BACKGROUND
plant stems represent a major contribution to crop biomass.
they contain a large proportion of cell walls  <cit> .
cell walls play different roles in growth, development and transportation of nutrients and water.
they also provide a mechanical support for plants as well as an efficient protection against environmental stresses  <cit> .
cell walls mainly consist of large biopolymers such as cellulose, hemicelluloses, pectins and lignins.
the structure and composition of these polymers vary during development not only between different tissues of the same plant, but also from one plant to another  <cit> .
although plant cell walls are mainly composed of carbohydrates and lignins, cell wall proteins  make up approximately 10% of their mass.
cwps can become n- and/or o-glycosylated during their transit through the secretory pathway  <cit> .
a recent proteomic analysis using cona sepharose affinity chromatography on arabidopsis stems showed that the majority of the trapped proteins were predicted to be secreted  <cit> .
in addition, some cwps receive a glycosylphosphatidylinositol  anchor  <cit> .
classical approaches for the study and identification of cwps are based on cell wall purification, protein extraction, separation by electrophoresis and identification of proteins by mass spectrometry.
many proteomic analyses were performed on arabidopsis cell suspension cultures or organs including roots, stems, leaves and etiolated hypocotyls  <cit> .
to better understand the biological processes encompassed by the proteins identified by proteomic approaches, cwps were classified according to their known or predicted functions  <cit> .
however, several problems are encountered during extraction, detection and identification of cwps by proteomic approaches  <cit> .
it means that all available cell wall proteomes are actually sub-proteomes.
on the other hand, they only provide information on extracellular proteins.
all the intracellular proteins as well as many membrane proteins contributing to cell wall biogenesis are not taken into consideration.
the availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies  <cit> .
global gene expression as well as regulation at different physiological stages or in response to changes in the environment can be analyzed.
the expression patterns of arabidopsis genes that are supposed to be involved in cell wall dynamics were analyzed  <cit> .
molecular events involved in the transition from primary to secondary growth were studied in arabidopsis and hybrid aspen trees  <cit> .
genes encoding proteins involved in the synthesis of cell wall components, cell death proteins, transporters, cytoskeleton-interacting proteins, and transcription factors were identified.
finally, the expression of over  <dig> genes encoding carbohydrate-active enzymes  was analyzed in populus trichocarpa  <cit> .
this work describes the identification of arabidopsis genes expressed at different developmental stages of stems.
the aims were  to get an overview of the transcriptional activity of genes possibly involved in cell wall biogenesis and of genes encoding cell wall proteins;  to compare the results from this transcriptome analysis to those of a previous proteomic analysis performed on the same material  <cit> .
new genes that are possibly involved in cell wall biogenesis during stem elongation were identified and the importance of post-transcriptional events in regulation of genes encoding cwps was shown.
RESULTS
in order to analyse the expression pattern of genes encoding cwps, complete arabidopsis transcriptome microarrays  were used  <cit> .
catma contains  <dig>  gene specific tags  for known or predicted genes.
three developmental stages of stems were studied: young elongating stems, intermediate stage, and mature stems as described in methods.
in stems, two biological processes are of high importance  <cit> .
cell elongation and synthesis of primary cell walls occur until the final size of the stems is reached.
at the same time, differentiation of conducting tissues occurs.
some vessels at the bottom of young stems already have thickened cell walls and lignin deposition is starting.
first signs of fiber differentiation appear at the intermediate stage while thickened secondary cell walls undergo lignification.
in both cases, synthesis of cell wall components as well as their rearrangements are major events.
<dig>  sugar composition of stems at various stages of development
cell wall polysaccharide biogenesis includes polymer synthesis, secretion, assembly, and rearrangement during development  <cit> .
consequently, the sugar composition of the cell wall polysaccharides is in relation with development stage of the cells.
for this reason analysis of sugar composition of cell wall materials from stems of three developmental stages were performed .
this analysis was performed as previously described  <cit> .
the results revealed a drastic increase in xyl amount from young elongating to mature stems.
little or no significant changes in the amounts of the other sugars were observed.
these results strongly suggested that xylan is mostly deposited at intermediate and mature stages of stem development.
in order to confirm the presence of xylan and to determine its structure, additional analyses were performed.
the alcohol-insoluble material from arabidopsis mature stems was sequentially extracted with ammonium oxalate,  <dig> m and  <dig> m koh.
the glycosidic composition of these three fractions was determined by gas chromatography.
analysis of the ammonium oxalate extract revealed the presence of gala, ara, rha, and gal suggesting that this fraction was mainly composed of pectic material.
in contrast, xyl was the main monosaccharide  of the  <dig> m and  <dig> m koh extracts, together with small amount of glc and traces of other sugars including glca and 4-o-methyl glucuronic acid , indicating that xylan was the main alkali-extracted polymer of arabidopsis stems.
to investigate the structure of xylan present in the alkali extracts, the fractions were treated with an endo-xylanase and the resulting fragments were analysed by high performance anion exchange chromatography-pulse amperometric detection  and matrix-assisted laser desorption ionization-time of flight mass spectrometry .
similar data were obtained for  <dig> m and  <dig> m koh extracts.
the hpaec elution profile of the endo-xylanase-generated fragments indicated the presence of xyl and xylobiose , as well as peaks arising from xylan-derived acidic oligosaccharides that are eluted with high concentration of sodium acetate .
for structural identification of the acidic fragments, the endo-xylanase-generated oligomers were analysed by maldi-tof ms .
the main ion at m/z  <dig> corresponded to the + adduct of xyl4-4omeglca.
the ion at m/z  <dig> was assigned to the + adduct of xyl4-glca.
other ions corresponded to + and + adducts of the two acidic xylan fragments.
these data are in agreement with recent results reported in papers describing the biosynthesis of arabidopsis xylan  <cit> .
acidic fragments were isolated and were analysed by proton nuclear magnetic resonance .
by comparison with previous nmr data on glucuronoxylan fragments  <cit> , the spectrum was fully in agreement with the presence of a mixture of a β-linked xylopyranose residues regularly substituted on c- <dig> by α-4omeglca or α-glca in a 2/ <dig> ratio .
the xyl to uronic acid ratio was estimated to be about 6– <dig> on the basis of the peak integration of the hpaec profile of endo-xylanase-generated oligomers .
in conclusion, these data indicated that a glucuronoxylan exhibiting a 4omeglca/α-glca in a ratio of 2/ <dig> is deposited during arabidopis stem development and along with cellulose and lignin represents one of the main polymers in the mature stem.
<dig>  transcriptome microarrays analyses
in order to analyse the expression pattern of genes encoding cwps, complete arabidopsis transcriptome microarrays  were used  <cit> .
catma contains  <dig>  gene specific tags  for known or predicted genes.
results are expressed as log <dig> of signal mean intensity.
expression level is considered as high when values are higher than  <dig>  moderate for values between  <dig> and  <dig> and low for values between  <dig> and background level estimated at  <dig>  .
in young stems,  <dig>   <dig> and 48% of the probes give signals corresponding to high, moderate and low levels of transcripts, respectively; whereas 39% of the probes give a signal lower than background.
in mature stems,  <dig>   <dig> and 48% of the probes give signals corresponding to high, moderate and low levels of transcripts, respectively; whereas 42% of the probes give a signal lower than background.
a modulation of level of transcripts is revealed with  <dig> probes at least at one of the three stages of development.
among those,  <dig> probes reveal changes during the transition from young stems to intermediate stage, and  <dig> probes during the transition from intermediate stage to mature stems.
this transcriptome has been analyzed in more details and is described in the following sections.
<dig> .
levels of transcripts of genes involved in cell wall biogenesis and of genes encoding secreted proteins during stem development
the levels of transcripts of two groups of genes involved in the synthesis of cell wall components were analyzed.
these genes encode intracellular proteins or proteins located at the plasma membrane .
first group comprises genes encoding glycosyl transferases  involved in the synthesis of cell wall polysaccharides .
genes from all gt families analyzed had detectable levels of transcripts in all  <dig> samples.
the levels of transcripts of only a few genes were modulated during stem development, i.e.
