BACKGROUND
although the form and function of plant cells are strongly correlated, the processes that determine the cell shape remain largely unknown.
plant cell morphogenesis is regulated in a non-cell-autonomous fashion by the surrounding tissues  <cit> , hormone interference during ontogenesis, and sometimes by polyploidy as a consequence of endoreduplication  <cit> .
in contrast, in unicellular relatives of land plants, it is possible to study the endogenous controls of cell morphogenesis without the interference by interacting cells and to better understand how these mechanisms have evolved in the green lineage.
the desmid micrasterias denticulata is a member of the conjugating green algae  that comprise the closest extant unicellular relatives of land plants  <cit> .
m. denticulata cells consist of two bilaterally symmetrical flat semicells, notched deeply around their perimeter into one polar lobe and four main lateral lobes.
following cell division, each semicell builds a new one through a process of septum bulging and symmetrical local growth cessations to form the successive lobes .
after completion of the primary wall , a rigid cellulosic secondary cell wall pierced by pores is deposited, followed by shedding of it.
this peculiar growth mechanism makes micrasterias an ideal model to study the spatial and temporal patterning of cell wall biogenesis  <cit> .
ultimately, the plant cell morphology is determined by the composition and structure of the cell wall that governs the cell expansion direction and rate.
as in land plants, the primary cell wall of m. denticulata bréb.
consists mainly of pectins  <cit> , cellulose microfibrils  <cit> , hemicelluloses  <cit>  and arabinogalactan proteins   <cit> .
the secondary cell wall owes it rigidness to cellulose microfibrils originating from rosettes organized as hexagonal arrays  <cit> , whereas mixed-linked glucan is the dominant hemicellulose  <cit> .
in land plants, expansins are important regulators of turgor-driven cell wall expansion.
these cell wall proteins comprise a large multigene superfamily consisting of four families  of which the evolutionary relationships are well characterized  <cit> .
they are unique in their ability to loosen the cell wall non-enzymatically by disrupting hydrogen bonds that link the cellulose and hemicellulose wall components  <cit> .
land plant expansins consist of two domains and a secretion signal.
the n-terminal expansin domain  <dig> and the c-terminal expansin domain  <dig> are homologous to the catalytic domain of glycoside hydrolase family  <dig>  proteins and a domain present in a family of grass pollen allergens, identified as a putative cellulose binding site  <cit> , respectively.
expansins play a role in tissue development  <cit>  and in growth of suspension-cultured cells  <cit> .
although genes encoding expansin-like proteins have been recently identified in green algae transcriptomes  <cit> , their physiological function and phylogenetic relationships with land plant expansins remain unknown.
here, we explore the molecular basis of cell morphogenesis and cell wall formation in synchronized m. denticulata cells by means of a cdna-amplified fragment length polymorphism -based quantitative transcriptome analysis  <cit> .
several cell wall-related genes, among which expansins, were identified.
examination of the expansins provided the first structural, phylogenetic and functional data on green algal homologues within this gene family.
RESULTS
cdna-aflp expression profiling
first we developed a synchronization protocol to monitor the cell morphogenesis-related gene expression in m. denticulata.
the protocol was based on the observation that the majority of the cells grown in a 14-h light/10-h dark regime divided during the second dark period, after the growth medium of a stationary culture  had been refreshed and, concomitantly, the cell density reduced at the start of the light period.
replacing the dark period by a light period enhanced the amount of synchronically dividing cells .
the effect of cell density on synchronization was significant , with an optimal cell density below  <dig> cells ml- <dig>  following synchronization, up to 85% of the cell population divided during an 8- to 9-h period, showing a sigmoid course .
by sampling this period at five consecutive time points we obtained samples with different proportions of cells at the major morphogenetic stages .
cdna-aflp expression profiling of these samples allowed the assignment of differentially expressed genes to either the onset of cell division ), the bulge ), the lobe ), or the doublet stage, during which the secondary cell wall is formed ).
in total, the relative abundance was monitored of  <dig> transcript-derived fragments  during the cell growth of m. denticulata , for which the expression patterns were altered visibly across time in  <dig> and significantly  in  <dig> tdfs.
