BACKGROUND
bambusa balcooa is an abundant tropical species and recognized by food and agriculture organization of the united nations as a priority bamboo species amongst eighteen other bamboo species distributed globally  <cit> .
it can quickly restore forest flora and the pre-existing environmental milieu due to its faster growth rate.
b. balcooa has better physical and mechanical properties and is used as raw material for paper and pulp industries.
in india alone, about  <dig>  million tonnes of bamboo are used in paper industries  <cit> .
the superior commercially useful traits of b. balcooa demanded isolation and cloning of gene contributing towards superior fiber quality production.
bioprospecting of genes from any non-model tree species for biotechnological applications is mainly focused on the development of molecular markers.
molecular markers allow rapid identification of the gene or genes of interest from genomic dna.
in our laboratory, we had developed a genotype specific scar  marker, balco <dig>   <cit>  with 375 bp long orf, having high similarities with a protein of arabidopsis thaliana  and a putative protein kinase of oryza sativa cv.
japonica .
the marker, balco <dig> was found to be associated with high cellulose containing fibers of b. balcooa genotypes  <cit> .
subsequently the marker, balco <dig> was employed to screen elite genotypes of b. balcooa for the efficient commercial utilization of the available superior natural germplasm resources.
there are limited reports on fiber related kinases.
it was shown that a receptor-like kinase  gene of cotton  participates in cotton fiber development and fiber cell wall synthesis  <cit> .
subsequently, one kinase est   <cit>  has been identified from bamboo which has higher expression during fiber cell initiation and elongation.
protein kinases represent a biologically significant group of plant proteins as they are implicated in several cellular functions including cell proliferation, differentiation and cell death mediating cell signaling pathways.
of these, the receptor-like protein kinases  form one of the largest and most diverse super-families of plant proteins  <cit> .
rlks have diverse biological roles, such as plant growth and development, hormonal response, cell differentiation, self-incompatibility, stress response and pathogen recognition  <cit> .
the receptor like cytoplasmic kinase  gene family codes for proteins lacking the transmembrane domain found in the typical rlks  <cit> .
the annotated arabidopsis genome shows around  <dig> rlk-coding genes out of which around  <dig> have no extracellular domain; suggesting involvement of rlck family in growth and developmental processes of plants  <cit> .
plant rlks are classified into several subclasses, the largest of which is the sub-family of lrr receptor-like kinases  <cit> .
lrr-rlks were studied extensively and it was found that this group of proteins plays important roles in diverse processes of plant development  <cit> .
under the above backdrop, in the present study we made an attempt to isolate full length sequence of this putative leucine rich kinase gene employing balco <dig> sequence information, rna ligase mediated - rapid amplification of cdna ends  and genome walking.
in silico sequence analysis confirms the presence of kinase and lrr domains within bbkst.
the protein encoded by the kinase domain of bbkst gene was expressed in escherichia coli, purified to determine the nature of this putative protein kinase.
biochemical analyses of the protein kinase were conducted and the nature and kinase activity of the protein has been deciphered.
subsequently, transgenic tobacco plants were raised and molecular analyses were conducted.
transcripts of bbkst were quantified in gus positive transgenic t <dig> tobacco plants using qpcr.
cellulose contents of the fibers isolated from transgenics and that of empty-vector transformed control tobacco plants were analysed.
physical parameters of fibers including flexibility coefficient, slenderness ratio and runkel ratios of transgenics were compared with that of vector-transformed tobacco plants to reveal whether bbkst confers any additive value in fiber quality as noted in superior genotypes of b. balcooa.
confocal laser scanning microscopy  with cellulose specific staining of stem cross sections of transgenics and that of vector-transformed plants was undertaken at the t <dig> and t <dig> generations to demonstrate in situ distribution of cellulose.
the present finding demonstrated that bbkst  encodes a serine/threonine protein kinase that enhances higher cellulose deposition/synthesis, an important attribute that confers superior fiber quality.
this was envisaged in our previous study  <cit> , wherein we showed that the presence of balco <dig>  which is a part of bbkst gene, is associated only with high cellulose containing b. balcooa genotypes.
the present study also has shown that bbkst induced formation of expanded xylary zone and enhanced cellulose synthesis in the transgenic tobacco plants bestowing additional mechanical strength.
thus, this finding revealed why the marker ‘balco1128’ is genotypically specific and present only in high cellulose containing superior-fiber-yielding genotypes as reported by pal et al.
<cit> .
RESULTS
analysis of the bbkst gene architecture
the full length bbkst gene  was isolated using race and genome walking.
a representative gel documenting race products and genome walked pcr products are shown in additional file 1: figure s <dig> ai, aii.
the bbkst sequence  was subjected to in silico analysis, which showed a 3342 bp long coding region with three exons of  <dig> ,  <dig>  and  <dig>  bp sizes, separated by two introns  as shown in figure  <dig>  the putative start  and stop  codons were positioned at  <dig> and 7033 bp, from the transcription start site, respectively, and a poly-adenylation motif aataaa was found at the end of the 3′ utr sequence.
a schematic diagram of the bbkst sequence organization is shown in figure 1a.
a putative promoter sequence, 840 bp in length, was analysed by using plantcare  and plantpan , and contains several specific structural elements including a putative tata box , caat box , kozak sequence  at the positions - <dig> to +3; with a cluster of important plant specific key regulatory cis-elements .
dnaman based analysis of 3951 bp long cdna sequence revealed that it contains  <dig> and 342 bp long utr regions at the 5′ and 3′ ends, respectively.
further analysis of bbkst gene using ncbi-cds software has clearly shown that the predicted bbkst protein has distinct pkc domain and lrr regions.