<dig> out of  <dig>  most remarkable changes were noted for genes encoding cellulose synthases related to secondary wall formation  and callose synthases .
the second group comprises genes encoding proteins putatively involved in the biosynthesis of monolignols that are lignin precursors .
a greater proportion of genes had modulated levels of transcripts: either a decrease during the transition from young elongating stems to intermediate stage , or an increase during transitions from young elongating stems to intermediate stage , or intermediate stage to mature stems .
this analysis revealed that synthesis of the cell wall components required for biogenesis of primary and secondary walls is an active process throughout stem development.
genes encoding secreted proteins were sorted using bioinformatics softwares as described in methods.
genes expressed at moderate or high level have been further annotated for experimentally proven or predicted biological functions.
a total of  <dig> genes were classified into  <dig> functional categories according to jamet et al.
<cit>  .
a large proportion of the genes encode proteins with unknown functions , miscellaneous functions  and proteins acting on carbohydrates .
other genes encode proteins predicted to be involved in signalling , proteases , proteins with interaction domains , oxido-reductases , and structural proteins .
bioinformatic softwares were used to search for functional domains in the encoded proteins.
the proteins were distributed in eight functional classes.
percentages of genes in each class are indicated
overall and as shown in figure  <dig>  this transcriptome analysis revealed that  <dig> genes were up-regulated during the transition from young stems to intermediate stage whereas  <dig> genes were up-regulated during the transition from intermediate stage to mature stems.
on the contrary,  <dig> genes were down-regulated during the transition from young stems to intermediate stage, whereas  <dig> genes were down-regulated during the transition from intermediate stage to mature stems.
finally,  <dig> genes were up-regulated throughout the development of stems whereas  <dig> genes were down-regulated.
all these changes probably correspond to major changes in cell walls during stem development either through rearrangements of polysaccharide networks or other biological processes not yet described.
<dig> .
genes involved in cell wall biogenesis
genes involved in cell wall biogenesis per se encode proteins that can be intracellular, extracellular, or located at the plasma membrane.
intracellular proteins are those performing reactions necessary for the synthesis of all cell wall polysaccharides except cellulose and monolignols.
cellulose and callose synthases are at the plasma membrane.
extracellular proteins play roles in cell wall biogenesis and rearrangements of cell wall polymers including polysaccharides , lignins  and structural proteins .
glycosyl transferases
genes encoding enzymes belonging to several gt families such as gt <dig>  gt <dig>  gt <dig>  gt <dig>  gt <dig> and gt <dig>  have detectable levels of transcripts in stems .
two genes of the gt <dig> family have high or moderate levels of transcripts .
they were recently assumed to be associated with cesa <dig>  and cesa <dig>  in a tissue-specific way to form cellulose-synthase complexes  <cit> .
at3g <dig> and at5g <dig> are cellulose synthase-like  proteins assumed to be involved in synthesis of non-cellulosic polysaccharides such as mannan, glucomannan and xylan  <cit> .
four out of  <dig> genes of the gt <dig> family have high or moderate levels of transcripts at intermediate stage of stem development .
gatl <dig>  gaut <dig>  and gaut <dig> have been predicted to encode proteins with putative galacturonosyltransferase activity, whereas at3g <dig> is a glycogen starch initiation protein.
three genes of gt <dig> and gt <dig> families have high or moderate levels of transcripts.
enzymes in these families have not been experimentally characterized, but their putative functions are galactosyl transferases and xyloglucan 6-xylosyltransferases, and they could be involved in glycan processing  <cit> .
in addition, the gt <dig> family was proposed to be involved in biosynthesis of galactomannan and xyloglucan  <cit> .
four genes of the gt <dig> family have high or moderate levels of transcripts at intermediate stage of stem development.
at2g <dig>  encoding a putative glucuronyltransferase plays a role in secondary wall synthesis.
fra <dig> mutants have short stems, thinner fiber walls with reduced amounts of glucuronoxylan in cell walls, and lack glucuronic acid residues in the xylans  <cit> .
at1g <dig>  and at5g <dig>  were supposed to be involved in the synthesis of rg-ii  <cit> .
recent reinvestigation of these transferases demonstrated that are rather related to the elongation of the backbone of xylan in stems .
finally, at5g <dig> encodes a putative xyloglucan galactosyltransferase.
two highly expressed putative callose synthases  from the gt <dig> family were identified.
taken together, genes having high or moderate levels of transcripts identified in  <dig> gt families could be involved in the synthesis of cell wall polysaccharides, i.e.
gt <dig>  gt <dig>  gt <dig>  gt <dig> and gt <dig>  other functions include biosynthesis of glycan , and compounds involved in mobilisation of energy in the form of sucrose .
several genes were found to be homologous to poplar xylem-specific gt genes  <cit> .
extracellular proteins acting on carbohydrates
ghs belong to  <dig> different families whose functions were recently reviewed  <cit> .
fourty-five genes encoding proteins acting on carbohydrates were identified among which  <dig> are cazys  and  <dig> are expansins.
cazys include glycoside hydrolases , carbohydrate esterases , and polysaccharide lyases  .
about half of the identified ghs  such as those belonging to gh <dig>  gh <dig>  gh <dig>  gh <dig>  gh <dig>  gh <dig>  gh <dig> and gh <dig> families could be involved in cell wall modification.
their substrates are assumed to be pectins , xyloglucans , or xylans, arabinoxylans and arabinans .
the most represented families are gh <dig>  gh <dig>  and gh <dig>  seven xyloglucan endotransglucosylase-hydrolases  of the gh <dig> family were identified.
three of them  were expressed at high levels in young stems and at intermediate stage, but were down-regulated in mature stems.
some xths have been shown to function in cell elongation by loosening of cell walls  <cit> .
it has also been shown that xths can be involved in formation of secondary cell walls of vascular tissues  <cit> .
seven genes of the gh <dig> family encoding putative β- <dig> -glucanases were expressed at different stages of stem development.
only one was down-regulated in mature stems .
five of these enzymes are gpi-anchored.
β- <dig> -glucanases catalyze the hydrolysis of β- <dig> -glucan linkages of callose in plants, and play a role in various important physiological processes such as regulating pollen tube growth or defence against pathogen attack during fertilization  <cit> .
four genes of the gh <dig> family were identified by this transcriptomic analysis.
at1g <dig> and at1g <dig> were up-regulated during stem growth whereas at3g <dig> and at3g <dig> were down-regulated.
the enzymes of this family are known as β-glucosidases and are involved in diverse processes such as cell wall remodelling, formation of secondary walls, and activation of phytohormones  <cit> .
the other ghs such as putative chitinases , β-d-galactosidases , and β-d-mannosidases  could be involved in post-translational modifications such as glycosylations of proteins  <cit> .
it should also be noted that a mutation of atctl <dig>  causes ectopic deposition of lignin and aberrant shapes of cells with incomplete cell walls in the pith of inflorescence stems  <cit> .
one enzyme of the gh <dig> family encodes an invertase  possibly involved in mobilisation of energy in form of sucrose  <cit> .
a previously published article demonstrated the presence of various exo-glycoside hydrolases in the mature stems  <cit> .
here, stems at three different stages of development were analyzed to determine the potential changes in the levels of activity of these enzymes.
the results obtained  demonstrated the presence of all the gh activities tested at the three stages of stem development.
decreasing enzyme activities from young to mature protein extracts were observed for β-d-xylosidase, α-l-arabinofuranosidase, β-d-glucuronidase and β-d-manosidase.
however, enzyme activities for β-d-glucosidase, α-d-galactosidase, β-d-galactosidase and α-d-glucosidase remained unchanged.