according to other studies  <cit> , we estimate that two-thirds of the mrna population was sampled, implying that the real number of genes differentially expressed during cell growth of m. denticulata could be ~ <dig>  a high similarity  to database entries with assigned identities and unknown or hypothetical genes was found for  <dig> and  <dig> tdfs, respectively, mostly with embryophytes and not with chlorophyta.
however, the majority of the tdfs  showed no sequence similarity to any database entry .
plausible explanations might be sequences too short to reveal any significant identity, short sequences representing non-conserved portions of genes, tdfs originating from the 3'-untranslated region of a gene, or tdfs representing genes specific to m. denticulata or streptophytic algae.
of the  <dig> annotated genes,  <dig> clustered into six groups   according to the timing of their highest expression .
except for one cluster consisting of six genes , the expression profiles were reproducible in the two independent sampling series.
the few genes not included in one of the described clusters typically showed narrow temporal expression patterns.
based on their annotation, the tdfs were classified into  <dig> functional categories, named according to the gene ontology terminology  .
the association between the functional category and the tdf clustering was not significant .
the major group with a significant hit was involved in cell wall metabolism.
the second largest category corresponded to sequences sharing significant similarity to unknown or hypothetical proteins.
of  <dig> tdfs with similarity to genes involved in cell wall biogenesis or cell pattern formation, the rna samples of the second cdna-aflp replication series and on an independently sampled series  were analyzed by real-time quantitative reverse-transcription -pcr.
in general, the expression profiles obtained by cdna-aflp and qrt-pcr  corresponded well , confirming the obtained expression results.
genes relevant for cell pattern formation
seven tdfs could be identified that might be relevant for cell pattern formation in m. denticulata, among which two members of the rab gtpase cycle and two members of the snare cycle of membrane fusion reactions.
rab <dig>  similar to md <dig>  is known to be involved in post-golgi transport to the plasma membrane, inducing the formation of new surface extensions and believed to be regulated by a guanine nucleotide dissociation inhibitor  <cit>  possibly corresponding to md <dig>  both md <dig> and md <dig> belonged to cluster c1a and, thus, had increased mrna levels before the onset of mitosis.
this observation might be related to the determination of the basic symmetry of a m. denticulata cell before mitosis, indicated by the development of a three-lobed semicell after removal of the nucleus  <cit> .
in contrast, the snare cycle members were highly expressed in cluster c <dig>  pointing to a role in further differentiation during the lobe stages for md <dig>  and md <dig> .
two tdfs were identified encoding putative glycophosphatidylinositol  anchors: md <dig> and md <dig>  belonging to clusters c1a, and c <dig>  respectively.
among other properties, the function of a gpi anchor might be its dominant targeting to a specific membrane domain  <cit> , possibly establishing a membrane template for morphogenesis.
md <dig> turned out to be a 179-amino-acid protein containing a signal peptide and a fasciclin domain  , with similarity to a fasciclin-like and an agp-like protein from brachypodium sylvaticum  and arabidopsis thaliana , respectively .
md <dig> , similar to a very-long-chain fatty acid-condensing enzyme, might be involved in morphogenesis in accordance to the essential role in cell expansion during plant morphogenesis of arabidopsis  <cit> .
genes involved in cell wall metabolism
a total of  <dig> cell wall-related genes were identified.
six tdfs operating in the monosaccharide metabolism, evenly distributed over c <dig> and c <dig>  could be identified as udp-pyrophosphorylases , a phosphoglucomutase , a rhamnose synthase , and a gdp-mannose  <dig> -epimerase .
nine polysaccharide synthesis enzymes all nearly clustered in c <dig>  among which two cellulose synthases, md <dig>  and md <dig>  and one cellulose synthase-like  gene of the cslc family, md <dig>  the exostosin family glycosyltransferases md <dig>  md <dig>  md <dig>  and the glycosyltransferase md <dig> might synthesize the hemicellulosic or pectinous part of the cell wall and mucilage as well that is pectic in nature  <cit>  and secreted simultaneously with cell wall material during cell growth  <cit> .
