BACKGROUND
plant growth occurs mainly by division and expansion of cells.
a meristematic cell might enlarge as much as 50000-fold its initial volume.
in this process, membrane surface area and amount of cell wall material increase.
the primary cell wall plays an essential role since it should allow turgor-driven increase in cell volume by permitting the incorporation of new cell wall material and rearrangement of the existing cell wall.
several plant organs including coleoptiles , internodes , and hypocotyls  were used to study cell elongation  <cit> .
environmental signals such as light, temperature, and hormones, regulate hypocotyl growth  <cit> .
a. thaliana seedlings grown in continuous darkness are a material of choice to analyze the cell elongation process.
indeed, cells of hypocotyls undergo a 100-fold length increase compared to embryo cells  <cit> .
growth occurs mostly by cell expansion, with little cell division  <cit> .
changes in wall thickness during elongation of a. thaliana hypocotyls were investigated using cryo-field-emission scanning electron microscopy  <cit> .
at the germination stage, cell wall thickening occurs and involves high rates of biosynthesis and deposition of cell wall components.
during the elongation stage, cell walls undergo remarkable thinning, requiring extensive polymer disassembly and rearrangement.
many genes are assumed to be involved in cell wall synthesis and rearrangement to support growth of plant cell walls  <cit> .
they encode cellulose synthases , cellulose synthases-like , endo-glucanases, xyloglucan endotransglucosylase/hydrolases  and expansins.
they belong to multigenic families, but the members of each family involved in elongation of hypocotyl cells were not precisely identified.
it is also likely that other genes are important for cell elongation.
in this paper, the transcriptomes of a. thaliana etiolated hypocotyls were compared at two developmental stages, half-grown  and fully-grown .
the transcriptome analysis was focused on genes possibly involved in cell wall biogenesis and on genes encoding secreted proteins.
transcript profiling was carried out using catma   <cit> :  to look at the level of transcripts of cell wall genes  belonging to families known to be involved in cell wall biogenesis;  to identify genes encoding secreted proteins  having high or moderate level of transcripts;  to reveal differential gene expression affecting cwgs and spgs between half- and fully-grown etiolated hypocotyls;  and to look at the correlation between transcript abundance and protein presence as revealed by a proteomic study performed on the same material  <cit> .
RESULTS
levels of transcripts of cell wall genes  during hypocotyl elongation
etiolated hypocotyls were compared at two developmental stages.
five-day-old hypocotyls were approximately half the final size .
growth followed an acropetal gradient.
after 5-days, the bottom cells were fully elongated, whereas the top cells were only starting elongation  <cit> .
eleven-day-old hypocotyls had reached their maximum size  <cit> .
catma was used for mrna profiling.
since one of the major modifications during cell elongation is the addition and rearrangement of cell wall components, a selection of genes possibly involved in cell wall biogenesis was done .
this selection was called "cell wall genes" .
it was mainly based on the knowledge of gene families known to be involved in biogenesis of cell walls, i.e.
synthesis and transport of cell wall components and their assembly or rearrangement in cell walls .
representing  <dig> gene families, it includes genes encoding proteins involved in substrate generation , polysaccharide synthesis , vesicle trafficking, assembly/disassembly of the wall , structural proteins, oxido-reductases involved in cross-linking of wall components .
few other gene families encoding cell wall proteins were also included such as arabinogalactan proteins , fasciclin agps , phytocyanins, multicopper oxidases, pectin methylesterase inhibitors , and subtilases.
only genes annotated by experts were retained .
altogether,  <dig> genes were selected among which  <dig> were analyzed through catma, while the remaining genes were not analyzed for technical reasons.
some genes had no gene-specific tag  on the microarray, others were not considered because of a poor signal of hybridization to the rna probe, or of inaccurate duplicates.
the level of transcripts was expressed as log <dig> of the mean signal intensity.
values of log <dig> below  <dig>  were considered as background as defined in methods.
three groups of genes were considered after taking into account the dynamic range of catma arrays  <cit> .
values between  <dig>  and  <dig> corresponded to low level of transcripts , values between  <dig> and  <dig> to moderate level  and values higher than  <dig> to high level .
among the  <dig> genes analyzed, the level of transcripts at one or both stages was below background for  <dig> genes , low for  <dig> , moderate for  <dig>  and high for  <dig> .
overall, among the analyzed genes,  <dig> % of those related to synthesis or transport of cell wall components, and  <dig> % of those involved in their modifications in cell walls have detectable level of transcripts.
among the  <dig> genes with high levels of transcripts at one or both stages ,  <dig> are involved in the synthesis or transport of cell wall components and  <dig> are involved in modifications of cell wall components.
two separate phases of growth were described in dark-grown a. thaliana hypocotyls: an early phase of active synthesis of cell wall polysaccharides up to 3-days after beginning of germination, and a late phase of cell expansion  <cit> .
the former phase results in thicker cell walls which later on become thinner as hypocotyls elongate  <cit> .
our results suggest that both synthesis and rearrangement of cell wall components are required throughout hypocotyl elongation, and even after growth arrest.
most of the cwgs were expected to be transcribed during cell elongation.
