aggregation, it is very essential to elucidate the role of responsible regulatory proteins that participate in other forms of cytoplasmic streaming, as well as additional factors specific for the localized defense response. proteins those generate intracellular motility are likely to be important components of such regulations. in addition, several crp-like plant lim proteins have been shown to function as abps, and lilim has been proposed as an effective abp, that plays an important role in pollen tube growth of l. longiflorum. in vitro, tobacco wlim binds directly to afs and triggers the formation of thick actin bundles. importantly, overexpression of wlim proteins was sufficient to significantly increase the bundling of afs in tobacco cells. thus, actin binding and bundling function of lim genes could also be an important factor for actin-dependent resistance mechanism against pathogen infection. in this study, high expression of brwlim2c gene against pathogen infection is tempting to speculate that brlim protein may also be implicated in the perception of pathogen resistance mechanism. genes in the second subfamily encode da proteins, which are specific to plants. the arabidopsis lim domain protein da was characterized to function as an ubiquitin receptor. ubiquitin is a highly conserved and wide-spread small protein modifier that is engaged in a wide range of cellular and biological processes, such as resistance to disease and abiotic stresses. the consequences of ubiquitination are accusing the target protein to proteolysis or relocalization or endocytosis. da is inevitably involved in ubiquitination resulting extension of the cellular proliferation period, thus increasing cell numbers and ultimately plant organ size. our highly expressed brdar in response to pathogen infection might be involved in biotic stress resistance of b. rapa following aforesaid mechanism. another member of this family chs3/atdar containing tir-nbs-lrr domain is associated with resistance signaling and cold response and atdar with controlling of root meristem size. alongside, no brdar protein contained tir-nbs-lrr domain like as chs3/atdar however, reports on the responses of lim genes to biotic stresses are still lacking, and this is the first report of such possible role of lim domain containing genes against biotic stress caused by f. oxysporum f.sp. conglutinans in chinese cabbage. thus, brlim and brdar genes might be involved in biotic stress resistance related functions in b. rapa. abiotic stresses in addition to biotic stresses, expression of brlims was also analyzed in response to abiotic stress conditions such as cold, aba and ph stresses. commonly ten brlims among them, showed differential expressions in response to the abiotic stresses applied in this study. specifically, brplim2d, brwlim2a, b, c, d, e, brwlim1a, b, brdar and showed differential expression after cold, aba and ph stress treatments compared to mock treated samples of chinese cabbage. brplim2d, brwlim2a, b and brdar were down-regulated in response to cold stress throughout the treatment period, while brwlim2c, d, e, brwlim1a, b and brdar were up-regulated during the first 8 h of treatment, after which they were down-regulated. following aba stress treatments, brplim2d, brwlim2b, e and brdar were also down-regulated throughout the treatment period, while brwlim2a, c, d, brwlim1a, b and brdar were up-regulated during the first 8 h of treatment, after which they were down-regulated. moreover, brplim2d, brwlim2a and brdar were down-regulated and brwlim1a, b brwlim2b and c were up-regulated throughout the treatment period in response to ph  and stress treatments compared to mock treated samples which were maintained after transferring into a fresh medium with ph . brwlim2d and brwlim2e were down-regulated in response to ph  and while was up-regulated in response to ph  brdar was down-regulated in response to ph  and up-regulated in response to ph  and interestingly, brplim2d and brdar were down-regulated and brwlim1a, b and brwlim2c were up-regulated in response to all abiotic stress treatments applied in this study. atwlim atwlim2a, atwlim2b, dar dar dar and dar genes also showed responses after cold stress treatments in a. thaliana during microarray expressions study collected from public database.figure real-time quantitative pcr expression analysis of brlim and brdar genes after a) cold, b) aba and c) ph stress treatments in chinese cabbage. relative gene expressions in treated samples are presented as fold change compared to mock treated samples. at each time point gene expression values in mock treated samples are set to plants actin cytoskeleton is a major signaling target and in response to numerous abiotic and biotic stimuli, it changes dramatically in various ways, ranging from filament bundling, to massive actin depolymerization, to assembly of new filament arrays. plant cells can detect mechanical stress on the epidermal cell surface and respond by reorganizing subcellular components like, actin microfilaments, endoplasmic reticulum and peroxisomes in a manner similar to that induced during attack by potential fungal or oomycete pathogens. expression analysis of poplar lims in tension