md <dig> was the α- <dig> -xylosyltransferase, typical of the hemicellulose biosynthetic pathway, whereas md <dig> was the xyloglucan endotransglycosylase/hydrolase  that is a xyloglucan-modifying enzyme.
the open reading frame  of md <dig> encoded a 277-amino-acid protein with a signal peptide and a gh16-xet domain  and therefore designated mdxth <dig>  the catalytic site deidfeflg, conserved among gh <dig> family members  <cit>  and most seed plant xths  <cit>  was present in mdxth <dig> as xexdxefxg and immediately followed by a potential n-glycosylation site nxt/s  <cit>  .
the other  <dig> identified tdfs were involved in wall assembly, reorganization, and selective degradation.
four of them gave significant hits with expansins: mdexp <dig> , mdexp <dig> , mdexp <dig> , and mdexp <dig> .
whereas mdexp <dig> and mdexp <dig> were expressed during the early morphogenetic stages , mdexp <dig> and mdexp <dig> were up-regulated during later stages  .
changes in the internal structure of the cell walls, required for cell expansion, might be achieved by the release of hydroxyl radicals mediated by the class-iii peroxidases md <dig> and md <dig>  peroxidase-generated hydroxyl radicals could cause non-enzymatic wall loosening by cleavage of various polysaccharides  <cit> .
the orf of md <dig> contained a secretion signal peptide and a pfam peroxidase domain  .
the h2o <dig> substrate for the peroxidase activity was probably generated by the glyoxal oxidases md <dig>  md <dig>  and md <dig>  hydrolytic enzymes included the pectinesterase md <dig>  the endo-β- <dig> -galactanase md <dig>  and two members of cluster c5: the polygalacturonidase md <dig> and the β-glucosidase md <dig>  possibly involved in degradation of a connecting zone between the primary and the secondary cell wall, thereby enabling shedding of the primary cell wall  <cit> .
phylogenetic relationship of m. denticulata expansin-resembling proteins
as the involvement of expansins in cell growth of green algae had not been examined previously, we concentrated the experiments on this class of proteins.
the full length characteristics of the m. denticulata expansin-resembling proteins  are given in additional file  <dig>  mdexp <dig> and mdexp <dig> exhibited the highest sequence similarity  .
phylogenetic analysis of the first dataset revealed that all mdexps were recovered as a monophyletic group with high support  .
the micrasterias and spirogyra sequences fell within the plant expansins and were most closely related to the expa family, with which they formed a well supported clade .
the mdexps are recovered sister to the expa clade and the spirogyra sequences form a paraphyletic assemblage, but the relationships between the micrasterias and spirogyra expansins and the expa clade are poorly supported.
the high sequence divergence of expansins within and among micrasterias and spirogyra is shown by the relatively longer branches than those within the expa clade.
in the second dataset, the putative expansin sequences of chlorophyta formed a highly divergent clade, separated from the plant expansins by a very long branch .
although the relationships between the chlorophyta clade, the dictyostelium clade and the plant expansin families were poorly resolved, the phylogenetic position of the micrasterias clade, closely allied to the expa family, was well supported.
domain organization of the m. denticulata expansin-resembling proteins
the structural domain organization of the different mdexps was compared with the characteristic structural features of plant expansins .
a secretion signal peptide was present in all of them .
while the pollen-allerg- <dig> domain occurred in all proteins, except mdexp <dig>  the gh <dig> domain was found in mdexp <dig> and mdexp <dig> only, albeit with insignificant e-values.
nevertheless, in all sequences, a dpbb- <dig> domain was present, a rare lipoprotein a-like double-psi beta-barrel, to which gh <dig> belongs, and even twice in mdexp <dig> .
the eight cystenyl residues forming disulfide bridges in fungal gh <dig> enzymes and maintaining their folded structure  <cit>  were conserved in the expansin domain  <dig> of some of the plant expansin groups  <cit>  and also in the mdexps .