exact orientation of the kinase domain and lrr repeats were recognized with a pfam search .
positions of six lrr repeat regions, atp binding site, active sites and nucleotide phosphate-binding region were determined by prosite and results are depicted in figure  <dig>  domain analysis also indicates that bbkst protein has no extracellular domain indicating that it belongs to rlck subfamily.
analysis of biochemical properties of bbkst-kinase
the kinase domain of the bbkst protein was expressed in e. coli bl <dig> cells as described in the methods section.
expression of bbkst-kinase was found in both soluble and insoluble fractions of total cell lysate .
subsequently, the purified protein was confirmed by western blot .
bbkst-kinase phosphorylates all the substrates tested in the present study viz., casein, myelin basic protein , histone , ‘lrrktide’ and ‘gs’ peptide .
the respective phosphorylated products were electrophoresed and the auto-radiographed image is shown in figure 4aii.
among these substrates, the protein showed higher specific activity with casein and mbp than others .
therefore, we used casein as a substrate in the subsequent experiments to study the biochemical properties of bbkst-kinase.
casein phosphorylation was determined over a ph range from  <dig> to  <dig> and subsequent specific activity showed a bell shaped pattern within the above said ph range .
the bbkst kinase showed highest activity at ph  <dig>  when casein was used as the in vitro substrate .
the effect of temperature on casein phosphorylation by the bbkst-kinase was also determined.
the substrate phosphorylation activity of the bbkst-kinase showed a gradual increase in activity from  <dig> to 15°c, followed by an optimum stationary stage up to 40°c and then gradually decreased with increasing temperature .
bbkst-kinase showed highest activity in presence of mg2+.
it was found that casein phosphorylation by bbkst-kinase increased along with increased concentration of mg2+ from  <dig>  or  <dig>  mm, while, in the case of mn2+ a weak phosphorylation of casein was noted only at  <dig>  mm  indicating that bbkst-kinase prefer mg2+ over mn2+ for its enzyme activity.
phosphoamino acid analysis of autophosphorylated bbkst revealed that bbkst phosphorylated only serine and threonine residues but not tyrosine .
enzyme kinetics of bbkst-kinase
bbkst exhibited michaelis-menten kinetics with respect to casein as substrate.
the vmax and km value of bbkst-kinase for casein determined from eadie-hofstee plots of v <dig> versus v0/ were  <dig>  ±  <dig>  pmol/min/μg and  <dig>  ±  <dig>  μm, respectively .
the kcat  was determined and the value was found to be ~  <dig>  min- <dig>  casein-phosphorylation of bbkst-kinase showed a steady increase along with time upto 3 min, which remain steady upto 5 min.
the dissociation constant  for substrate phosphorylation was computed as  <dig>  ±  <dig>  pmol .
effect of protein kinase inhibitors on specific activity of bbkst
none of the three inhibitors  tested could inhibit completely the activity of bbkst-kinase .
of these three, quercetin showed significantly higher inhibition than h- <dig> and staurosporine.
the ic <dig> value for quercetin was estimated at  <dig> nm.
anova results showed there is significant difference  in ic <dig> values of these three kinase inhibitors.
the products were electrophoresed in sds-page and a corresponding autoradiogram of the gel is presented in figure 7b.
spatial expression of bbkst transcripts in b. balcooa
differential expression of the bbkst transcripts in the stem, internode, rhizome and leaf of b. balcooa was noted and confirmed through three independent rna slot blot hybridization as described in the method section.
results obtained were found to be highly reproducible.
the strongest expression of the bbkst transcripts was found in mature stem while weaker expression was observed in rhizome.
leaf and internodal tissue showed moderate expression of bbkst transcript .
all the above findings were further confirmed by qpcr analysis, wherein a weaker expression of the transcript in the rhizome was detected .
differential expression of bbkst transcript at different fiber developmental stages of b. balcooa
expression analyses of the bbkst transcript at different internodal tissue  revealed highest expression of bbkst in the 10th internode followed by 8th .
expression level gradually decreased with fiber maturity and lowest expression level was noted in the 15th internode.
results obtained were consistent in three independent experiments.
the expression level of the bbkst was found to be  <dig>  fold higher during fiber elongation  and  <dig>  fold higher during fiber maturation  than in the fiber initiation stage.
all the above findings were further confirmed by qpcr analysis .
analysis of transient expressions of the bbkst in plant cell
transient expression of a bbkst-gfp fusion was analysed using onion peels bombarded with plant expression vector pcambia  <dig> carrying total cdna sequence of bbkst, designated as js <dig> .
onion epidermal cells exhibited expression of the gfp-fusion protein in the cytoplasmic region of the js <dig> transformed onion cell after 24 hr .
expression was not found in the non-transformed tissue .
this observation indicates constitutive expression of gfp reporter in the cytoplasm.
development of transgenic tobacco plants with bbkst gene
tobacco leaf discs were transformed with pcambia  <dig> carrying js <dig> and transformants were selected under hygromycin containing medium.
putative transgenic plants were then transferred to soil and t <dig> plants took nearly 6 months to produce flowers and seeds.
the comparative phenotypic features of empty-vector transformed and transgenic plants are shown in figure 11a-f. it was noted that empty vector-transformed plants were gradually stooping from one month onwards after soil transfer , but the transgenic plants were strong and upright .
the stem of vector-transformed plants were comparatively weak than the transgenic plants containing bbkst .
similar observations were also noted in case of t <dig> transgenic tobacco plants .
selection of gus positive plants
gus expression was studied in thirty day old transgenic tobacco seedlings  in presence of x-gluc as stated in method section.
prominent blue colouration was observed in transgenic seedlings .