this result as well as transcriptomic analyses  strongly suggested that the expression of ghs is regulated in relation to stem development.
six carbohydrate esterases  were identified, these are the pectin methylesterases  or pectin acetylesterases.
five of these genes were down-regulated in mature stems.
activities of these enzymes can cause major changes in the physical, chemical, and biological properties of pectins  <cit> .
at acidic ph, demethylesterification by pmes can promote the activity of polygalacturonases, thus contributing to cell wall loosening.
on the contrary, pme activity was shown to be inversely correlated with the rate of growth of expanding tissues, suggesting possible involvement in wall rigidification.
two genes encoding polysaccharide lyases  were down-regulated either during the whole development of stems , or during the transition from young stems to intermediate stage .
pls catalyze the cleavage of pectate, the de-esterified product of pectins, which is the major component maintaining the structural integrity of cell walls in higher plants  <cit> .
five α-expansins and one β-expansin are expressed at high or moderate levels during stem development.
two α-expansin genes  were repressed during stem development whereas the gene encoding a β-expansin was up-regulated during the transition from intermediate stage to mature stems.
interaction of xyloglucans, xylans and other hemicelluloses with cellulose is modulated by expansins that presumably disrupt hydrogen bonds between these components  <cit> .
genes involved in the synthesis of monolignols
monolignol biosynthetic pathway involves many different steps  <cit> .
the first steps are part of the more general phenylpropanoid pathway including synthesis of soluble phenolics such as flavonoids and sinapic esters.
based on bioinformatics and functional studies, complete inventories of genes involved in this pathway have been published for arabidopsis and a few genes of each multigene family were demonstrated to contribute to the phenylpropanoid pathway  <cit> .
the level of transcripts of all these genes has been analyzed in the stem transcriptomes .
several genes were found to be expressed throughout stem development: hydroxycinnamoyl coa:shikinate/quinase hydrocinnamoyl transferase , p-coumarate 3-hydroxylase  <dig> , ferulate 5-hydroxylase  and caffeic acid methyltransferase .
changes in the level of transcripts were observed for other genes.
level of transcripts of pal <dig> was higher in young stems than in mature stems, whereas levels of transcripts of pal <dig> and pal <dig> increased at the mature stage.
among the four genes encoding the phenylalanine ammonia-lyase , only pal <dig>  , pal <dig>  and pal <dig>  have been shown to be important in the phenylpropanoid pathway  <cit> .
concerning the next step of the pathway, level of transcripts of the trans-cinnamate 4-hydroxylase gene  was found to be higher in mature stems.
several 4-coumarate coa ligase genes  were shown to be involved to lignification  <cit> .
the increase of the level of transcripts of 4cl <dig> during stem development could be related to this process.
in contrast, nothing is known about the function of 4cl-like <dig> , whose level of transcripts was found to be modulated during stem development.
the levels of transcripts of caffeoyl-coa reductase genes  strongly increased during stem development.
however, no hypothesis on their role in lignification could be proposed since only ccoaomt <dig>  has been shown to be related to lignification  <cit> .
the two last steps of the pathway are specific to lignification  <cit> .
they involve cinnamoyl coa reductase  and cinnamyl alcohol dehydrogenase  genes.
level of transcript of ccr <dig>  was higher at the young elongating stems.
studies using knockout mutants showed that ccr <dig> is the major expressed gene in the biosynthetic pathway of lignin  <cit> .
nine genes belong to the cad multigene family  <cit> .
the levels of transcripts of three cad genes  decreased during the transition from young elongating stems to mature stems.
only cad <dig>  and cad <dig> have been shown to play major role in lignification  <cit> .
a minor role of cad <dig> in lignification of elongating stems has been shown  <cit> .
the level of transcripts of all the other cad genes which have not been demonstrated to be linked to lignification  <cit>  remained the same throughout stem development.
in conclusion, most genes already shown to contribute to monolignol biosynthesis were found to be expressed in stems.
a few of them like pal <dig> and 4-cl <dig> showed increase in their level of transcripts during stem development as expected from their demonstrated roles in monolignol biosynthesis.
however, the function of genes such as pal <dig>  4cl-like <dig>  ccoaomt- <dig>  ccoaomt- <dig> and cad <dig> should be investigated since their level of transcripts is modulated during stem development.
the genes identified as highly expressed in stems are the same as those reported in previous studies  <cit>  although the sampling and the growth conditions of the plants were different.
this demonstrates the importance of these genes in the monolignol metabolic pathway.
in addition, expression data bases such as genecat  using affimetrix information and rt-pcr  <cit>  indicate the same trend, that is high expression in the first and the second internodes.
extracellular oxido-reductases
four genes encoding peroxidases are expressed at moderate or high levels in developing stems .
one of them is up-regulated in mature stems  whereas one is down-regulated .
in addition,  <dig> peroxidase genes have low levels of transcripts at the three stages of stem development analyzed.
this study identified peroxidase genes that were not previously shown to be differentially expressed in developing stems  <cit> .
peroxidases belong to a large multigene family of  <dig> members in arabidopsis and play many roles during development and in response to environmental changes  <cit> .
two peroxidases of arabidopsis were shown to play a role in promoting root elongation  <cit> .
conversely, peroxidases can cause reduction in cell wall extensibility by formation of diferuloyl bridges between pectin residues, isodityrosine bridges between hydroxyproline-rich proteins like extensins, and covalent links between lignin precursors  <cit> .
eight genes encoding proteins with predicted multicopper oxidase domains have been identified.
such enzymes catalyse full four-electron reduction of dioxygen  to water  using a variety of substrates  <cit> .
four genes encoding putative laccases have moderate or high level of expression in developing stems .
laccase activity in plants has been assumed to participate in cell wall lignification  <cit> .
all four genes are up-regulated during the transition from young stems to intermediate stage as previously described  <cit> .
the expression of these genes was also reported to cluster with most monolignol biosynthetic genes in stems  <cit> .
three genes encoding multicopper oxidases of the sks family were identified .
two of them were down-regulated during stem development .
it was shown that sku <dig>  is involved in the control of root growth  <cit> , and that sks <dig>  contributes to cotyledon vascular patterning during development  <cit> .
three genes encoding germin-like proteins  were expressed at high levels in young stems and at intermediate stage of development.
two of them were strongly down-regulated in mature stems .
although the functions of glps are not clearly understood, it was shown that a glp gene is highly expressed during cotton fiber elongation, but is repressed once the rate of growth slows down  <cit> .
considering the variety of gene families involved in modification of carbohydrates and in oxido-reduction reactions, these results show that all cell wall components probably undergo modifications during stem growth.
even if many of these gene families were known to be involved in cell wall assembly or remodelling, this transcriptomic analysis allows the identification of genes involved in stem development processes.