in m. denticulata, the ggacgy motif was present as ggscgy/f, whereas the gxxcgxcf/y motif in the same expansin domain  <dig> was fully conserved.
a third motif characteristic for this domain, the y/frrvpc motif, varied among the mdexps .
the key residues of the gh <dig> catalytic site, conserved among expa and expb proteins , were absent.
in land plant expansins, the pollen-allergen domain contains four conserved tryptophan residues that form part of the hydrophobic core of this domain  <cit>  .
in the mdexps up to two of these residues occurred and were fully conserved, when the structurally related amino acids phenylalanine and tyrosine are taken into account .
although the highly conserved hatfyg motif near the n-terminus is characteristic of expa proteins  <cit> , this motif could not be found in the mdexps.
the expa and expb proteins were distinguished by the presence or absence of short stretches of amino acids at conserved positions at either side of the hfdl motif in the gh <dig> active site   <cit> .
according to the phylogeny, the mdexps contained an α-insertion characteristic of expas, but they lacked the four highly conserved n-terminal residues 'gwcn' found in other expas  <cit> .
of the hfdl motif, only the leucine residue was conserved .
however, the long c-terminal extension of mdexp <dig> was typical for exla proteins  <cit> .
although mdexps were heterogeneous and divergent, they clearly shared several characteristics of the expa protein domains, supporting our phylogenetic results.
when a domain is present, its position is given .
a, unique characteristic of the expa family; b, unique characteristic of the expb family; la, unique characteristic of the exla family; lb, unique characteristic of the exlb family
subcellular localization of the expansin-resembling mdexp <dig> and phenotypic changes due to its overexpression
the orf of the m. denticulata expansin-resembling protein with the highest mrna levels during cell growth, namely mdexp <dig>  was cloned into an overexpression vector to allow c-terminal fusions to the green fluorescence protein   <cit> .
as observed by confocal laser scanning microscopy of transiently mdexp2-gfp-overexpressing interphase cells, the mdexp2-gfp fluorescence occurred as motile cytoplasmic dots  but could not be observed in the secondary cell wall itself, probably because of quenching due to a low apoplast ph  <cit> .
therefore, mdexp2-gfp-overexpressing interphase cells were processed for transmission electron microscopy  and stained with gfp antibodies and protein a-gold to investigate whether the mdexp2-gfp protein localizes into the secondary cell wall.
indeed, a positive signal was observed in the secondary cell wall, albeit not abundantly , probably due to the instability of the gfp protein in this acid compartment  <cit> .
in addition, mucilage vesicles still attached to distal golgi cisternae  and some released from the dictyosome  were stained.
this immunogold labelling indicated that the punctate pattern of the gfp fluorescence  could correspond to golgi-derived mucilage vesicles and that the fusion protein was directed to the wall via the endoplasmic reticulum-golgi secretory pathway.
no staining was observed in experiments for specificity control consisting of sections treated with protein a-gold alone .
in control sections of transgenic cells producing the free gfp, labelling occurred in the cytoplasm and was absent from the cell wall and cell organelles .
next,  <dig> independent transient transgenic cells were isolated and further analysed .
a group of cells lost the gfp-fluorescence within a few days and divided, resulting in normal daughter cells, while the majority of the cells died, possibly because of strong mdexp <dig> overexpression as indicated by their bright gfp fluorescence.
however, in eight independent cell lines, a range of phenotypes related to mdexp <dig> overexpression during cell division and growth could be observed.
line  <dig> exhibited strong lobe elongation without loss of growth polarity after the first cell division .
the lobes were stretched and rounded instead of flattened at their tips.
after the second cell division of line  <dig> and in all other cases , the growth polarity was altered.
line  <dig> lost its planarity upon cell division and, thus, had the most severe phenotype.
new semicells, without the characteristically lobed morphology, but almost without indentations, grew out three-dimensionally.
upon a new cell division of one of the daughter cells, the same phenotype was observed, whereas the newly formed semicells were also fused with each other .
in lines  <dig>   <dig>   <dig>   <dig> ,  <dig>   <dig>  and  <dig> axial but not radial elongation was impaired, resulting in semicells with a stunted polar lobe and fused lateral lobes .