genes involved in rearrangement of cell wall components encode glycoside hydrolases  such as endoglucanases, xths, and beta-galactosidases; carbohydrate esterases  such as pectin methylesterases ; pectin acylesterases; polysaccharide lyases ; expansins of the alpha- or beta-type; and peroxidases.
however,  <dig> genes encoding pmes and  <dig> genes encoding pectin acylesterases have detectable levels of transcripts among which  <dig> genes have high levels of transcripts in elongating hypocotyls .
this result seems contradictory with previous results showing that a low level of pectin esterification, associated to abundance of pmes and acylesterases, restricted cell elongation in a. thaliana hypocotyls  <cit> .
it should be noted that  <dig> genes encoding pmeis also have detectable levels of transcripts.
the interplay between pmes and their inhibitors could regulate the activity of pmes.
fifteen genes encoding proteins possibly involved in oxido-reduction reactions have high levels of transcripts .
seven peroxidase genes fall in this category, among which at2g <dig>  has one of the highest levels of transcripts.
the multiple roles of peroxidases during growth and growth arrest were reviewed  <cit> .
other cwgs are also well-represented such as those encoding arabinogalactan proteins , fasciclin agps  and subtilases .
even if agps and flas were shown to be associated with wood formation in poplar  <cit> , their role in cell expansion is not very clear at present.
likewise, nothing is known about the role of proteases during cell elongation.
finally, the cobra gene  has a high level of transcripts.
it has been shown to play an important role in microfibril deposition during rapid elongation and in the orientation of cellulose microfibrils  <cit> .
this work gives clues for understanding the function and possible involvement in multiple processes of members of multigene families either during cell elongation or after its arrest.
indeed, general functions were proposed for most of these gene families, but only scarce information is available for specific members.
genes encoding secreted proteins with high or moderate level of transcripts in etiolated hypocotyls
most of the gene families described above was already known to be involved in cell wall biogenesis.
in order to identify new genes encoding secreted proteins that might be involved in cell expansion, a second selection was carried out.
based on results of proteomic studies, some proteins without a predicted signal peptide were assumed to be secreted.
however, the sub-cellular localization of such proteins was never shown in another way  <cit> .
for this study, only the genes encoding proteins with a predicted signal peptide were selected.
the  <dig> selected genes were ranked by level of transcripts, producing a profile similar to the one obtained with cwgs at 5-days;  <dig> genes  above background level,  <dig>  with a low transcript level,  <dig>  with a moderate level, and  <dig>  with a high level.
same results were obtained at 11-days.
from this selection, only genes encoding proteins predicted to be located either outside the cell or in the plasma membrane were retained .
in additional file  <dig>   <dig> genes named "secretory pathway genes"  with moderate or high levels of transcripts are listed and grouped in families according to their predicted functional domains .
such protein families were already described in cell wall proteomic studies  <cit> : proteins acting on carbohydrates ; proteases ; proteins possibly involved in signaling ; structural proteins ; proteins possibly involved in oxido-reduction reactions ; proteins with interacting domains ; proteins related to lipid metabolism ; miscellaneous proteins ; proteins of unknown function .
main differences between transcriptomic and proteomic data lie in the genes encoding proteins possibly involved in signaling since they comprise agps, flas, and plasma membrane proteins that are difficult to isolate, separate or identify through proteomics  <cit> .
in the same way, the group of proteins of unknown function is very important because 48% of them are predicted to have trans-membrane domains.
on the contrary, the group of structural proteins is probably under-represented because of the lack of appropriate gsts for many of them.
indeed, their repetitive amino acid sequences make the design of specific probes difficult.
one should note the abundance of proteases that can be assumed to be essential for protein turnover in tissues undergoing rapid elongation followed by elongation arrest within a short time.
they may also be involved in signaling  <cit>  or in protein maturation  <cit> .
in addition, there are probably interactions between proteases and protease inhibitors to regulate the proteolytic activities in cell walls.
among the  <dig> proteins of yet unknown function,  <dig> have known structural domains.
others share domains with other proteins, such as domains of unknown function , or belong to the so-called uncharacterized protein families .
many are of particular interest, since they are only present in plants.
among these  <dig> spgs, only the  <dig> encoding proteins acting on carbohydrates, and the  <dig> encoding peroxidases or laccases were shown or assumed to contribute to assembly or rearrangement of cell wall components.
it means that this study allowed identifying about  <dig> genes encoding secreted proteins that are candidates to play roles during growth of a. thaliana etiolated hypocotyls.
their functional characterization will be paramount to understand cell wall architecture and assembly during an elongation process.
are there variations in the level of transcripts between half- and fully-grown hypocotyls?
in 5-day-old hypocotyls, apical cells are elongating whereas basal cells are fully elongated.
when compared to 11-day-old hypocotyls, where all the cells are fully elongated, the observed differences in levels of transcripts should mainly come from the cells which are at a different developmental stage, namely growing cells.
altogether,  <dig> genes are differentially expressed between 5- and 11-day-old hypocotyls.
among these genes,  <dig> encode proteins predicted to be secreted .
in addition, 32% of the genes having levels of transcripts modified by a factor  <dig> between the two developmental stages encode secreted proteins.
the following detailed analysis will be focused on these genes.
sixty-three and  <dig> genes have a higher level of transcripts in 5- and 11-day-old hypocotyls respectively.