<dig> .
genes encoding secreted proteins involved in other cell wall functions
proteins with interaction domains
several families of genes encoding proteins with putative domains of interaction with polysaccharides and/or proteins were identified.
these include proteins with leucine-rich repeat  domains , lectins  and enzyme inhibitors .
due to their high specificity towards carbohydrates, lectins may be involved in various physiological functions  <cit> .
they could be involved in the recognition between cells or between cells and various carbohydrate-containing molecules or in assembly of the polysaccharide matrix.
other possible functions of lectins in plants include: transport and packaging of carbohydrates, and mobilization of storage materials.
inhibitors of pmes regulate plant pme activity.
five genes encoding pme inhibitors were identified among which  <dig> are down-regulated in mature stems .
this down-regulation seems to be correlated with that of  <dig> genes encoding pmes mentioned above.
proteins involved in signaling
nine genes encoding arabinogalactan proteins  and  <dig> genes encoding fasciclin-like agps  had high or moderate levels of transcripts in developing stems.
in addition,  <dig> and  <dig> genes encoding agps and flas, respectivement, had low levels of transcripts.
five fla genes are down-regulated in mature stems.
agps belong to a class of hyp-rich glycoproteins that are highly glycosylated and abundant in plant cell walls  <cit> .
agps have been involved in plant development such as cell fate determination, somatic embryogenesis, and cell proliferation  <cit> .
agps are assumed to be signal molecules  <cit> .
the association of agps with pectic polysaccharides has also been suggested  <cit> .
flas constitute a distinct class of proteins.
proteins containing fasciclin domains have been shown to function as adhesion molecules in a broad spectrum of organisms  <cit> .
more recently, agps and flas were found to be associated with wood formation in poplar  <cit> .
twenty-two genes encoding putative receptor kinases are expressed at high or moderate levels in developing stems.
only a few of them are up- or down-regulated.
protein kinases play a very significant role in signal transduction.
our screening revealed that  <dig> genes are lrr-rlks  <cit> .
their extracellular domain is composed of tandem repeats of a well-conserved leucine-rich motif and their intracellular domain consists in a protein kinase with ser/thr specificity.
lrr-rlks are involved in development, hormone perception, and pathogen response  <cit> .
proteases
twenty-four genes encoding putative proteinases were identified.
these enzymes belong to asp-, ser- and cys-protease families.
proteinases may be involved in processing and/or turnover of cwps, as well as in peptide signal transduction  <cit> .
several proteomic analyses of plant cell walls show a large discrepancy between the observed and the expected molecular weights of proteins, suggesting the existence of a turnover of cwps  <cit> .
expression of a cys protease was shown to be associated with programmed cell death  <cit> .
the occurrence of many proteases and their high expression levels during stem development suggests that these enzymes are actively involved in cell expansion and/or in secondary wall formation.
however, their activity could be modulated by the presence of many protease inhibitors since  <dig> genes encoding such inhibitors were found to have moderate or high levels of transcripts.
structural proteins
only a few genes encoding structural cwps were found to have moderate or high levels of transcripts in developing stems.
this low number might be the consequence of the difficulty to design specific probes for genes encoding repetitive sequences of amino acids such as structural cwps.
indeed, catma does not offer a good coverage of such gene families, e.g.
only  <dig> extensin genes out of  <dig> were analyzed.
identified genes belong to the major classes of cell wall structural proteins, e.g.
extensins, proline rich-proteins , glycine rich-proteins  and lrr-extensins .
five out of thirteen genes are down-regulated in mature stems whereas two genes encoding grps are up-regulated .
structural cwps such as extensins and prps are assumed to be insolubilized when cell elongation ceases  <cit> .
extensins, grps, and prps were shown to be expressed in specific cell types including xylem and phloem tissues by tissue-printing of stems  <cit> .
the role of grps in the building of the cell walls of protoxylem elements has been shown by immunological studies  <cit> .
miscellaneous proteins
a number of genes  encoding miscellaneous proteins were identified by transcriptomic analysis.
a large proportion of these genes encode proteins predicted to be protease inhibitors  or are involved in lipid metabolism .
lipids are essential for cell growth  <cit>  and results of these analyses suggest that lipid metabolism is important for stem development.
five genes encoding thaumatin-like proteins among which  <dig> are repressed during stem development were identified.
they encode pathogenesis-related proteins that are expressed constitutively at certain stages of plant development  <cit> .
identification of several zn transporters suggests that zn is an essential micronutrient for stem development.
four genes encode phytocyanins .
three of these genes are down-regulated in mature stems.
it should be noted that  <dig> genes of the same family are expressed at low levels in developing stems.
phytocyanins are assumed to play a role in oxido-reduction processes in cell walls as electron transfer proteins  <cit> .
in addition, stellacyanins and uclacyanins have structural domain possibly interacting with structural cwps.
proteins of yet unknown function
numerous genes  encoding proteins with unknown function were identified in this transcriptomic analysis.
some of which share structural domains or the so-called domains of unknown function .
about half of them encode proteins predicted to have trans-membrane domains or gpi anchors.
the encoded proteins may play house-keeping functions or be specifically involved in stem development.
altogether, many gene families encoding secreted cell wall proteins were found to have members expressed at high or moderate levels during stem development.
proteinase genes are actively transcribed, suggesting essential roles in protein processing, turnover, or signalling.
finally, many genes have yet unknown function during stem development.
validation of expression microarray results by real-time-qpcr
in order to validate the results from microarray analysis, transcripts of  <dig> selected genes, which were significantly expressed in the microarray studies, were analyzed by real-time-qpcr .
the majority of these selected genes were related to cell wall biogenesis.
these included 4-coumarate-coa ligase , caffeoyl-coa 3-o-methyltransferase , cellulose synthase subunit , cellulose synthase, catalytic subunit , cellulose synthase, catalytic subunit , putative laccase , gh of family  <dig> , pectate lyase , phosphate responsive protein , plasma membrane intrinsic protein 2c , putative expansin , chlorophyll a-b binding protein , catalase  <dig>  and putative raffinose synthase .
two genes, actin  <dig>  and 26s proteasome regulatory subunit , were selected as endogenous control.
compared with transcriptomic analyses, significant correlation was confirmed by rt-qpcr according to the calculated average in transcript levels of all the tested genes.
<dig>  transcriptomics vs proteomics
in order to better understand gene regulation in stems, data from transcriptomics were compared to those of a previous proteomic study performed using the same biological material, i.e.
mature arabidopsis stems  <cit> .
this proteomic study was designed to identify n-glycoproteins trapped on cona sepharose and allowed the identification of  <dig> genes encoding secreted proteins.
their levels of transcripts were searched in the stem transcriptome.
results were obtained for  <dig> genes on the catma hybridized with total rnas from mature stems .
eighteen genes were found to have levels of transcripts lower than background,  <dig> had weak levels of transcripts,  <dig> moderate levels of transcripts and  <dig> high levels of transcripts.
altogether, 25% of the genes identified through proteomics have levels of expression below background and 58% have low level of transcripts.
the percentage of genes having detectable levels of expression decreases from about 82% to 75% during stem growth.
the encoded proteins might have long half-lifes and remain in cell walls after the disappearance of the mrnas.
it means that transcriptomics fails to give an overall picture of proteins present in cell walls at a given time.
conversely, only  <dig> genes identified through proteomics are found among the  <dig> genes encoding secreted proteins expressed at high or moderate level.
this means that many proteins escaped this proteomic analyses.
one explanation might be the partial analysis of proteins separated by 2-d electrophoresis  <cit> .
in addition, low abundant proteins are not usually identified in proteomic studies, and structural proteins are difficult to extract from cell walls since they form insoluble networks  <cit> .
proteins with transmembrane domains are also not easily extracted by salts.
agps and flas are difficult to identify because of their high level of glycosylation.
however, these explanations are not valid for proteins like ghs that are soluble.
alternatively, the numerous proteases present in cell walls probably play a great role in protein turnover, although it was shown that glycoproteins are more resistant against digestion by proteases  <cit> .
in addition, some regulatory mechanisms preventing the translation of certain mrnas may also exist.
such discrepancies between proteomics and transcriptomics have been described in yeast  <cit> .
finally, proteome and transcriptome approaches appear to be complementary to get an overview of genes expressed in developing stems.
the observed discrepancies show the importance of post-transcriptional regulations.