sometimes, the second division gave rise to a similar morphology , but in most cases the phenotype was lost over one to two subsequent generations .
that all phenotypes still had the gfp signal and none of them resulted from control experiments with transgenic cells expressing only the gfp  <cit>  suggests that they were related to the expression of the transgene.
discussion
genome-wide expression analysis revealed a role for rab and snare cycles in membrane fusions and for agp-like proteins in cell pattern establishment.
agps, differing in composition from land plants, had recently been found to be present in the growing primary cell wall of micrasterias  <cit> .
our analysis further suggests an involvement of class-iii peroxidases, xth and expansins in cell wall growth.
class-iii peroxidases had been considered absent in green algae  <cit> , although a  mrna occurred in the desmid closterium  <cit> .
here, a full length algal class-iii peroxidase is linked to cell growth.
furthermore, despite their supposed lack of xyloglucans  <cit> , xet activity was found in the streptophyte chara and the chlorophyte ulva  <cit> .
recently, the -β-glucan  has been determined as the main constituent of the secondary cell wall of micrasterias  <cit>  and in this study, the first algal xth was identified.
the green algae valonia  and nitella  exhibit acid-induced wall extension, but this response is seemingly not mediated by proteins  <cit> .
contrary to the assumption of a land plant specific mechanism  <cit> , four genes with significant similarity to expansins were up-regulated during cell growth of micrasterias, in agreement with a presumed ancient evolutionary origin  <cit> .
based on significant blast similarities with the expansin domains  <cit> , global pairwise alignment and phylogeny, and structural features like the presence of a secretion signal, mdexp <dig>  mdexp <dig>  and mdexp <dig> are considered expansins, but considerably diverge in gene architecture from embryophytic expansins, as indicated by both domain analysis and phylogeny.
these results add to the evidence that expansins are not strongly conserved through evolution  <cit> .
the key residues of the gh <dig> domain catalytic site and the hfdl motif, which are present in land plants and spirogyra, do not occur in micrasterias.
the hfdl motif is present in most groups of plant expansins, but is absent in a few plant expa and expb proteins  <cit> .
the eight n-terminal cysteines required for protein folding  <cit>  and the four c-terminal tryptophans or related residues involved in cellulose binding  <cit>  are conserved between micrasterias, spirogyra and land plants and can be considered as key characteristics of plant expansins.
the ggxcgy/f and the gxxcgxcf/y motifs in the gh <dig> domain are conserved as well.
the only constant difference in the conserved amino acid residues in micrasterias when compared to land plants is the occurrence of a serine residue instead of an alanine residue in the ggacgy motif of the gh <dig> domain.
as expansins disrupt noncovalent bonding between cellulose microfibrils and matrix glucans that stick to the microfibril  <cit> , we hypothesize that the characteristics of the mdexps might be related to the dominant mlg in the secondary cell walls of micrasterias  <cit>  instead of the -β-glucan backbone present in dicotyledonous plants.
the occurrence of mlg in lichens  <cit> , fungi  <cit> , green algae   <cit> , horsetails  <cit> , and poales  <cit>  has been suggested to result from convergent evolution  <cit> , whereas the occurrence of distinct mdexps might be connected to two different  cell wall types, implied by their different temporal expression patterns.
based on the present expansin phylogeny, combined with current hypotheses on the evolution of the closest relatives to land plants  <cit> , expansins can fairly be assumed to have evolved before the origin of land plants.
however, the unresolved relationships between embryophytes and the streptophyte lineages zygnematophyceae, coleochaetophyceae and charophyceae  <cit>  hamper a solid reconstruction of expansin gene history.
assuming that the zygnematophyceae form the closest living relatives to land plants  <cit> , a possible scenario would be that expansins evolved into two lineages  in a common ancestor of embryophytes and zygnematophyceae .
the apparent lack of expb and exl in micrasterias and spirogyra might be due to gene loss, early in the evolution of the zygnematophyceae.