the highest difference  was found at 11-days for a gene encoding a glycine-rich protein .
conversely, the largest decrease  was observed at 11-days for a gene encoding a putative asp protease .
the number of genes of selected families expressed differentially in both samples is represented in figure  <dig>  for comparison, the number of genes of the same families having high or moderate levels of transcripts is also represented.
all the selected gene families are represented by almost the same number of genes at both developmental stages .
however, there are striking differences when the comparison is done with genes showing significant variation in transcript level .
several genes encoding glycosyl transferases , ghs, and pmes have higher levels of transcripts at 5-days than at 11-days, i.e.
at a time hypocotyls undergo active elongation.
although gts and ghs are expected to be expressed during elongation, pmes could play roles during both elongation and growth arrest  <cit> .
the proportion of cells already elongated could also be significant after 5-days of growth.
alternatively, there might be a delay between synthesis of mrnas, and production of a functional protein.
this might be the case for some pmes that are produced as polyproteins comprising an inhibitor at their n-terminus and an active enzyme at their c-terminus  <cit> .
other genes having higher levels of transcripts at 5-days encode peroxidases, proteases, and proteins homologous to gdsl lipases/acylhydrolases.
the multiple functions of peroxidases were mentioned above.
the role of proteases in cell walls during active elongation has not yet been described.
it should be noted that  <dig> genes encoding protease inhibitors are up-regulated at 11-days, suggesting complex regulations of proteolytic activities in cell walls after the arrest of hypocotyl elongation.
genes belonging to other gene families have higher levels of transcripts at 11-days.
they code for protease inhibitors, agps, grps, late abundant embryogenesis  proteins, phytosulfokines , and gibberellic acid-stimulated arabidopsis proteins .
agp and fla genes are very well-represented in the transcriptome of hypocotyls with  <dig> agp genes and  <dig> fla genes having detectable levels of transcripts .
agps are candidates for cell-to-cell communication  <cit> , and flas were found to be associated to wood formation in poplar  <cit> .
concerning grps, they were shown to be associated to cell walls of xylem and phloem by tissue printing  <cit> .
the great increase in the amount of transcripts of at2g <dig> is consistent with the development of protoxylem elements that contain grps  <cit> .
nothing is known about the role leas could play in fully-developed hypocotyls.
psks were shown to promote tracheary element differentiation in zinnia cell suspension cultures, and to play roles during growth  <cit> .
five gasa genes have higher levels of transcripts at 11-days than at 5-days.
a. thaliana gasa <dig> and gasa <dig> were shown to play roles in flowering regulation and seed development  <cit> , as well as in stem growth and flowering time  <cit> .
increase in the level of transcripts of the gasa genes in 11-day-old hypocotyls thus points at their role in elongation arrest.
finally,  <dig> genes encoding secreted proteins of unknown function are transcribed at higher level at 11-days.
exploring the function of these proteins will be one of the major tasks for the future.
these differences in transcript abundance between the two stages of hypocotyl growth should be taken carefully with regard to the possible functions carried out by the proteins, since many other genes from the same families are transcribed in half- and fully-grown hypocotyls, but without significant differences.
transcriptome vs proteome
in order to look for the consistency between levels of mrnas and presence of the corresponding proteins in cell walls, a proteomic analysis was performed on cell walls  <cit> , and the results were compared to those of this transcriptomic analysis.
the cell wall proteomes of 5- and 11-days-old hypocotyls were achieved and a total of  <dig> proteins predicted to be secreted were identified .
when these  <dig> proteins were compared to the  <dig> spgs with moderate and high levels of transcripts , only  <dig> proteins matched .
conversely, from the  <dig> spgs having high levels of transcripts in etiolated hypocotyls, only  <dig>  showed the corresponding proteins .
it was expected that proteomic profiling identified at least the proteins encoded by the highly-transcribed genes.
the great inconsistency between the abundance of mrnas and the presence of the corresponding proteins was surprising, but several reasons may explain this disparity.
it is known that cwp extraction and identification can be challenging  <cit> .
many proteins can remain linked to the polysaccharide matrix, such as the structural proteins  <cit> , or some peroxidases that might be strongly bound to pectins  <cit> .
others are difficult to identify because of their structure, e.g.
highly o-glycosylated agps, which requires a special deglycosylation step  <cit> .
some proteins contain few linkages sensitive to tryptic digestion, and can escape identification by peptide mass mapping.
finally, low-abundant proteins elude proteomic analyses.
for the proteins that were identified without particular problems such as ghs, expansins and proteases, only a few of them correspond to highly-transcribed genes.
it indicates that a high level of transcripts is not always correlated with the presence of the protein in sufficient amount to be identified in proteomic approaches.
genes are classified by predicted functional domains as described in methods.
the intensity of the signal is expressed as log <dig>  the log <dig> of ratios between the levels of transcripts at 11-days and 5-days as well as the bonferroni p-values are indicated.
in a second step, the level of transcripts of the  <dig> genes encoding the proteins identified through proteomics was analyzed .
the transcript data of  <dig> genes was not found in the catma experiment since some of them have no gst or were eliminated because of poor signals of hybridization to the rna probe.
the levels of transcripts of the  <dig> remaining genes were surprising, for 5-days and 11-days,  <dig> % and  <dig> % respectively had low level of transcript while  <dig> % and  <dig> % respectively had levels of transcripts below background.