blue colour did not appear in empty vector-transformed seedling .
copy number determination of bbkst
in most of the t <dig> events the transgene was found to be stably integrated into the genome as a single copy, confirming perfect transgene transmission in the next generation .the gus expressive and single copy bbkst integrated lines were selected for further study.
heterologous expression of bbkst in transgenic tobacco
qpcr detected the expression level of bbkst in different t <dig> progeny lines.
different transgenic lines exhibited 150- <dig> fold enhanced expression level than that of the empty vector control tobacco plants.
among five lines of transgenic bbkst plants, line  <dig> on an average showed maximum expression followed by line  <dig> and  <dig> .
physical and anatomical characteristics of fibers of transgenic plants
stem diameters of five transgenic plants  expressing bbkst gene and empty vector-transformed tobacco plants  were measured and data are represented in figure 13a.
stem thickness of independent transgenic plants varied from  <dig>  cm to  <dig>  cm with an average diameter of  <dig>  cm.
no significant increase in stem diameter of transgenic plants expressing bbkst gene over vector-transformed plants was noted.
a comparative account between xylem width and stem width of five independent transgenic events at the t <dig> generation is presented in figure 13b.
the standardized maceration technique proved useful in isolating intact, individual fibers from the internodal tissues of tobacco plants.
the computed flexibility co-efficient of fibers of transgenic t <dig> plants were significantly higher than those of control transformed plants .
similarly, slenderness ratios of transgenic fibers were significantly higher than empty vector-transformed tobacco plants .
while, the lowest runkel ratio was found in transgenic line  <dig> followed by  <dig> and  <dig> and the highest in vector transformed tobacco plants .
*fibers isolated from transgenic  and vector transformed  tobacco plants.
mean value followed by same superscripts within a column do not differ significantly  according to dmrt.
anatomical changes concomitant with transgene expression in different transgenic lines  revealed significant increase in the number of xylary fibers of vascular bundles compared to the empty-vector transformed tobacco stems .
heterologous expression of bbkst in transgenic tobacco plants
stem cross sections of transgenic tobacco plants carrying bbkst were subjected to congo-red staining as described in methods and subsequently the stained sections were studied under clsm.
enhanced level of cellulose depositions in the walls of xylary fibers in the stems of transgenic plants was evident.
the vascular zone was more pronounced in transgenic than empty vector-transformed t <dig> plants .
similar observations were also noted in t <dig> transgenic tobacco plants .
biochemical quantification of cellulose contents
cellulose contents of five independent transgenic plants  expressing bbkst gene and empty vector transformed tobacco plants were analysed as stated in method and the quantitative data are represented in table  <dig>  cellulose content of these transgenic lines varied from  <dig> - <dig> % fresh wt.
with an average of  <dig> % fresh wt, whereas  <dig> % cellulose content was noted in the vector-transformed tobacco plants.
overall a significant   <dig>  fold increase of cellulose contents in transgenic tobacco plants expressing bbkst gene was noted, compared to that of vector-transformed plant .
discussion
in a previous study, a species specific sequence characterized amplified region  marker was developed that discriminates b. balcooa from other bamboo species  <cit> .
further investigations with different natural genetic resources of b. balcooa yielded a second dna marker, balco <dig>  that has been successfully employed to identify the elite genotypes of b. balcooa endowed with high cellulose contents and possessing superior fiber qualities with respect to tensile strength and durability  <cit> .
however, the genetic linkage between balco <dig> and high cellulose containing trait could not be demonstrated due to the monocarpic nature and unusually long sexual cycle of bamboo  <cit> .
nevertheless, in silico analysis revealed the presence of an open reading frame within the marker sequence which prompted us to identify the full length sequence of bbkst gene.
the 7375 bp long full length bbkst consists of  <dig> introns,  <dig> exons, 5′-3′ utr and an upstream promoter like region.
database searches revealed the presence of key promoter-specific structural elements and many cis-acting elements in the upstream region of bbkst gene ; among these, the rss <dig> site regulates phloem specific sucrose synthesis by enhancing sucrose synthase production  <cit> .
the deduced amino acid sequence of bbkst showed high sequence homology with other reported protein kinases from various plant taxa.
the complete orf of the protein upon blastp illustrated that bbkst has more than 70% homology with arabidopsis thaliana , hypothetical protein of vitis vinifera , an atp binding protein of ricinus communis , predicted protein of populus trichocarpa  and hypothetical protein sorbidraft_01g <dig> of sorghum bicolor  and putative protein kinase of oryza sativa var.
japonica  .
5′ of the bbkst protein sequence contains six lrr motifs which may act as substrate binding sites and the 3′ end contains a serine-threonine protein kinase domain, devoid of any transmembrane domains.
pfam domain search predicted that bbkst is a receptor like cytoplasmic kinase.
lrr-rlks possess different numbers of lrr variously arranged in their extracellular domains, which are involved in protein-protein interaction and in targeting the receptors  <cit> .
plant rlcks usually comprised of a carboxyl-terminal kinase domain but no apparent signal sequence or transmembrane domain are present.
although a large number of rlks are present in plant genomes, function of most of these rlks and rlcks are not known  <cit> .
a limited number of rlcks are functionally characterized, for example, brassica  m-locus protein kinase mlpk associated with self-incompatibility  <cit> , tobacco  ntpk <dig> and ntpk <dig> involved in pollen germination and pollen tube elongation  <cit> , tomato  pto and pti <dig> in disease resistance  <cit> , soybean  gmpti <dig> in coordinating plant defense responses  <cit>  and arabidopsis  cdg <dig> related to br signaling  <cit> .