CONCLUSIONS
here we have examined the expression patterns of the genes encoding cell wall proteins.
some of the genes identified in this work were previously identified by various microarray analyses of arabidopsis stems.
these include xth  <cit> ,  <dig> -β-glucanase and pectin esterase  <cit> , pectate lyase, agp, glycine rich and proline rich proteins  <cit> .
however, we identified and established expression patterns of many new genes possibly involved in primary and secondary cell wall biogenesis.
many genes involved in the synthesis of cell wall components were found to be transcribed as well as many genes encoding secreted proteins.
the latter genes encode proteins acting on carbohydrates, oxido-reductases, proteins with interaction domains and structural proteins.
among secreted proteins, proteins acting on carbohydrates represent the most abundant class after proteins of unknown function.
in addition, genes encoding proteins such as agp, flas, grps and prps not reported by proteomic analyses, had high levels of transcripts.
this transcriptomic analysis allowed identification of many gene candidates that play a role in cell wall construction during stem development.
both primary and secondary walls are drastically modified thus allowing cells to undergo elongation during early steps of development.
these modifications are part of the differentiation processes during late stages of development.
the enzymes which catalyze glucan-chain elongation in cellulose, belonging to the family  <dig> of glycoside transferase , are crucial for primary and secondary wall formations  <cit> .
in plants this family of gts, designated as the cellulose synthase catalytic subunits , has multiple members.
in the present work several highly expressed genes of this family of enzymes have been identified .
comparison of the gene expression profile of cesa genes obtained in the present study with those of genecat database () showed similar high expression profile of  <dig> genes with exception of at2g <dig> which is not expressed according to genecat database.
though good correlation in expression scores of these genes were obtained, some differences were observed concerning expression level or difference in expression.
these differences could be consequences of developmental stage of plant, condition of growth, environment and from minor unavoidable experimental errors during extraction of rnas and measurement.
finally, comparison of transcriptomic and proteomic analyses showed that about 25% of the genes encoding proteins identified by proteomics had level of transcripts above background in the catma analysis.
conversely, only a small proportion of genes identified by proteomics were identified by transcriptomics, i.e.
only  <dig> out of the  <dig> genes encoding secreted proteins were expressed at high or moderate levels.
such discrepancies between proteomics and transcriptomics suggest complex regulatory mechanisms which occur at post-transcriptional levels.

 plant stems represent a major contribution to crop biomass. they contain a large proportion of cell walls. cell walls play different roles in growth, development and transportation of nutrients and water. they also provide a mechanical support for plants as well as an efficient protection against environmental stresses. cell walls mainly consist of large biopolymers such as cellulose, hemicelluloses, pectins and lignins. the structure and composition of these polymers vary during development not only between different tissues of the same plant, but also from one plant to another. although plant cell walls are mainly composed of carbohydrates and lignins, cell wall proteins make up approximately 10% of their mass. cwps can become n- and/or o-glycosylated during their transit through the secretory pathway. a recent proteomic analysis using cona sepharose affinity chromatography on arabidopsis stems showed that the majority of the trapped proteins were predicted to be secreted. in addition, some cwps receive a glycosylphosphatidylinositol anchor. classical approaches for the study and identification of cwps are based on cell wall purification, protein extraction, separation by electrophoresis and identification of proteins by mass spectrometry. many proteomic analyses were performed on arabidopsis cell suspension cultures or organs including roots, stems, leaves and etiolated hypocotyls. to better understand the biological processes encompassed by the proteins identified by proteomic approaches, cwps were classified according to their known or predicted functions. however, several problems are encountered during extraction, detection and identification of cwps by proteomic approaches. it means that all available cell wall proteomes are actually sub-proteomes. on the other hand, they only provide information on extracellular proteins. all the intracellular proteins as well as many membrane proteins contributing to cell wall biogenesis are not taken into consideration. the availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. global gene expression as well as regulation at different physiological stages or in response to changes in the environment can be analyzed. the expression patterns of arabidopsis genes that are supposed to be involved in cell wall dynamics were analyzed. molecular events involved in the transition from primary to secondary growth were studied in arabidopsis and hybrid aspen trees. genes encoding proteins involved in the synthesis of cell wall components, cell death proteins, transporters, cytoskeleton-interacting proteins, and transcription factors were identified. finally, the expression of over genes encoding carbohydrate-active enzymes was analyzed in populus trichocarpa. this work describes the identification of arabidopsis genes expressed at different developmental stages of stems. the aims were to get an overview of the transcriptional activity of genes possibly involved in cell wall biogenesis and of genes encoding cell wall proteins; to compare the results from this transcriptome analysis to those of a previous proteomic analysis performed on the same material. new genes that are possibly involved in cell wall biogenesis during stem elongation were identified and the importance of post-transcriptional events in regulation of genes encoding cwps was shown.  in order to analyse the expression pattern of genes encoding cwps, complete arabidopsis transcriptome microarrays were used. catma contains gene specific tags for known or predicted genes. three developmental stages of stems were studied: young elongating stems, intermediate stage, and mature stems as described in methods. in stems, two biological processes are of high importance. cell elongation and synthesis of primary cell walls occur until the final size of the stems is reached. at the same time, differentiation of conducting tissues occurs. some vessels at the bottom of young stems already have thickened cell walls and lignin deposition is starting. first signs of fiber differentiation appear at the intermediate stage while thickened secondary cell walls undergo lignification. in both cases, synthesis of cell wall components as well as their rearrangements are major events. sugar composition of stems at various stages of development cell wall polysaccharide biogenesis includes polymer synthesis, secretion, assembly, and rearrangement during development. consequently, the sugar composition of the cell wall polysaccharides is in relation with development stage of the cells. for this reason analysis of sugar composition of cell wall materials from stems of three developmental stages were performed. this analysis was performed as previously described. the results revealed a drastic increase in xyl amount from young elongating to mature stems. little or no significant changes in the amounts of the other sugars were observed. these results strongly suggested that xylan is mostly deposited at intermediate and mature stages of stem development. in order to confirm the presence of xylan and to determine its structure, additional analyses were performed. the alcohol-insoluble material from arabidopsis mature stems was sequentially extracted with ammonium oxalate, m and m koh. the glycosidic composition of these three fractions was determined by gas chromatography. analysis of the ammonium oxalate extract revealed the presence of gala, ara, rha, and gal suggesting that this fraction was mainly composed of pectic material. in contrast, xyl was the main monosaccharide of the m and m koh extracts, together with small amount of glc and traces of other sugars including glca and 4-o-methyl glucuronic acid, indicating that xylan was the main alkali-extracted polymer of arabidopsis stems. to investigate the structure of xylan present in the alkali extracts, the fractions were treated with an endo-xylanase and the resulting fragments were analysed by high performance anion exchange chromatography-pulse amperometric detection and matrix-assisted laser desorption ionization-time of flight mass spectrometry. similar data were obtained for m and m koh extracts. the hpaec elution profile of the endo-xylanase-generated fragments indicated the presence of xyl and xylobiose, as well as peaks arising from xylan-derived acidic oligosaccharides that are eluted with high concentration of sodium acetate. for structural identification of the acidic fragments, the endo-xylanase-generated oligomers were analysed by maldi-tof ms. the main ion at m/z corresponded to the + adduct of xyl4-4omeglca. the ion at m/z was assigned to the + adduct of xyl4-glca. other ions corresponded to + and + adducts of the two acidic xylan fragments. these data are in agreement with recent results reported in papers describing the biosynthesis of arabidopsis xylan. acidic fragments were isolated and were analysed by proton nuclear magnetic resonance. by comparison with previous nmr data on glucuronoxylan fragments, the spectrum was fully in agreement with the presence of a mixture of a β-linked xylopyranose residues regularly substituted on c- by α-4omeglca or α-glca in a 2/ ratio. the xyl to uronic acid ratio was estimated to be about 6– on the basis of the peak integration of the hpaec profile of endo-xylanase-generated oligomers. in conclusion, these data indicated that a glucuronoxylan exhibiting a 4omeglca/α-glca in a ratio of 2/ is deposited during arabidopis stem development and along with cellulose and lignin represents one of the main polymers in the mature stem. transcriptome microarrays analyses in order to analyse the expression