however, all the identified proteins are assumed to be the most abundant.
this suggests that the transcripts could have short half-lifes and/or that the proteins could have a low turnover.
this is the case of several genes encoding proteins acting on carbohydrates  or oxido-reductases .
additional experiments will be necessary to determine the half-lifes of the transcripts and of the proteins in etiolated hypocotyls.
the results obtained with the catma analysis were confirmed by quantitative rt-pcr  analysis .
several genes corresponding to the three cases described were chosen: high or moderate level of transcripts and proteins identified; high level of transcripts and proteins not identified; low or below background level of transcripts and proteins identified.
note that genes having very low levels of transcripts give signals below the sensitivity of the catma analysis.
altogether, these results show that there is not a clear correlation between the presence of cwps as shown by cell wall proteomic analysis and the amount of transcripts of the corresponding genes.
the quality of this correlation may depend on genes and/on environmental conditions.
for example, the quantification of soluble proteins of yeast at mid-log phase showed that for a given transcript level, protein levels were found to vary by more than 20-fold, whereas for a given protein level, transcript levels were found to vary 30-fold  <cit> .
however, up-regulation of yeast genes in response to glucose or nitrogen limitation was found to be controlled at the transcriptional or post-transcriptional level respectively  <cit> .
in a. thaliana and rice, changes observed in the soluble proteome in response to bacterial challenge were not strictly correlated to changes in transcript levels  <cit> .
these results show that quantitative analysis of transcript levels is not sufficient to infer protein levels.
multilevel analysis must take into account the stability of transcripts, their availability for active translation, as well as the stability of proteins, which is certainly essential considering the high number of proteases in cell walls.
with regard to transcript stability, data from a recent study aiming at measuring mrna decay rates in a. thaliana cell suspension cultures  <cit>  were used to look for half-lifes of gene transcripts identified through proteomics .
it can be seen that more than half of the proteins  identified by cell wall proteomics correspond to genes having transcripts with rather long half-lifes .
conversely, no gene corresponding to proteins identified by cell wall proteomics has transcripts with half-lifes shorter than  <dig> h. this distribution differs from that of transcripts of genes having high or moderate level of transcripts since  <dig> % of these genes have half-lifes shorter than  <dig> h.
as a particular case, etiolated hypocotyls of a. thaliana at 5-days and 11-days,  <dig> and  <dig> genes respectively showed both high level of transcripts, and encoded proteins identified by cell wall proteomics.
they might be considered as good markers for cell elongation of dark-grown hypocotyls.
CONCLUSIONS
mrna profiling of the genes potentially involved in cell wall biogenesis  in etiolated hypocotyls showed that more than half of them present a detectable level of transcripts.
all gene families are expressed.
the results suggest that both synthesis and rearrangement of wall components are required throughout hypocotyl elongation.
the transcriptomic analysis of genes encoding secreted proteins showed that around  <dig> new genes might be implicated in this process.
understanding the biochemical and biological functions of these genes might reveal new mechanisms of cell wall expansion or of growth arrest, or new functions for the cell wall.
around  <dig> genes encoding secreted proteins had significantly different levels of transcripts between growing and fully-elongated hypocotyls.
as expected, genes acting on polysaccharides  had higher levels of transcripts at 5-days, whereas others encoding pmes or peroxidases were not supposed to have higher level of transcripts during active elongation.
their function during cell elongation should be reexamined.
several genes encoding proteases also have higher level of transcripts at 5-days and could play roles in protein maturation or turn over.
on the contrary, several genes encoding agps, protease inhibitors, and proteins homologous to gibberellin regulated proteins had higher levels of transcripts at 11-days.
their functions after the end of the elongation process remain to be found.
as expected, some genes encoding grps were found to have much higher levels of transcripts in fully-grown hypocotyls at a time lignification is an active process.
however, since all these genes belong to multigene families, one cannot rule out the fact that a similar function can be shared by several genes.
finally, looking into the transcript level of the genes corresponding to the  <dig> proteins identified by proteomic analysis of the cell walls of half- and fully-grown hypocotyls, 15% were below the catma background.
on the contrary, only 13% of the genes encoding secreted proteins with high or moderate levels of transcripts corresponded to proteins identified through proteomics.
thus, the comparison between transcript levels and presence of the corresponding proteins suggested that many genes encoding proteins secreted in cell walls are regulated at a post-transcriptional level.
in conclusion, transcriptomic and proteomic data appeared to be complementary to describe the regulation of gene activity during the elongation of etiolated hypocotyls.