there are few reports available in monocotyledonous plants, for example, zmpti1a in maize  involved in pollen callose deposition, and zmpti1b, in pathogen defense  <cit>  and another reported from rice  osrlck <dig>  involved in pollen performance during pollination  <cit> .
there are very few evidences presuming that kinases of the rlk family have an additive role in superior fiber development.
however no comprehensive study has yet been made to elucidate the biochemical nature underpinning their biological activity.
in this communication we have analyzed biochemical properties of the bbkst-protein kinase, including the substrate specificity, divalent cation preference, phosphate donor specificity and potential inhibitors of bbkst-kinase activity.
it was found that bbkst-kinase is a monomeric 42-kd serine/threonine protein kinase enzyme, phophorylating a broad spectrum of proteins .
all kinases require divalent cations  to coordinate the phosphate groups of the phosphate donor.
bbkst, like most serine/threonine kinases, exhibited a strong preference for mg+ <dig> over mn+ <dig> in the phosphorylation reaction.
bbkst lack any regulatory domains hence phosphorylation and dephophorylation appear to be main regulatory mechanisms of its activity as proposed by kobayashi et al.
<cit> .
bamboo fibers are highly specialized cells with three distinct stages of differentiation  <cit> .
stage i consists of fiber cell differentiation/initiation; stage ii comprises of fiber cell elongation and at this stage secondary cell wall deposition starts; stage iii includes maturation of fiber, in which fiber cell elongation ceases and gradually additional wall layers are deposited.
there is a solitary published report in the literature demonstrating genes that are differentially expressed at these three stages of fiber development  <cit> , characterizing the cellular functions of these genes that are specifically expressed at a certain stage of fiber development.
bbkst transcript expression was noted from stage i of fiber development.
expression of the gene gradually increases during stage ii and declined after fiber maturation.
transformed onion epidermal cells expressing the js <dig> construct carrying bbkst gene coupled with gfp showed expression of gfp in the cytoplasm indicating the proper orientation of the construct and confirms that the js <dig> is functional yet in a heterologous system.
localization of gfp implied co-expression of bbkst in the cytoplasmic region.
further investigations by co-expressing other sub-cellular markers would reveal precise location of bbkst within the cell.
southern hybridization study revealed mostly single copy insertion in the gus positive seedlings.
heterologous expression of bbkst transcripts in t <dig> lines was remarkably high in tested transgenic tobacco.
the vascular region of transgenic tobacco containing bbkst gene was more pronounced than in the vector-transformed tobacco plants endowing additional tensile strength, which is also substantiated by their strong upright phenotypes, significantly higher flexibility coefficient, slenderness ratio, and lower runkel ratio of the fibers.
the slenderness ratio or felting power is the physical indicator for fiber durability, while the magnitude of tensile strength is usually proportional to the flexibility coefficient .
increase in the numbers of xylary fibers in the vascular bundles of transgenic plants was noted compared to stems of vector-transformed plants.
enhanced level of cellulose depositions in the stems of t <dig> and t <dig> transgenic plants were apparent and confirmed by quantitative analyses of cellulose contents indicating enhanced cellulose synthase activity.
such augmented cellulose deposition resulting in thicker xylem secondary cell wall formation has also been reported in the transgenic lines of hybrid poplar  <cit> .
plasma membrane-associated cellulose synthase  is the catalytic unit of cellulose synthesis  <cit>  that triggers the functional cellulose-synthesis machinery during fiber development.
cels have udp-glucose binding activity and β- <dig>  4-glucan  synthesis activity.
fujii and colleagues  <cit>  have demonstrated that sucrose synthase  is an integral component of the cellulose synthesis machinery.
in a contemporary investigation coleman et al.
<cit>  demonstrated close association of susy with cellulose synthesis during secondary wall formation.
suppression of susy gene expression inhibited cotton fiber cell initiation and elongation  <cit> .
in our previous study we reported expression of susy gene  in all the three stages of fiber development, but the highest expression was noted during fiber elongation concomitant with highest cellulose deposition  <cit> .
however, to date there is no evidence of enhanced cellulose synthesis associated with increased number of xylem fibers.
nevertheless, cellulose-rich g -layer in the walls of fiber cells generates tensile strength in the secondary xylem  <cit> .
it is presumed from the present finding that bbkst has an active role in triggering susy production which stimulates cels resulting in cellulose overproduction, and providing extra tensile strength of the fiber in certain genotypes of b. balcooa endowed with the marker/gene  <cit> .
to the best of our knowledge, the results presented here are the first data describing the biochemical features of a receptor like cytoplasmic protein kinase involved in higher cellulose production and conferring superior fiber quality with high tensile strength and durability in transgenic plants.
presumably bbkst plays an active role in triggering sucrose synthase production which stimulates cellulose synthase resulting in cellulose overproduction, consequently providing extra physical strengths to fibers of transgenic tobacco plants endowed with bbkst gene.
CONCLUSIONS
this is the first attempt to characterize a protein comprehensively at the biochemical and molecular level encoded by a gene associated with superior fiber quality in bamboo.
further comprehensive study on all interacting proteins involved in superior quality fiber formation will aid in manipulating fiber compositions required for the paper, textile or timber industries, as well as to comprehend multifunctional activities of fiber cells.