pattern of genes encoding cwps, complete arabidopsis transcriptome microarrays were used. catma contains gene specific tags for known or predicted genes. results are expressed as log of signal mean intensity. expression level is considered as high when values are higher than moderate for values between and and low for values between and background level estimated at. in young stems, and 48% of the probes give signals corresponding to high, moderate and low levels of transcripts, respectively; whereas 39% of the probes give a signal lower than background. in mature stems, and 48% of the probes give signals corresponding to high, moderate and low levels of transcripts, respectively; whereas 42% of the probes give a signal lower than background. a modulation of level of transcripts is revealed with probes at least at one of the three stages of development. among those, probes reveal changes during the transition from young stems to intermediate stage, and probes during the transition from intermediate stage to mature stems. this transcriptome has been analyzed in more details and is described in the following sections.. levels of transcripts of genes involved in cell wall biogenesis and of genes encoding secreted proteins during stem development the levels of transcripts of two groups of genes involved in the synthesis of cell wall components were analyzed. these genes encode intracellular proteins or proteins located at the plasma membrane. first group comprises genes encoding glycosyl transferases involved in the synthesis of cell wall polysaccharides. genes from all gt families analyzed had detectable levels of transcripts in all samples. the levels of transcripts of only a few genes were modulated during stem development, i.e. out of most remarkable changes were noted for genes encoding cellulose synthases related to secondary wall formation and callose synthases. the second group comprises genes encoding proteins putatively involved in the biosynthesis of monolignols that are lignin precursors. a greater proportion of genes had modulated levels of transcripts: either a decrease during the transition from young elongating stems to intermediate stage, or an increase during transitions from young elongating stems to intermediate stage, or intermediate stage to mature stems. this analysis revealed that synthesis of the cell wall components required for biogenesis of primary and secondary walls is an active process throughout stem development. genes encoding secreted proteins were sorted using bioinformatics softwares as described in methods. genes expressed at moderate or high level have been further annotated for experimentally proven or predicted biological functions. a total of genes were classified into functional categories according to jamet et al.. a large proportion of the genes encode proteins with unknown functions, miscellaneous functions and proteins acting on carbohydrates. other genes encode proteins predicted to be involved in signalling, proteases, proteins with interaction domains, oxido-reductases, and structural proteins. bioinformatic softwares were used to search for functional domains in the encoded proteins. the proteins were distributed in eight functional classes. percentages of genes in each class are indicated overall and as shown in figure this transcriptome analysis revealed that genes were up-regulated during the transition from young stems to intermediate stage whereas genes were up-regulated during the transition from intermediate stage to mature stems. on the contrary, genes were down-regulated during the transition from young stems to intermediate stage, whereas genes were down-regulated during the transition from intermediate stage to mature stems. finally, genes were up-regulated throughout the development of stems whereas genes were down-regulated. all these changes probably correspond to major changes in cell walls during stem development either through rearrangements of polysaccharide networks or other biological processes not yet described.. genes involved in cell wall biogenesis genes involved in cell wall biogenesis per se encode proteins that can be intracellular, extracellular, or located at the plasma membrane. intracellular proteins are those performing reactions necessary for the synthesis of all cell wall polysaccharides except cellulose and monolignols. cellulose and callose synthases are at the plasma membrane. extracellular proteins play roles in cell wall biogenesis and rearrangements of cell wall polymers including polysaccharides, lignins and structural proteins. glycosyl transferases genes encoding enzymes belonging to several gt families such as gt gt gt gt gt and gt have detectable levels of transcripts in stems. two genes of the gt family have high or moderate levels of transcripts. they were recently assumed to be associated with cesa and cesa in a tissue-specific way to form cellulose-synthase complexes. at3g and at5g are cellulose synthase-like proteins assumed to be involved in synthesis of non-cellulosic polysaccharides such as mannan, glucomannan and xylan. four out of genes of the gt family have high or moderate levels of transcripts at intermediate stage of stem development. gatl gaut and gaut have been predicted to encode proteins with putative galacturonosyltransferase activity, whereas at3g is a glycogen starch initiation protein. three genes of gt and gt families have high or moderate levels of transcripts. enzymes in these families have not been experimentally characterized, but their putative functions are galactosyl transferases and xyloglucan 6-xylosyltransferases, and they could be involved in glycan processing. in addition, the gt family was proposed to be involved in biosynthesis of galactomannan and xyloglucan. four genes of the gt family have high or moderate levels of transcripts at intermediate stage of stem development. at2g encoding a putative glucuronyltransferase plays a role in secondary wall synthesis. fra mutants have short stems, thinner fiber walls with reduced amounts of glucuronoxylan in cell walls, and lack glucuronic acid residues in the xylans. at1g and at5g were supposed to be involved in the synthesis of rg-ii. recent reinvestigation of these transferases demonstrated that are rather related to the elongation of the backbone of xylan in stems. finally, at5g encodes a putative xyloglucan galactosyltransferase. two highly expressed putative callose synthases from the gt family were identified. taken together, genes having high or moderate levels of transcripts identified in gt families could be involved in the synthesis of cell wall polysaccharides, i.e. gt gt gt gt and gt other functions include biosynthesis of glycan, and compounds involved in mobilisation of energy in the form of sucrose. several genes were found to be homologous to poplar xylem-specific gt genes. extracellular proteins acting on carbohydrates ghs belong to different families whose functions were recently reviewed. fourty-five genes encoding proteins acting on carbohydrates were identified among which are cazys and are expansins. cazys include glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases. about half of the identified ghs such as those belonging to gh gh gh gh gh gh gh and gh families could be involved in cell wall modification. their substrates are assumed to be pectins, xyloglucans, or xylans, arabinoxylans and arabinans. the most represented families are gh gh and gh seven xyloglucan endotransglucosylase-hydrolases of the gh family were identified. three of them were expressed at high levels in young stems and at intermediate stage, but were down-regulated in mature stems. some xths have been shown to function in cell elongation by loosening of cell walls. it has also been shown that xths can be involved in formation of secondary cell walls of vascular tissues. seven genes of the gh family encoding putative β- -glucanases were expressed at different stages of stem development. only one was down-regulated in mature stems. five of these enzymes are gpi-anchored. β- -glucanases catalyze the hydrolysis of β- -glucan linkages of callose in plants, and play a role in various important physiological processes such as regulating pollen tube growth or defence against pathogen attack during fertilization. four genes of the gh family were identified by this transcriptomic analysis. at1g and at1g were up-regulated during stem growth whereas at3g and at3g were down-regulated. the enzymes of this family are known as β-glucosidases and are involved in diverse processes such as cell wall remodelling, formation of secondary walls, and activation of phytohormones. the other ghs such as putative chitinases, β-d-galactosidases, and β-d-mannosidases could be involved in post-translational modifications such as glycosylations of proteins. it should also be noted that a mutation of atctl causes ectopic deposition of lignin and aberrant shapes of cells with incomplete cell walls in the pith of inflorescence stems. one enzyme of the gh family encodes an invertase possibly involved in mobilisation of energy in form of sucrose. a previously published article demonstrated the presence of various exo-glycoside hydrolases in the mature stems. here, stems at three different stages of development were analyzed to determine the potential changes in the levels of activity of these enzymes. the results obtained demonstrated the presence of all the gh activities tested at the three stages of stem development. decreasing enzyme activities from young to mature protein extracts were observed for β-d-xylosidase, α-l-arabinofuranosidase, β-d-glucuronidase and β-d-manosidase. however, enzyme activities for β-d-glucosidase, α-d-galactosidase, β-d-galactosidase and α-d-glucosidase remained unchanged. this result as well as transcriptomic analyses strongly suggested that the expression of ghs is regulated in relation to stem development. six carbohydrate esterases were identified, these are the pectin methylesterases or pectin acetylesterases. five of these genes were down-regulated in mature stems. activities of these enzymes can cause major changes in the physical, chemical, and biological properties of pectins. at acidic ph, demethylesterification by pmes can promote the activity of polygalacturonases, thus contributing to cell wall loosening. on the contrary, pme activity was shown to be inversely correlated with the rate of growth of expanding tissues, suggesting possible involvement in wall rigidification. two genes encoding polysaccharide lyases were down-regulated either