 plant growth occurs mainly by division and expansion of cells. a meristematic cell might enlarge as much as 50000-fold its initial volume. in this process, membrane surface area and amount of cell wall material increase. the primary cell wall plays an essential role since it should allow turgor-driven increase in cell volume by permitting the incorporation of new cell wall material and rearrangement of the existing cell wall. several plant organs including coleoptiles, internodes, and hypocotyls were used to study cell elongation. environmental signals such as light, temperature, and hormones, regulate hypocotyl growth. a. thaliana seedlings grown in continuous darkness are a material of choice to analyze the cell elongation process. indeed, cells of hypocotyls undergo a 100-fold length increase compared to embryo cells. growth occurs mostly by cell expansion, with little cell division. changes in wall thickness during elongation of a. thaliana hypocotyls were investigated using cryo-field-emission scanning electron microscopy. at the germination stage, cell wall thickening occurs and involves high rates of biosynthesis and deposition of cell wall components. during the elongation stage, cell walls undergo remarkable thinning, requiring extensive polymer disassembly and rearrangement. many genes are assumed to be involved in cell wall synthesis and rearrangement to support growth of plant cell walls. they encode cellulose synthases, cellulose synthases-like, endo-glucanases, xyloglucan endotransglucosylase/hydrolases and expansins. they belong to multigenic families, but the members of each family involved in elongation of hypocotyl cells were not precisely identified. it is also likely that other genes are important for cell elongation. in this paper, the transcriptomes of a. thaliana etiolated hypocotyls were compared at two developmental stages, half-grown and fully-grown. the transcriptome analysis was focused on genes possibly involved in cell wall biogenesis and on genes encoding secreted proteins. transcript profiling was carried out using catma: to look at the level of transcripts of cell wall genes belonging to families known to be involved in cell wall biogenesis; to identify genes encoding secreted proteins having high or moderate level of transcripts; to reveal differential gene expression affecting cwgs and spgs between half- and fully-grown etiolated hypocotyls; and to look at the correlation between transcript abundance and protein presence as revealed by a proteomic study performed on the same material.  levels of transcripts of cell wall genes during hypocotyl elongation etiolated hypocotyls were compared at two developmental stages. five-day-old hypocotyls were approximately half the final size. growth followed an acropetal gradient. after 5-days, the bottom cells were fully elongated, whereas the top cells were only starting elongation. eleven-day-old hypocotyls had reached their maximum size. catma was used for mrna profiling. since one of the major modifications during cell elongation is the addition and rearrangement of cell wall components, a selection of genes possibly involved in cell wall biogenesis was done. this selection was called "cell wall genes". it was mainly based on the knowledge of gene families known to be involved in biogenesis of cell walls, i.e. synthesis and transport of cell wall components and their assembly or rearrangement in cell walls. representing gene families, it includes genes encoding proteins involved in substrate generation, polysaccharide synthesis, vesicle trafficking, assembly/disassembly of the wall, structural proteins, oxido-reductases involved in cross-linking of wall components. few other gene families encoding cell wall proteins were also included such as arabinogalactan proteins, fasciclin agps, phytocyanins, multicopper oxidases, pectin methylesterase inhibitors, and subtilases. only genes annotated by experts were retained. altogether, genes were selected among which were analyzed through catma, while the remaining genes were not analyzed for technical reasons. some genes had no gene-specific tag on the microarray, others were not considered because of a poor signal of hybridization to the rna probe, or of inaccurate duplicates. the level of transcripts was expressed as log of the mean signal intensity. values of log below were considered as background as defined in methods. three groups of genes were considered after taking into account the dynamic range of catma arrays. values between and corresponded to low level of transcripts, values between and to moderate level and values higher than to high level. among the genes analyzed, the level of transcripts at one or both stages was below background for genes, low for, moderate for and high for. overall, among the analyzed genes, % of those related to synthesis or transport of cell wall components, and % of those involved in their modifications in cell walls have detectable level of transcripts. among the genes with high levels of transcripts at one or both stages, are involved in the synthesis or transport of cell wall components and are involved in modifications of cell wall components. two separate phases of growth were described in dark-grown a. thaliana hypocotyls: an early phase of active synthesis of cell wall polysaccharides up to 3-days after beginning of germination, and a late phase of cell expansion. the former phase results in thicker cell walls which later on become thinner as hypocotyls elongate. our results suggest that both synthesis and rearrangement of cell wall components are required throughout hypocotyl elongation, and even after growth arrest. most of the cwgs were expected to be transcribed during cell elongation. genes involved in rearrangement of cell wall components encode glycoside hydrolases such as endoglucanases, xths, and beta-galactosidases; carbohydrate esterases such as pectin methylesterases; pectin acylesterases; polysaccharide lyases; expansins of the alpha- or beta-type; and peroxidases. however, genes encoding pmes and genes encoding pectin acylesterases have detectable levels of transcripts among which genes have high levels of transcripts in elongating hypocotyls. this result seems contradictory with previous results showing that a low level of pectin esterification, associated to abundance of pmes and acylesterases, restricted cell elongation in a. thaliana hypocotyls. it should be noted that genes encoding pmeis also have detectable levels of transcripts. the interplay between pmes and their inhibitors could regulate the activity of pmes. fifteen genes encoding proteins possibly involved in oxido-reduction reactions have high levels of transcripts. seven peroxidase genes fall in this category, among which at2g has one of the highest levels of transcripts. the multiple roles of peroxidases during growth and growth arrest were reviewed. other cwgs are also well-represented such as those encoding arabinogalactan proteins, fasciclin agps and subtilases. even if agps and