 bambusa balcooa is an abundant tropical species and recognized by food and agriculture organization of the united nations as a priority bamboo species amongst eighteen other bamboo species distributed globally. it can quickly restore forest flora and the pre-existing environmental milieu due to its faster growth rate. b. balcooa has better physical and mechanical properties and is used as raw material for paper and pulp industries. in india alone, about million tonnes of bamboo are used in paper industries. the superior commercially useful traits of b. balcooa demanded isolation and cloning of gene contributing towards superior fiber quality production. bioprospecting of genes from any non-model tree species for biotechnological applications is mainly focused on the development of molecular markers. molecular markers allow rapid identification of the gene or genes of interest from genomic dna. in our laboratory, we had developed a genotype specific scar marker, balco with 375 bp long orf, having high similarities with a protein of arabidopsis thaliana and a putative protein kinase of oryza sativa cv. japonica. the marker, balco was found to be associated with high cellulose containing fibers of b. balcooa genotypes. subsequently the marker, balco was employed to screen elite genotypes of b. balcooa for the efficient commercial utilization of the available superior natural germplasm resources. there are limited reports on fiber related kinases. it was shown that a receptor-like kinase gene of cotton participates in cotton fiber development and fiber cell wall synthesis. subsequently, one kinase est has been identified from bamboo which has higher expression during fiber cell initiation and elongation. protein kinases represent a biologically significant group of plant proteins as they are implicated in several cellular functions including cell proliferation, differentiation and cell death mediating cell signaling pathways. of these, the receptor-like protein kinases form one of the largest and most diverse super-families of plant proteins. rlks have diverse biological roles, such as plant growth and development, hormonal response, cell differentiation, self-incompatibility, stress response and pathogen recognition. the receptor like cytoplasmic kinase gene family codes for proteins lacking the transmembrane domain found in the typical rlks. the annotated arabidopsis genome shows around rlk-coding genes out of which around have no extracellular domain; suggesting involvement of rlck family in growth and developmental processes of plants. plant rlks are classified into several subclasses, the largest of which is the sub-family of lrr receptor-like kinases. lrr-rlks were studied extensively and it was found that this group of proteins plays important roles in diverse processes of plant development. under the above backdrop, in the present study we made an attempt to isolate full length sequence of this putative leucine rich kinase gene employing balco sequence information, rna ligase mediated - rapid amplification of cdna ends and genome walking. in silico sequence analysis confirms the presence of kinase and lrr domains within bbkst. the protein encoded by the kinase domain of bbkst gene was expressed in escherichia coli, purified to determine the nature of this putative protein kinase. biochemical analyses of the protein kinase were conducted and the nature and kinase activity of the protein has been deciphered. subsequently, transgenic tobacco plants were raised and molecular analyses were conducted. transcripts of bbkst were quantified in gus positive transgenic t tobacco plants using qpcr. cellulose contents of the fibers isolated from transgenics and that of empty-vector transformed control tobacco plants were analysed. physical parameters of fibers including flexibility coefficient, slenderness ratio and runkel ratios of transgenics were compared with that of vector-transformed tobacco plants to reveal whether bbkst confers any additive value in fiber quality as noted in superior genotypes of b. balcooa. confocal laser scanning microscopy with cellulose specific staining of stem cross sections of transgenics and that of vector-transformed plants was undertaken at the t and t generations to demonstrate in situ distribution of cellulose. the present finding demonstrated that bbkst encodes a serine/threonine protein kinase that enhances higher cellulose deposition/synthesis, an important attribute that confers superior fiber quality. this was envisaged in our previous study, wherein we showed that the presence of balco which is a part of bbkst gene, is associated only with high cellulose containing b. balcooa genotypes. the present study also has shown that bbkst induced formation of expanded xylary zone and enhanced cellulose synthesis in the transgenic tobacco plants bestowing additional mechanical strength. thus, this finding revealed why the marker ‘balco1128’ is genotypically specific and present only in high cellulose containing superior-fiber-yielding genotypes as reported by pal et al..  analysis of the bbkst gene architecture the full length bbkst gene was isolated using race and genome walking. a representative gel documenting race products and genome walked pcr products are shown in additional file 1: figure s ai, aii. the bbkst sequence was subjected to in silico analysis, which showed a 3342 bp long coding region with three exons of, and bp sizes, separated by two introns as shown in figure  the putative start and stop codons were positioned at and 7033 bp, from the transcription start site, respectively, and a poly-adenylation motif aataaa was found at the end of the 3′ utr sequence. a schematic diagram of the bbkst sequence organization is shown in figure 1a. a putative promoter sequence, 840 bp in length, was analysed by using plantcare and plantpan, and contains several specific structural elements including a putative tata box, caat box, kozak sequence at the positions - to +3; with a cluster of important plant specific key regulatory cis-elements. dnaman based analysis of 3951 bp long cdna sequence revealed that it contains and 342 bp long utr regions at the 5′ and 3′ ends, respectively. further analysis of bbkst gene using ncbi-cds software has clearly shown that the predicted bbkst protein has distinct pkc domain and lrr regions. exact orientation of the kinase domain and lrr repeats were recognized with a pfam search. positions of six lrr repeat regions, atp binding site, active sites and nucleotide phosphate-binding region were determined by prosite and results are depicted in figure  domain analysis also indicates that bbkst protein has no extracellular domain indicating that it belongs to rlck subfamily. analysis of biochemical properties of bbkst-kinase the kinase domain of the bbkst protein was expressed in e. coli bl cells as described in the methods section. expression of bbkst-kinase was found in both soluble and insoluble fractions of total cell lysate. subsequently, the purified protein was confirmed by western blot. bbkst-kinase phosphorylates all the substrates tested in the present