it should be emphasized however, that the ancient relationships among expansin families are difficult to resolve.
therefore the phylogenetic positions of the green algal expansin-resembling genes should be interpreted with care, hinting at a complete divergence of the plant expansin families within the embryophytic lineage.
distinct differences in gene architecture between micrasterias and embryophytic expansins have raised the question whether the biochemical functions of mdexps and embryophytic expansins are similar.
to this end, we studied functionally mdexp <dig>  the mdexp with the highest expression levels during growth, through localization and overexpression.
a gfp antibody detecting the mdexp2-gfp fusion protein was used, because the sequence conservation was too low for the available plant expansin antibodies.
unfortunately, currently, because only transient genetic transformation of micrasterias is possible  <cit> , immunoelectron microscopic detection in the growing cell walls is unfeasible.
nevertheless, the ectopically produced protein was targeted to the fully-grown secondary cell wall.
in addition, the phenotypic results obtained from its overexpression suggest that mdexp <dig> can alter the cell wall shape, but this effect on growth cannot be excluded to result from saturation or blockage of the membrane trafficking of other essential proteins.
the phenotypes were remarkably variable, whereby the phenotype severity did not seem to directly correlate with expansin abundance , as reported previously  <cit> .
although a phenotype could be observed corresponding to the expected enhanced wall extensibility due to increased expansin levels  <cit> , the elongation growth impaired in most cases, but not the lateral expansion, resulting in the fusion of the lateral lobes.
a number of factors might explain the reduced growth of tomato  overexpressing an expansin  <cit> .
all together, the growth phase-specific expression, the accumulation in the cell walls, and its overexpression phenotype, allow us to to hypothesize that mdexp <dig> might have a biochemical function related to that of land plant expansins.
CONCLUSIONS
our study provides novel data on gene expression during morphogenesis and cell growth in the desmid micrasterias denticulata and adds to our understanding of the evolution of genes involved in cell wall formation in green algae and land plants.
cell walls have played crucial roles in the colonization of land by plants  <cit> .
for a detailed understanding of how cell walls have evolved, cell wall components and cell wall-related genes in land plants and their closest relatives, the streptophyte green algae need to be analyzed comprehensively.
although some cell wall components appear to be adaptations of land plants, cell wall evolution after the colonization of land is seemingly characterized by the elaboration of a pre-existing set of genes and polysaccharides rather than by substantial innovations  <cit> .
the data add to the growing body of evidence that the evolutionary origins of many cell wall components and regulating genes in embryophytes antedate the colonization of land.

 although the form and function of plant cells are strongly correlated, the processes that determine the cell shape remain largely unknown. plant cell morphogenesis is regulated in a non-cell-autonomous fashion by the surrounding tissues, hormone interference during ontogenesis, and sometimes by polyploidy as a consequence of endoreduplication. in contrast, in unicellular relatives of land plants, it is possible to study the endogenous controls of cell morphogenesis without the interference by interacting cells and to better understand how these mechanisms have evolved in the green lineage. the desmid micrasterias denticulata is a member of the conjugating green algae that comprise the closest extant unicellular relatives of land plants. m. denticulata cells consist of two bilaterally symmetrical flat semicells, notched deeply around their perimeter into one polar lobe and four main lateral lobes. following cell division, each semicell builds a new one through a process of septum bulging and symmetrical local growth cessations to form the successive lobes. after completion of the primary wall, a rigid cellulosic secondary cell wall pierced by pores is deposited, followed by shedding of it. this peculiar growth mechanism makes micrasterias an ideal model to study the spatial and temporal patterning of cell wall biogenesis. ultimately, the plant cell morphology is determined by the composition and structure of the cell wall that governs the cell expansion direction and rate. as in land plants, the primary cell wall of m. denticulata bréb. consists mainly of pectins, cellulose microfibrils, hemicelluloses and arabinogalactan proteins. the secondary cell wall owes it rigidness to cellulose microfibrils originating from rosettes organized as hexagonal arrays, whereas mixed-linked glucan is the dominant hemicellulose. in land plants, expansins are important regulators of turgor-driven cell wall expansion. these cell wall proteins comprise a large multigene superfamily consisting of four families of