flas were shown to be associated with wood formation in poplar, their role in cell expansion is not very clear at present. likewise, nothing is known about the role of proteases during cell elongation. finally, the cobra gene has a high level of transcripts. it has been shown to play an important role in microfibril deposition during rapid elongation and in the orientation of cellulose microfibrils. this work gives clues for understanding the function and possible involvement in multiple processes of members of multigene families either during cell elongation or after its arrest. indeed, general functions were proposed for most of these gene families, but only scarce information is available for specific members. genes encoding secreted proteins with high or moderate level of transcripts in etiolated hypocotyls most of the gene families described above was already known to be involved in cell wall biogenesis. in order to identify new genes encoding secreted proteins that might be involved in cell expansion, a second selection was carried out. based on results of proteomic studies, some proteins without a predicted signal peptide were assumed to be secreted. however, the sub-cellular localization of such proteins was never shown in another way. for this study, only the genes encoding proteins with a predicted signal peptide were selected. the selected genes were ranked by level of transcripts, producing a profile similar to the one obtained with cwgs at 5-days; genes above background level, with a low transcript level, with a moderate level, and with a high level. same results were obtained at 11-days. from this selection, only genes encoding proteins predicted to be located either outside the cell or in the plasma membrane were retained. in additional file genes named "secretory pathway genes" with moderate or high levels of transcripts are listed and grouped in families according to their predicted functional domains. such protein families were already described in cell wall proteomic studies: proteins acting on carbohydrates; proteases; proteins possibly involved in signaling; structural proteins; proteins possibly involved in oxido-reduction reactions; proteins with interacting domains; proteins related to lipid metabolism; miscellaneous proteins; proteins of unknown function. main differences between transcriptomic and proteomic data lie in the genes encoding proteins possibly involved in signaling since they comprise agps, flas, and plasma membrane proteins that are difficult to isolate, separate or identify through proteomics. in the same way, the group of proteins of unknown function is very important because 48% of them are predicted to have trans-membrane domains. on the contrary, the group of structural proteins is probably under-represented because of the lack of appropriate gsts for many of them. indeed, their repetitive amino acid sequences make the design of specific probes difficult. one should note the abundance of proteases that can be assumed to be essential for protein turnover in tissues undergoing rapid elongation followed by elongation arrest within a short time. they may also be involved in signaling or in protein maturation. in addition, there are probably interactions between proteases and protease inhibitors to regulate the proteolytic activities in cell walls. among the proteins of yet unknown function, have known structural domains. others share domains with other proteins, such as domains of unknown function, or belong to the so-called uncharacterized protein families. many are of particular interest, since they are only present in plants. among these spgs, only the encoding proteins acting on carbohydrates, and the encoding peroxidases or laccases were shown or assumed to contribute to assembly or rearrangement of cell wall components. it means that this study allowed identifying about genes encoding secreted proteins that are candidates to play roles during growth of a. thaliana etiolated hypocotyls. their functional characterization will be paramount to understand cell wall architecture and assembly during an elongation process. are there variations in the level of transcripts between half- and fully-grown hypocotyls? in 5-day-old hypocotyls, apical cells are elongating whereas basal cells are fully elongated. when compared to 11-day-old hypocotyls, where all the cells are fully elongated, the observed differences in levels of transcripts should mainly come from the cells which are at a different developmental stage, namely growing cells. altogether, genes are differentially expressed between 5- and 11-day-old hypocotyls. among these genes, encode proteins predicted to be secreted. in addition, 32% of the genes having levels of transcripts modified by a factor between the two developmental stages encode secreted proteins. the following detailed analysis will be focused on these genes. sixty-three and genes have a higher level of transcripts in 5- and 11-day-old hypocotyls respectively. the highest difference was found at 11-days for a gene encoding a glycine-rich protein. conversely, the largest decrease was observed at 11-days for a gene encoding a putative asp protease. the number of genes of selected families expressed differentially in both samples is represented in figure for comparison, the number of genes of the same families having high or moderate levels of transcripts is also represented. all the selected gene families are represented by almost the same number of genes at both developmental stages. however, there are striking differences when the comparison is done with genes showing significant variation in transcript level. several genes encoding glycosyl transferases, ghs, and pmes have higher levels of transcripts at 5-days than at 11-days, i.e. at a time hypocotyls undergo active elongation. although gts and ghs are expected to be expressed during elongation, pmes could play roles during both elongation and growth arrest. the proportion of cells already elongated could also be significant after 5-days of growth. alternatively, there might be a delay between synthesis of mrnas, and production of a functional protein. this might be the case for some pmes that are produced as polyproteins comprising an inhibitor at their n-terminus and an active enzyme at their c-terminus. other genes having higher levels of transcripts at 5-days encode peroxidases, proteases, and proteins homologous to gdsl lipases/acylhydrolases. the multiple functions of peroxidases were mentioned above. the role of proteases in cell walls during active elongation has not yet been described. it should be noted that genes encoding protease inhibitors are up-regulated at 11-days, suggesting complex regulations of proteolytic activities in cell walls after the arrest of hypocotyl elongation. genes belonging to other gene families have higher levels of transcripts at 11-days. they code for protease inhibitors, agps, grps, late abundant embryogenesis proteins, phytosulfokines, and gibberellic acid-stimulated