study viz., casein, myelin basic protein, histone, ‘lrrktide’ and ‘gs’ peptide. the respective phosphorylated products were electrophoresed and the auto-radiographed image is shown in figure 4aii. among these substrates, the protein showed higher specific activity with casein and mbp than others. therefore, we used casein as a substrate in the subsequent experiments to study the biochemical properties of bbkst-kinase. casein phosphorylation was determined over a ph range from to and subsequent specific activity showed a bell shaped pattern within the above said ph range. the bbkst kinase showed highest activity at ph  when casein was used as the in vitro substrate. the effect of temperature on casein phosphorylation by the bbkst-kinase was also determined. the substrate phosphorylation activity of the bbkst-kinase showed a gradual increase in activity from to 15°c, followed by an optimum stationary stage up to 40°c and then gradually decreased with increasing temperature. bbkst-kinase showed highest activity in presence of mg2+. it was found that casein phosphorylation by bbkst-kinase increased along with increased concentration of mg2+ from or  mm, while, in the case of mn2+ a weak phosphorylation of casein was noted only at  mm indicating that bbkst-kinase prefer mg2+ over mn2+ for its enzyme activity. phosphoamino acid analysis of autophosphorylated bbkst revealed that bbkst phosphorylated only serine and threonine residues but not tyrosine. enzyme kinetics of bbkst-kinase bbkst exhibited michaelis-menten kinetics with respect to casein as substrate. the vmax and km value of bbkst-kinase for casein determined from eadie-hofstee plots of v versus v0/ were  ±  pmol/min/μg and  ±   μm, respectively. the kcat was determined and the value was found to be ~   min- casein-phosphorylation of bbkst-kinase showed a steady increase along with time upto 3 min, which remain steady upto 5 min. the dissociation constant for substrate phosphorylation was computed as  ±  pmol. effect of protein kinase inhibitors on specific activity of bbkst none of the three inhibitors tested could inhibit completely the activity of bbkst-kinase. of these three, quercetin showed significantly higher inhibition than h- and staurosporine. the ic value for quercetin was estimated at nm. anova results showed there is significant difference in ic values of these three kinase inhibitors. the products were electrophoresed in sds-page and a corresponding autoradiogram of the gel is presented in figure 7b. spatial expression of bbkst transcripts in b. balcooa differential expression of the bbkst transcripts in the stem, internode, rhizome and leaf of b. balcooa was noted and confirmed through three independent rna slot blot hybridization as described in the method section. results obtained were found to be highly reproducible. the strongest expression of the bbkst transcripts was found in mature stem while weaker expression was observed in rhizome. leaf and internodal tissue showed moderate expression of bbkst transcript. all the above findings were further confirmed by qpcr analysis, wherein a weaker expression of the transcript in the rhizome was detected. differential expression of bbkst transcript at different fiber developmental stages of b. balcooa expression analyses of the bbkst transcript at different internodal tissue revealed highest expression of bbkst in the 10th internode followed by 8th. expression level gradually decreased with fiber maturity and lowest expression level was noted in the 15th internode. results obtained were consistent in three independent experiments. the expression level of the bbkst was found to be fold higher during fiber elongation and fold higher during fiber maturation than in the fiber initiation stage. all the above findings were further confirmed by qpcr analysis. analysis of transient expressions of the bbkst in plant cell transient expression of a bbkst-gfp fusion was analysed using onion peels bombarded with plant expression vector pcambia carrying total cdna sequence of bbkst, designated as js. onion epidermal cells exhibited expression of the gfp-fusion protein in the cytoplasmic region of the js transformed onion cell after 24 hr. expression was not found in the non-transformed tissue. this observation indicates constitutive expression of gfp reporter in the cytoplasm. development of transgenic tobacco plants with bbkst gene tobacco leaf discs were transformed with pcambia carrying js and transformants were selected under hygromycin containing medium. putative transgenic plants were then transferred to soil and t plants took nearly 6 months to produce flowers and seeds. the comparative phenotypic features of empty-vector transformed and transgenic plants are shown in figure 11a-f. it was noted that empty vector-transformed plants were gradually stooping from one month onwards after soil transfer, but the transgenic plants were strong and upright. the stem of vector-transformed plants were comparatively weak than the transgenic plants containing bbkst. similar observations were also noted in case of t transgenic tobacco plants. selection of gus positive plants gus expression was studied in thirty day old transgenic tobacco seedlings in presence of x-gluc as stated in method section. prominent blue colouration was observed in transgenic seedlings. blue colour did not appear in empty vector-transformed seedling. copy number determination of bbkst in most of the t events the transgene was found to be stably integrated into the genome as a single copy, confirming perfect transgene transmission in the next generation.the gus expressive and single copy bbkst integrated lines were selected for further study. heterologous expression of bbkst in transgenic tobacco qpcr detected the expression level of bbkst in different t progeny lines. different transgenic lines exhibited 150- fold enhanced expression level than that of the empty vector control tobacco plants. among five lines of transgenic bbkst plants, line on an average showed maximum expression followed by line and. physical and anatomical characteristics of fibers of transgenic plants stem diameters of five transgenic plants expressing bbkst gene and empty vector-transformed tobacco plants were measured and data are represented in figure 13a. stem thickness of independent transgenic plants varied from  cm to  cm with an average diameter of  cm. no significant increase in stem diameter of transgenic plants expressing bbkst gene over vector-transformed plants was noted. a comparative account between xylem width and stem width of five independent transgenic events at the t generation is presented in figure 13b. the standardized maceration technique proved useful in isolating intact, individual fibers from the internodal tissues of tobacco plants. the computed flexibility co-efficient of fibers of transgenic t plants were significantly higher than those of control transformed plants. similarly, slenderness ratios of transgenic fibers were significantly higher than empty vector-transformed tobacco plants. while, the lowest runkel ratio was found in transgenic line followed