during the whole development of stems, or during the transition from young stems to intermediate stage. pls catalyze the cleavage of pectate, the de-esterified product of pectins, which is the major component maintaining the structural integrity of cell walls in higher plants. five α-expansins and one β-expansin are expressed at high or moderate levels during stem development. two α-expansin genes were repressed during stem development whereas the gene encoding a β-expansin was up-regulated during the transition from intermediate stage to mature stems. interaction of xyloglucans, xylans and other hemicelluloses with cellulose is modulated by expansins that presumably disrupt hydrogen bonds between these components. genes involved in the synthesis of monolignols monolignol biosynthetic pathway involves many different steps. the first steps are part of the more general phenylpropanoid pathway including synthesis of soluble phenolics such as flavonoids and sinapic esters. based on bioinformatics and functional studies, complete inventories of genes involved in this pathway have been published for arabidopsis and a few genes of each multigene family were demonstrated to contribute to the phenylpropanoid pathway. the level of transcripts of all these genes has been analyzed in the stem transcriptomes. several genes were found to be expressed throughout stem development: hydroxycinnamoyl coa:shikinate/quinase hydrocinnamoyl transferase, p-coumarate 3-hydroxylase, ferulate 5-hydroxylase and caffeic acid methyltransferase. changes in the level of transcripts were observed for other genes. level of transcripts of pal was higher in young stems than in mature stems, whereas levels of transcripts of pal and pal increased at the mature stage. among the four genes encoding the phenylalanine ammonia-lyase, only pal, pal and pal have been shown to be important in the phenylpropanoid pathway. concerning the next step of the pathway, level of transcripts of the trans-cinnamate 4-hydroxylase gene was found to be higher in mature stems. several 4-coumarate coa ligase genes were shown to be involved to lignification. the increase of the level of transcripts of 4cl during stem development could be related to this process. in contrast, nothing is known about the function of 4cl-like, whose level of transcripts was found to be modulated during stem development. the levels of transcripts of caffeoyl-coa reductase genes strongly increased during stem development. however, no hypothesis on their role in lignification could be proposed since only ccoaomt has been shown to be related to lignification. the two last steps of the pathway are specific to lignification. they involve cinnamoyl coa reductase and cinnamyl alcohol dehydrogenase genes. level of transcript of ccr was higher at the young elongating stems. studies using knockout mutants showed that ccr is the major expressed gene in the biosynthetic pathway of lignin. nine genes belong to the cad multigene family. the levels of transcripts of three cad genes decreased during the transition from young elongating stems to mature stems. only cad and cad have been shown to play major role in lignification. a minor role of cad in lignification of elongating stems has been shown. the level of transcripts of all the other cad genes which have not been demonstrated to be linked to lignification remained the same throughout stem development. in conclusion, most genes already shown to contribute to monolignol biosynthesis were found to be expressed in stems. a few of them like pal and 4-cl showed increase in their level of transcripts during stem development as expected from their demonstrated roles in monolignol biosynthesis. however, the function of genes such as pal 4cl-like ccoaomt- ccoaomt- and cad should be investigated since their level of transcripts is modulated during stem development. the genes identified as highly expressed in stems are the same as those reported in previous studies although the sampling and the growth conditions of the plants were different. this demonstrates the importance of these genes in the monolignol metabolic pathway. in addition, expression data bases such as genecat using affimetrix information and rt-pcr indicate the same trend, that is high expression in the first and the second internodes. extracellular oxido-reductases four genes encoding peroxidases are expressed at moderate or high levels in developing stems. one of them is up-regulated in mature stems whereas one is down-regulated. in addition, peroxidase genes have low levels of transcripts at the three stages of stem development analyzed. this study identified peroxidase genes that were not previously shown to be differentially expressed in developing stems. peroxidases belong to a large multigene family of members in arabidopsis and play many roles during development and in response to environmental changes. two peroxidases of arabidopsis were shown to play a role in promoting root elongation. conversely, peroxidases can cause reduction in cell wall extensibility by formation of diferuloyl bridges between pectin residues, isodityrosine bridges between hydroxyproline-rich proteins like extensins, and covalent links between lignin precursors. eight genes encoding proteins with predicted multicopper oxidase domains have been identified. such enzymes catalyse full four-electron reduction of dioxygen to water using a variety of substrates. four genes encoding putative laccases have moderate or high level of expression in developing stems. laccase activity in plants has been assumed to participate in cell wall lignification. all four genes are up-regulated during the transition from young stems to intermediate stage as previously described. the expression of these genes was also reported to cluster with most monolignol biosynthetic genes in stems. three genes encoding multicopper oxidases of the sks family were identified. two of them were down-regulated during stem development. it was shown that sku is involved in the control of root growth, and that sks contributes to cotyledon vascular patterning during development. three genes encoding germin-like proteins were expressed at high levels in young stems and at intermediate stage of development. two of them were strongly down-regulated in mature stems. although the functions of glps are not clearly understood, it was shown that a glp gene is highly expressed during cotton fiber elongation, but is repressed once the rate of growth slows down. considering the variety of gene families involved in modification of carbohydrates and in oxido-reduction reactions, these results show that all cell wall components probably undergo modifications during stem growth. even if many of these gene families were known to be involved in cell wall assembly or remodelling, this transcriptomic analysis allows the identification of genes involved in stem development processes.. genes encoding secreted proteins involved in other cell wall functions proteins with interaction domains several families of genes encoding proteins with putative domains of interaction with polysaccharides and/or proteins were identified. these include proteins with leucine-rich repeat domains, lectins and enzyme inhibitors. due to their high specificity towards carbohydrates, lectins may be involved in various physiological functions. they could be involved in the recognition between cells or between cells and various carbohydrate-containing molecules or in assembly of the polysaccharide matrix. other possible functions of lectins in plants include: transport and packaging of carbohydrates, and mobilization of storage materials. inhibitors of pmes regulate plant pme activity. five genes encoding pme inhibitors were identified among which are down-regulated in mature stems. this down-regulation seems to be correlated with that of genes encoding pmes mentioned above. proteins involved in signaling nine genes encoding arabinogalactan proteins and genes encoding fasciclin-like agps had high or moderate levels of transcripts in developing stems. in addition, and genes encoding agps and flas, respectivement, had low levels of transcripts. five fla genes are down-regulated in mature stems. agps belong to a class of hyp-rich glycoproteins that are highly glycosylated and abundant in plant cell walls. agps have been involved in plant development such as cell fate determination, somatic embryogenesis, and cell proliferation. agps are assumed to be signal molecules. the association of agps with pectic polysaccharides has also been suggested. flas constitute a distinct class of proteins. proteins containing fasciclin domains have been shown to function as adhesion molecules in a broad spectrum of organisms. more recently, agps and flas were found to be associated with wood formation in poplar. twenty-two genes encoding putative receptor kinases are expressed at high or moderate levels in developing stems. only a few of them are up- or down-regulated. protein kinases play a very significant role in signal transduction. our screening revealed that genes are lrr-rlks. their extracellular domain is composed of tandem repeats of a well-conserved leucine-rich motif and their intracellular domain consists in a protein kinase with ser/thr specificity. lrr-rlks are involved in development, hormone perception, and pathogen response. proteases twenty-four genes encoding putative proteinases were identified. these enzymes belong to asp-, ser- and cys-protease families. proteinases may be involved in processing and/or turnover of cwps, as well as in peptide signal transduction. several proteomic analyses of plant cell walls show a large discrepancy between the observed and the expected molecular weights of proteins, suggesting the existence of a turnover of cwps. expression of a cys protease was shown to be associated with programmed cell death. the occurrence of many proteases and their high expression levels during stem development suggests that these enzymes are actively involved in cell expansion and/or in secondary wall formation. however, their activity could be modulated by the presence of many protease inhibitors since genes encoding such inhibitors were found to have moderate or high levels of transcripts. structural proteins only a few genes encoding structural cwps were found to have moderate or high levels of transcripts in developing stems. this low number might be the consequence of the difficulty to design specific probes for genes encoding repetitive sequences of amino acids such as structural cwps. indeed, catma