arabidopsis proteins. agp and fla genes are very well-represented in the transcriptome of hypocotyls with agp genes and fla genes having detectable levels of transcripts. agps are candidates for cell-to-cell communication, and flas were found to be associated to wood formation in poplar. concerning grps, they were shown to be associated to cell walls of xylem and phloem by tissue printing. the great increase in the amount of transcripts of at2g is consistent with the development of protoxylem elements that contain grps. nothing is known about the role leas could play in fully-developed hypocotyls. psks were shown to promote tracheary element differentiation in zinnia cell suspension cultures, and to play roles during growth. five gasa genes have higher levels of transcripts at 11-days than at 5-days. a. thaliana gasa and gasa were shown to play roles in flowering regulation and seed development, as well as in stem growth and flowering time. increase in the level of transcripts of the gasa genes in 11-day-old hypocotyls thus points at their role in elongation arrest. finally, genes encoding secreted proteins of unknown function are transcribed at higher level at 11-days. exploring the function of these proteins will be one of the major tasks for the future. these differences in transcript abundance between the two stages of hypocotyl growth should be taken carefully with regard to the possible functions carried out by the proteins, since many other genes from the same families are transcribed in half- and fully-grown hypocotyls, but without significant differences. transcriptome vs proteome in order to look for the consistency between levels of mrnas and presence of the corresponding proteins in cell walls, a proteomic analysis was performed on cell walls, and the results were compared to those of this transcriptomic analysis. the cell wall proteomes of 5- and 11-days-old hypocotyls were achieved and a total of proteins predicted to be secreted were identified. when these proteins were compared to the spgs with moderate and high levels of transcripts, only proteins matched. conversely, from the spgs having high levels of transcripts in etiolated hypocotyls, only showed the corresponding proteins. it was expected that proteomic profiling identified at least the proteins encoded by the highly-transcribed genes. the great inconsistency between the abundance of mrnas and the presence of the corresponding proteins was surprising, but several reasons may explain this disparity. it is known that cwp extraction and identification can be challenging. many proteins can remain linked to the polysaccharide matrix, such as the structural proteins, or some peroxidases that might be strongly bound to pectins. others are difficult to identify because of their structure, e.g. highly o-glycosylated agps, which requires a special deglycosylation step. some proteins contain few linkages sensitive to tryptic digestion, and can escape identification by peptide mass mapping. finally, low-abundant proteins elude proteomic analyses. for the proteins that were identified without particular problems such as ghs, expansins and proteases, only a few of them correspond to highly-transcribed genes. it indicates that a high level of transcripts is not always correlated with the presence of the protein in sufficient amount to be identified in proteomic approaches. genes are classified by predicted functional domains as described in methods. the intensity of the signal is expressed as log the log of ratios between the levels of transcripts at 11-days and 5-days as well as the bonferroni p-values are indicated. in a second step, the level of transcripts of the genes encoding the proteins identified through proteomics was analyzed. the transcript data of genes was not found in the catma experiment since some of them have no gst or were eliminated because of poor signals of hybridization to the rna probe. the levels of transcripts of the remaining genes were surprising, for 5-days and 11-days, % and % respectively had low level of transcript while % and % respectively had levels of transcripts below background. however, all the identified proteins are assumed to be the most abundant. this suggests that the transcripts could have short half-lifes and/or that the proteins could have a low turnover. this is the case of several genes encoding proteins acting on carbohydrates or oxido-reductases. additional experiments will be necessary to determine the half-lifes of the transcripts and of the proteins in etiolated hypocotyls. the results obtained with the catma analysis were confirmed by quantitative rt-pcr analysis. several genes corresponding to the three cases described were chosen: high or moderate level of transcripts and proteins identified; high level of transcripts and proteins not identified; low or below background level of transcripts and proteins identified. note that genes having very low levels of transcripts give signals below the sensitivity of the catma analysis. altogether, these results show that there is not a clear correlation between the presence of cwps as shown by cell wall proteomic analysis and the amount of transcripts of the corresponding genes. the quality of this correlation may depend on genes and/on environmental conditions. for example, the quantification of soluble proteins of yeast at mid-log phase showed that for a given transcript level, protein levels were found to vary by more than 20-fold, whereas for a given protein level, transcript levels were found to vary 30-fold. however, up-regulation of yeast genes in response to glucose or nitrogen limitation was found to be controlled at the transcriptional or post-transcriptional level respectively. in a. thaliana and rice, changes observed in the soluble proteome in response to bacterial challenge were not strictly correlated to changes in transcript levels. these results show that quantitative analysis of transcript levels is not sufficient to infer protein levels. multilevel analysis must take into account the stability of transcripts, their availability for active translation, as well as the stability of proteins, which is certainly essential considering the high number of proteases in cell walls. with regard to transcript stability, data from a recent study aiming at measuring mrna decay rates in a. thaliana cell suspension cultures were used to look for half-lifes of gene transcripts identified through proteomics. it can be seen that more than half of the proteins identified by cell wall proteomics correspond to genes having transcripts with rather long half-lifes. conversely, no gene corresponding to proteins identified by cell wall proteomics has transcripts with half-lifes shorter than h. this distribution differs from that of transcripts of genes having high or moderate level of transcripts since % of these genes have half-lifes shorter than h. as a particular case, etiolated hypocotyls of a. thaliana at 5-days and 11-days, and genes respectively showed both high level of transcripts, and encoded proteins identified by cell wall proteomics. they might be considered as good markers for cell elongation of dark-grown hypocotyls.  