by and and the highest in vector transformed tobacco plants. *fibers isolated from transgenic and vector transformed tobacco plants. mean value followed by same superscripts within a column do not differ significantly according to dmrt. anatomical changes concomitant with transgene expression in different transgenic lines revealed significant increase in the number of xylary fibers of vascular bundles compared to the empty-vector transformed tobacco stems. heterologous expression of bbkst in transgenic tobacco plants stem cross sections of transgenic tobacco plants carrying bbkst were subjected to congo-red staining as described in methods and subsequently the stained sections were studied under clsm. enhanced level of cellulose depositions in the walls of xylary fibers in the stems of transgenic plants was evident. the vascular zone was more pronounced in transgenic than empty vector-transformed t plants. similar observations were also noted in t transgenic tobacco plants. biochemical quantification of cellulose contents cellulose contents of five independent transgenic plants expressing bbkst gene and empty vector transformed tobacco plants were analysed as stated in method and the quantitative data are represented in table  cellulose content of these transgenic lines varied from - % fresh wt. with an average of % fresh wt, whereas % cellulose content was noted in the vector-transformed tobacco plants. overall a significant fold increase of cellulose contents in transgenic tobacco plants expressing bbkst gene was noted, compared to that of vector-transformed plant. discussion in a previous study, a species specific sequence characterized amplified region marker was developed that discriminates b. balcooa from other bamboo species. further investigations with different natural genetic resources of b. balcooa yielded a second dna marker, balco that has been successfully employed to identify the elite genotypes of b. balcooa endowed with high cellulose contents and possessing superior fiber qualities with respect to tensile strength and durability. however, the genetic linkage between balco and high cellulose containing trait could not be demonstrated due to the monocarpic nature and unusually long sexual cycle of bamboo. nevertheless, in silico analysis revealed the presence of an open reading frame within the marker sequence which prompted us to identify the full length sequence of bbkst gene. the 7375 bp long full length bbkst consists of introns, exons, 5′-3′ utr and an upstream promoter like region. database searches revealed the presence of key promoter-specific structural elements and many cis-acting elements in the upstream region of bbkst gene; among these, the rss site regulates phloem specific sucrose synthesis by enhancing sucrose synthase production. the deduced amino acid sequence of bbkst showed high sequence homology with other reported protein kinases from various plant taxa. the complete orf of the protein upon blastp illustrated that bbkst has more than 70% homology with arabidopsis thaliana, hypothetical protein of vitis vinifera, an atp binding protein of ricinus communis, predicted protein of populus trichocarpa and hypothetical protein sorbidraft_01g of sorghum bicolor and putative protein kinase of oryza sativa var. japonica. 5′ of the bbkst protein sequence contains six lrr motifs which may act as substrate binding sites and the 3′ end contains a serine-threonine protein kinase domain, devoid of any transmembrane domains. pfam domain search predicted that bbkst is a receptor like cytoplasmic kinase. lrr-rlks possess different numbers of lrr variously arranged in their extracellular domains, which are involved in protein-protein interaction and in targeting the receptors. plant rlcks usually comprised of a carboxyl-terminal kinase domain but no apparent signal sequence or transmembrane domain are present. although a large number of rlks are present in plant genomes, function of most of these rlks and rlcks are not known. a limited number of rlcks are functionally characterized, for example, brassica m-locus protein kinase mlpk associated with self-incompatibility, tobacco ntpk and ntpk involved in pollen germination and pollen tube elongation, tomato pto and pti in disease resistance, soybean gmpti in coordinating plant defense responses and arabidopsis cdg related to br signaling. there are few reports available in monocotyledonous plants, for example, zmpti1a in maize involved in pollen callose deposition, and zmpti1b, in pathogen defense and another reported from rice osrlck involved in pollen performance during pollination. there are very few evidences presuming that kinases of the rlk family have an additive role in superior fiber development. however no comprehensive study has yet been made to elucidate the biochemical nature underpinning their biological activity. in this communication we have analyzed biochemical properties of the bbkst-protein kinase, including the substrate specificity, divalent cation preference, phosphate donor specificity and potential inhibitors of bbkst-kinase activity. it was found that bbkst-kinase is a monomeric 42-kd serine/threonine protein kinase enzyme, phophorylating a broad spectrum of proteins. all kinases require divalent cations to coordinate the phosphate groups of the phosphate donor. bbkst, like most serine/threonine kinases, exhibited a strong preference for mg+ over mn+ in the phosphorylation reaction. bbkst lack any regulatory domains hence phosphorylation and dephophorylation appear to be main regulatory mechanisms of its activity as proposed by kobayashi et al.. bamboo fibers are highly specialized cells with three distinct stages of differentiation. stage i consists of fiber cell differentiation/initiation; stage ii comprises of fiber cell elongation and at this stage secondary cell wall deposition starts; stage iii includes maturation of fiber, in which fiber cell elongation ceases and gradually additional wall layers are deposited. there is a solitary published report in the literature demonstrating genes that are differentially expressed at these three stages of fiber development, characterizing the cellular functions of these genes that are specifically expressed at a certain stage of fiber development. bbkst transcript expression was noted from stage i of fiber development. expression of the gene gradually increases during stage ii and declined after fiber maturation. transformed onion epidermal cells expressing the js construct carrying bbkst gene coupled with gfp showed expression of gfp in the cytoplasm indicating the proper orientation of the construct and confirms that the js is functional yet in a heterologous system. localization of gfp implied co-expression of bbkst in the cytoplasmic region. further investigations by co-expressing other sub-cellular markers would reveal precise location of bbkst within the cell. southern hybridization study revealed mostly single copy insertion in the gus positive seedlings. heterologous expression of bbkst transcripts in t lines was remarkably high in tested transgenic tobacco. the vascular region