wood also suggested a connection between plant lims and mechanical stress. thus, rearrangement of cytoskeletal elements through actin binding and bundling may also be the mechanism of abiotic stress responses of the crp-like lim genes. alongside, aforementioned ubiquitination mechanism of da and related proteins might also be implicated in abiotic stress resistance function of brdar proteins. in a very recent study, expression of gmada gene of soybeans showed responses to salt, drought, acid and alkali stresses and aba treatment. atda expression occurs in response to aba trteatment and chs3/atdar is associated with resistance signaling and cold response in a. thaliana. supporting this report bi et al. hypothesized that disease resistance and environmental stress response pathways are intricately tied in plants, although the degree of overlap and the precise signaling events required for each pathway remain unclear. thus, the results of this study indicate the likely involvement of the analyzed genes towards abiotic stress tolerance in brassica.  this present study identified lim domain containing genes in b. rapa and classified into two distinct functional groups. one group is plant–specific and designated as brdar, and another is crp-like designated as brlim. these sequences were analyzed, compared them with lim domain containing genes of other plants and analyzed their expression after applying biotic and abiotic stresses in chinese cabbage. overall, brplim2d, brwlim2a, b, c, d, e, brwlim1a, b, brdar and responded after cold, aba and ph stress treatments in b. rapa, of which brwlim2c and brdar showed responses to f. oxysporum f.sp. conglutinans infection as well. thus, these eight brlims and two brdars might be promising candidates for multiple abiotic stresses resistance, while brwlim2c and brdar might be the most promising among them for both biotic and abiotic stresses resistance in brassica crops. the remaining brlims and brdars might also be useful resources for induction of resistance to other biotic and abiotic stresses and/or various developmental processes of brassica. 
all brdar genes showed higher expression in flower buds compared to other organs. taken together, the results of this study indicate that brlim and brdar genes may be involved in resistance against biotic and abiotic stresses in brassica. in this study, we collected lim genes designated as brassica rapa lim from the brassica database, analyzed the sequences, compared them with lim genes of other plants and analyzed their expression after applying biotic and abiotic stresses in chinese cabbage. in addition, brwlim2c and brdar responded to fusarium oxysporum f. sp. these genes showed distinct clusters when compared to other recognized lim proteins upon phylogenetic analysis. upon sequence analysis these genes were confirmed as lim genes and found to have a high degree of homology with lim genes of other species. brassica rapacharacterizationexpression analysislim genestress resistanceissue-copyright-statement© the author 2014 conglutinans infection, while commonly two brdars and eight brlims responded to cold, aba and ph stress treatments in chinese cabbage plants. additionally, organ specific expression of these genes was observed in chinese cabbage plants, with brplim2a, b, c, brdar brplim2e, f and g only being expressed in flower buds. these organ specific expressions were clearly correlated with the phylogenetic grouping.
 lim genes have been reported to trigger the formation of actin bundles, a major higher-order cytoskeletal assembly, in higher plants; however, the stress resistance related functions of these genes are still not well known. in this study, we collected lim genes designated as brassica rapa lim from the brassica database, analyzed the sequences, compared them with lim genes of other plants and analyzed their expression after applying biotic and abiotic stresses in chinese cabbage.  upon sequence analysis these genes were confirmed as lim genes and found to have a high degree of homology with lim genes of other species. these genes showed distinct clusters when compared to other recognized lim proteins upon phylogenetic analysis. additionally, organ specific expression of these genes was observed in chinese cabbage plants, with brplim2a, b, c, brdar brplim2e, f and g only being expressed in flower buds. furthermore, the expression of these genes was high in the early flowering stages. the remaining genes were expressed in almost all organs tested. all brdar genes showed higher expression in flower buds compared to other organs. these organ specific expressions were clearly correlated with the phylogenetic grouping. in addition, brwlim2c and brdar responded to fusarium oxysporum f. sp. conglutinans infection, while commonly two brdars and eight brlims responded to cold, aba and ph stress treatments in chinese cabbage plants.  taken together, the results of this study indicate that brlim and brdar genes may be involved in resistance against biotic and abiotic stresses in brassica. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords brassica rapacharacterizationexpression analysislim genestress resistanceissue-copyright-statement© the author 2014 