does not offer a good coverage of such gene families, e.g. only extensin genes out of were analyzed. identified genes belong to the major classes of cell wall structural proteins, e.g. extensins, proline rich-proteins, glycine rich-proteins and lrr-extensins. five out of thirteen genes are down-regulated in mature stems whereas two genes encoding grps are up-regulated. structural cwps such as extensins and prps are assumed to be insolubilized when cell elongation ceases. extensins, grps, and prps were shown to be expressed in specific cell types including xylem and phloem tissues by tissue-printing of stems. the role of grps in the building of the cell walls of protoxylem elements has been shown by immunological studies. miscellaneous proteins a number of genes encoding miscellaneous proteins were identified by transcriptomic analysis. a large proportion of these genes encode proteins predicted to be protease inhibitors or are involved in lipid metabolism. lipids are essential for cell growth and results of these analyses suggest that lipid metabolism is important for stem development. five genes encoding thaumatin-like proteins among which are repressed during stem development were identified. they encode pathogenesis-related proteins that are expressed constitutively at certain stages of plant development. identification of several zn transporters suggests that zn is an essential micronutrient for stem development. four genes encode phytocyanins. three of these genes are down-regulated in mature stems. it should be noted that genes of the same family are expressed at low levels in developing stems. phytocyanins are assumed to play a role in oxido-reduction processes in cell walls as electron transfer proteins. in addition, stellacyanins and uclacyanins have structural domain possibly interacting with structural cwps. proteins of yet unknown function numerous genes encoding proteins with unknown function were identified in this transcriptomic analysis. some of which share structural domains or the so-called domains of unknown function. about half of them encode proteins predicted to have trans-membrane domains or gpi anchors. the encoded proteins may play house-keeping functions or be specifically involved in stem development. altogether, many gene families encoding secreted cell wall proteins were found to have members expressed at high or moderate levels during stem development. proteinase genes are actively transcribed, suggesting essential roles in protein processing, turnover, or signalling. finally, many genes have yet unknown function during stem development. validation of expression microarray results by real-time-qpcr in order to validate the results from microarray analysis, transcripts of selected genes, which were significantly expressed in the microarray studies, were analyzed by real-time-qpcr. the majority of these selected genes were related to cell wall biogenesis. these included 4-coumarate-coa ligase, caffeoyl-coa 3-o-methyltransferase, cellulose synthase subunit, cellulose synthase, catalytic subunit, cellulose synthase, catalytic subunit, putative laccase, gh of family, pectate lyase, phosphate responsive protein, plasma membrane intrinsic protein 2c, putative expansin, chlorophyll a-b binding protein, catalase and putative raffinose synthase. two genes, actin and 26s proteasome regulatory subunit, were selected as endogenous control. compared with transcriptomic analyses, significant correlation was confirmed by rt-qpcr according to the calculated average in transcript levels of all the tested genes. transcriptomics vs proteomics in order to better understand gene regulation in stems, data from transcriptomics were compared to those of a previous proteomic study performed using the same biological material, i.e. mature arabidopsis stems. this proteomic study was designed to identify n-glycoproteins trapped on cona sepharose and allowed the identification of genes encoding secreted proteins. their levels of transcripts were searched in the stem transcriptome. results were obtained for genes on the catma hybridized with total rnas from mature stems. eighteen genes were found to have levels of transcripts lower than background, had weak levels of transcripts, moderate levels of transcripts and high levels of transcripts. altogether, 25% of the genes identified through proteomics have levels of expression below background and 58% have low level of transcripts. the percentage of genes having detectable levels of expression decreases from about 82% to 75% during stem growth. the encoded proteins might have long half-lifes and remain in cell walls after the disappearance of the mrnas. it means that transcriptomics fails to give an overall picture of proteins present in cell walls at a given time. conversely, only genes identified through proteomics are found among the genes encoding secreted proteins expressed at high or moderate level. this means that many proteins escaped this proteomic analyses. one explanation might be the partial analysis of proteins separated by 2-d electrophoresis. in addition, low abundant proteins are not usually identified in proteomic studies, and structural proteins are difficult to extract from cell walls since they form insoluble networks. proteins with transmembrane domains are also not easily extracted by salts. agps and flas are difficult to identify because of their high level of glycosylation. however, these explanations are not valid for proteins like ghs that are soluble. alternatively, the numerous proteases present in cell walls probably play a great role in protein turnover, although it was shown that glycoproteins are more resistant against digestion by proteases. in addition, some regulatory mechanisms preventing the translation of certain mrnas may also exist. such discrepancies between proteomics and transcriptomics have been described in yeast. finally, proteome and transcriptome approaches appear to be complementary to get an overview of genes expressed in developing stems. the observed discrepancies show the importance of post-transcriptional regulations.  here we have examined the expression patterns of the genes encoding cell wall proteins. some of the genes identified in this work were previously identified by various microarray analyses of arabidopsis stems. these include xth, -β-glucanase and pectin esterase, pectate lyase, agp, glycine rich and proline rich proteins. however, we identified and established expression patterns of many new genes possibly involved in primary and secondary cell wall biogenesis. many genes involved in the synthesis of cell wall components were found to be transcribed as well as many genes encoding secreted proteins. the latter genes encode proteins acting on carbohydrates, oxido-reductases, proteins with interaction domains and structural proteins. among secreted proteins, proteins acting on carbohydrates represent the most abundant class after proteins of unknown function. in addition, genes encoding proteins such as agp, flas, grps and prps not reported by proteomic analyses, had high levels of transcripts. this transcriptomic analysis allowed identification of many gene candidates that play a role in cell wall construction during stem development. both primary and secondary walls are drastically modified thus allowing cells to undergo elongation during early steps of development. these modifications are part of the differentiation processes during late stages of development. the enzymes which catalyze glucan-chain elongation in cellulose, belonging to the family of glycoside transferase, are crucial for primary and secondary wall formations. in plants this family of gts, designated as the cellulose synthase catalytic subunits, has multiple members. in the present work several highly expressed genes of this family of enzymes have been identified. comparison of the gene expression profile of cesa genes obtained in the present study with those of genecat database () showed similar high expression profile of genes with exception of at2g which is not expressed according to genecat database. though good correlation in expression scores of these genes were obtained, some differences were observed concerning expression level or difference in expression. these differences could be consequences of developmental stage of plant, condition of growth, environment and from minor unavoidable experimental errors during extraction of rnas and measurement. finally, comparison of transcriptomic and proteomic analyses showed that about 25% of the genes encoding proteins identified by proteomics had level of transcripts above background in the catma analysis. conversely, only a small proportion of genes identified by proteomics were identified by transcriptomics, i.e. only out of the genes encoding secreted proteins were expressed at high or moderate levels. such discrepancies between proteomics and transcriptomics suggest complex regulatory mechanisms which occur at post-transcriptional levels. 
particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. although the expression patterns of some arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. the discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. the availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. analysis of the genes predicted to encode cell wall proteins revealed that about genes had moderate or high levels of transcripts. many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. among them, we identified many new genes possibly involved in cell wall biogenesis. the encoded proteins were distributed into classes, based on the presence of predicted functional domains. a total of genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes.
 different strategies can be used to study proteins involved in cell biogenesis. the availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. although the expression patterns of some arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins.  transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. a total of genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. the encoded proteins were distributed into classes, based on the presence of predicted functional domains. proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics.  analysis of the genes predicted to encode cell wall proteins revealed that about genes had moderate or high levels of transcripts. among them, we identified many new genes possibly involved in cell wall biogenesis. the discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. 