mrna profiling of the genes potentially involved in cell wall biogenesis in etiolated hypocotyls showed that more than half of them present a detectable level of transcripts. all gene families are expressed. the results suggest that both synthesis and rearrangement of wall components are required throughout hypocotyl elongation. the transcriptomic analysis of genes encoding secreted proteins showed that around new genes might be implicated in this process. understanding the biochemical and biological functions of these genes might reveal new mechanisms of cell wall expansion or of growth arrest, or new functions for the cell wall. around genes encoding secreted proteins had significantly different levels of transcripts between growing and fully-elongated hypocotyls. as expected, genes acting on polysaccharides had higher levels of transcripts at 5-days, whereas others encoding pmes or peroxidases were not supposed to have higher level of transcripts during active elongation. their function during cell elongation should be reexamined. several genes encoding proteases also have higher level of transcripts at 5-days and could play roles in protein maturation or turn over. on the contrary, several genes encoding agps, protease inhibitors, and proteins homologous to gibberellin regulated proteins had higher levels of transcripts at 11-days. their functions after the end of the elongation process remain to be found. as expected, some genes encoding grps were found to have much higher levels of transcripts in fully-grown hypocotyls at a time lignification is an active process. however, since all these genes belong to multigene families, one cannot rule out the fact that a similar function can be shared by several genes. finally, looking into the transcript level of the genes corresponding to the proteins identified by proteomic analysis of the cell walls of half- and fully-grown hypocotyls, 15% were below the catma background. on the contrary, only 13% of the genes encoding secreted proteins with high or moderate levels of transcripts corresponded to proteins identified through proteomics. thus, the comparison between transcript levels and presence of the corresponding proteins suggested that many genes encoding proteins secreted in cell walls are regulated at a post-transcriptional level. in conclusion, transcriptomic and proteomic data appeared to be complementary to describe the regulation of gene activity during the elongation of etiolated hypocotyls. 
a significant proportion of them has detectable levels of transcripts, suggesting that these processes are important throughout hypocotyl elongation and after growth arrest. encoded proteins were proteases, protease inhibitors, proteins with interacting domains, and proteins involved in lipid metabolism. finally, comparison with results of a cell wall proteomic study on the same material revealed that out of the identified proteins were products of the genes having high or moderate level of transcripts. members of known multigenic families involved in cell wall biogenesis, and new genes that might participate in cell elongation were identified. cell enlargement implicates significant changes in the composition and structure of the cell wall. in order to understand cell wall biogenesis during cell elongation, mrna profiling was made on half- and fully-grown etiolated hypocotyls. the first set of genes named cell wall genes included genes known to be involved in cell wall biogenesis. arabidopsis thaliana hypocotyls undergo a 100-fold length increase mainly by cell elongation. no clear correlation was found between the abundance of transcripts and the presence of the proteins demonstrating the importance of post-transcriptional events for the regulation of genes during cell elongation and that transcriptomic and proteomic data are complementary. significant differences were shown in the expression of such genes in half- and fully-grown hypocotyls. genes encoding proteins involved in substrate generation or in synthesis of polysaccharides, and extracellular proteins were found to have high transcript levels. based on transcript level, genes were selected. genes not known to be involved in cell elongation were found to have high levels of transcripts. about 15% of the genes encoding proteins identified by proteomics showed levels of transcripts below background. in addition, of them encoded proteins with yet unknown function. transcriptomic analysis was focused on two sets of genes. a second set of genes labeled secretory pathway genes was studied to search for new genes encoding secreted proteins possibly involved in wall expansion.
 plant growth is a complex process involving cell division and elongation. arabidopsis thaliana hypocotyls undergo a 100-fold length increase mainly by cell elongation. cell enlargement implicates significant changes in the composition and structure of the cell wall. in order to understand cell wall biogenesis during cell elongation, mrna profiling was made on half- and fully-grown etiolated hypocotyls.  transcriptomic analysis was focused on two sets of genes. the first set of genes named cell wall genes included genes known to be involved in cell wall biogenesis. a significant proportion of them has detectable levels of transcripts, suggesting that these processes are important throughout hypocotyl elongation and after growth arrest. genes encoding proteins involved in substrate generation or in synthesis of polysaccharides, and extracellular proteins were found to have high transcript levels. a second set of genes labeled secretory pathway genes was studied to search for new genes encoding secreted proteins possibly involved in wall expansion. based on transcript level, genes were selected. genes not known to be involved in cell elongation were found to have high levels of transcripts. encoded proteins were proteases, protease inhibitors, proteins with interacting domains, and proteins involved in lipid metabolism. in addition, of them encoded proteins with yet unknown function. finally, comparison with results of a cell wall proteomic study on the same material revealed that out of the identified proteins were products of the genes having high or moderate level of transcripts. about 15% of the genes encoding proteins identified by proteomics showed levels of transcripts below background.  members of known multigenic families involved in cell wall biogenesis, and new genes that might participate in cell elongation were identified. significant differences were shown in the expression of such genes in half- and fully-grown hypocotyls. no clear correlation was found between the abundance of transcripts and the presence of the proteins demonstrating the importance of post-transcriptional events for the regulation of genes during cell elongation and that transcriptomic and proteomic data are complementary. 