of transgenic tobacco containing bbkst gene was more pronounced than in the vector-transformed tobacco plants endowing additional tensile strength, which is also substantiated by their strong upright phenotypes, significantly higher flexibility coefficient, slenderness ratio, and lower runkel ratio of the fibers. the slenderness ratio or felting power is the physical indicator for fiber durability, while the magnitude of tensile strength is usually proportional to the flexibility coefficient. increase in the numbers of xylary fibers in the vascular bundles of transgenic plants was noted compared to stems of vector-transformed plants. enhanced level of cellulose depositions in the stems of t and t transgenic plants were apparent and confirmed by quantitative analyses of cellulose contents indicating enhanced cellulose synthase activity. such augmented cellulose deposition resulting in thicker xylem secondary cell wall formation has also been reported in the transgenic lines of hybrid poplar. plasma membrane-associated cellulose synthase is the catalytic unit of cellulose synthesis that triggers the functional cellulose-synthesis machinery during fiber development. cels have udp-glucose binding activity and β- 4-glucan synthesis activity. fujii and colleagues have demonstrated that sucrose synthase is an integral component of the cellulose synthesis machinery. in a contemporary investigation coleman et al. demonstrated close association of susy with cellulose synthesis during secondary wall formation. suppression of susy gene expression inhibited cotton fiber cell initiation and elongation. in our previous study we reported expression of susy gene in all the three stages of fiber development, but the highest expression was noted during fiber elongation concomitant with highest cellulose deposition. however, to date there is no evidence of enhanced cellulose synthesis associated with increased number of xylem fibers. nevertheless, cellulose-rich g -layer in the walls of fiber cells generates tensile strength in the secondary xylem. it is presumed from the present finding that bbkst has an active role in triggering susy production which stimulates cels resulting in cellulose overproduction, and providing extra tensile strength of the fiber in certain genotypes of b. balcooa endowed with the marker/gene. to the best of our knowledge, the results presented here are the first data describing the biochemical features of a receptor like cytoplasmic protein kinase involved in higher cellulose production and conferring superior fiber quality with high tensile strength and durability in transgenic plants. presumably bbkst plays an active role in triggering sucrose synthase production which stimulates cellulose synthase resulting in cellulose overproduction, consequently providing extra physical strengths to fibers of transgenic tobacco plants endowed with bbkst gene.  this is the first attempt to characterize a protein comprehensively at the biochemical and molecular level encoded by a gene associated with superior fiber quality in bamboo. further comprehensive study on all interacting proteins involved in superior quality fiber formation will aid in manipulating fiber compositions required for the paper, textile or timber industries, as well as to comprehend multifunctional activities of fiber cells. 
number of xylary fibers was higher in transgenic t and t plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower runkel ratio of the fibers. heterologous expression of bbkst under control of 35s promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. southern analysis revealed single copy insertion of bbkst in most of the agrobacterium mediated transgenic tobacco plants. balco was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. tissue specific expression studies showed much higher expression of bbkst transcript in stems and internodes of b. balcooa than in leaves and rhizomes. previously we have reported a genotype specific molecular marker, balco of bambusa balcooa containing an orf having high similarity with receptor like cytoplasmic kinase of arabidopsis and oryza. bambusa balcooafiberreceptor like cytoplasmic kinasescar markerserine-threoninekinaseleucine rich repeat domainkinase domain the full length cdna of bbkst, a serine-threonine protein kinase was isolated from b. balcooa comprising of six lrr domains at the n-terminal end and a kinase domain at the c-terminal end. bbkst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant. bioinformatics study followed by phospho-amino analysis further confirmed that bbkst-kinase belongs to the serine/threonine protein kinase family. this finding clearly demonstrated that bbkst gene encodes a serine/threonine protein kinase. under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development. transcript analysis of the bbkst gene following rna slot blot hybridization and qpcr revealed higher expression of bbkst during initiation and elongation stages of fiber development. molecular markers allow rapid identification of biologically important germplasm/s having desired character. bacteria-expressed bbkst-kinase domain showed mg2+ dependent kinase activity at ph , 28°c.
 molecular markers allow rapid identification of biologically important germplasm/s having desired character. previously we have reported a genotype specific molecular marker, balco of bambusa balcooa containing an orf having high similarity with receptor like cytoplasmic kinase of arabidopsis and oryza. balco was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development.  the full length cdna of bbkst, a serine-threonine protein kinase was isolated from b. balcooa comprising of six lrr domains at the n-terminal end and a kinase domain at the c-terminal end. bacteria-expressed bbkst-kinase domain showed mg2+ dependent kinase activity at ph , 28°c. bioinformatics study followed by phospho-amino analysis further confirmed that bbkst-kinase belongs to the serine/threonine protein kinase family. transcript analysis of the bbkst gene following rna slot blot hybridization and qpcr revealed higher expression of bbkst during initiation and elongation stages of fiber development. tissue specific expression studies showed much higher expression of bbkst transcript in stems and internodes of b. balcooa than in leaves and rhizomes. southern analysis revealed single copy insertion of bbkst in most of the agrobacterium mediated transgenic tobacco plants. real-time pcr detected 150- fold enhanced expression of bbkst in different t tobacco lines than that of the vector transformed plants. heterologous expression of bbkst under control of 35s promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. number of xylary fibers was higher in transgenic t and t plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower runkel ratio of the fibers.  this finding clearly demonstrated that bbkst gene encodes a serine/threonine protein kinase. bbkst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant. bambusa balcooafiberreceptor like cytoplasmic kinasescar markerserine-threoninekinaseleucine rich repeat domainkinase domain 