which the evolutionary relationships are well characterized. they are unique in their ability to loosen the cell wall non-enzymatically by disrupting hydrogen bonds that link the cellulose and hemicellulose wall components. land plant expansins consist of two domains and a secretion signal. the n-terminal expansin domain and the c-terminal expansin domain are homologous to the catalytic domain of glycoside hydrolase family proteins and a domain present in a family of grass pollen allergens, identified as a putative cellulose binding site, respectively. expansins play a role in tissue development and in growth of suspension-cultured cells. although genes encoding expansin-like proteins have been recently identified in green algae transcriptomes, their physiological function and phylogenetic relationships with land plant expansins remain unknown. here, we explore the molecular basis of cell morphogenesis and cell wall formation in synchronized m. denticulata cells by means of a cdna-amplified fragment length polymorphism -based quantitative transcriptome analysis. several cell wall-related genes, among which expansins, were identified. examination of the expansins provided the first structural, phylogenetic and functional data on green algal homologues within this gene family.  cdna-aflp expression profiling first we developed a synchronization protocol to monitor the cell morphogenesis-related gene expression in m. denticulata. the protocol was based on the observation that the majority of the cells grown in a 14-h light/10-h dark regime divided during the second dark period, after the growth medium of a stationary culture had been refreshed and, concomitantly, the cell density reduced at the start of the light period. replacing the dark period by a light period enhanced the amount of synchronically dividing cells. the effect of cell density on synchronization was significant, with an optimal cell density below cells ml- following synchronization, up to 85% of the cell population divided during an 8- to 9-h period, showing a sigmoid course. by sampling this period at five consecutive time points we obtained samples with different proportions of cells at the major morphogenetic stages. cdna-aflp expression profiling of these samples allowed the assignment of differentially expressed genes to either the onset of cell division ), the bulge ), the lobe ), or the doublet stage, during which the secondary cell wall is formed ). in total, the relative abundance was monitored of transcript-derived fragments during the cell growth of m. denticulata, for which the expression patterns were altered visibly across time in and significantly in tdfs. according to other studies, we estimate that two-thirds of the mrna population was sampled, implying that the real number of genes differentially expressed during cell growth of m. denticulata could be ~ a high similarity to database entries with assigned identities and unknown or hypothetical genes was found for and tdfs, respectively, mostly with embryophytes and not with chlorophyta. however, the majority of the tdfs showed no sequence similarity to any database entry. plausible explanations might be sequences too short to reveal any significant identity, short sequences representing non-conserved portions of genes, tdfs originating from the 3'-untranslated region of a gene, or tdfs representing genes specific to m. denticulata or streptophytic algae. of the annotated genes, clustered into six groups according to the timing of their highest expression. except for one cluster consisting of six genes, the expression profiles were reproducible in the two independent sampling series. the few genes not included in one of the described clusters typically showed narrow temporal expression patterns. based on their annotation, the tdfs were classified into functional categories, named according to the gene ontology terminology. the association between the functional category and the tdf clustering was not significant. the major group with a significant hit was involved in cell wall metabolism. the second largest category corresponded to sequences sharing significant similarity to unknown or hypothetical proteins. of tdfs with similarity to genes involved in cell wall biogenesis or cell pattern formation, the rna samples of the second cdna-aflp replication series and on an independently sampled series were analyzed by real-time quantitative reverse-transcription -pcr. in general, the expression profiles obtained by cdna-aflp and qrt-pcr corresponded well, confirming the obtained expression results. genes relevant for cell pattern formation seven tdfs could be identified that might be relevant for cell pattern formation in m. denticulata, among which two members of the rab gtpase cycle and two members of the snare cycle of membrane fusion reactions. rab similar to md is known to be involved in post-golgi transport to the plasma membrane, inducing the formation of new surface extensions and believed to be regulated by a guanine nucleotide dissociation inhibitor possibly corresponding to md both md and md belonged to cluster c1a and, thus, had increased mrna levels before the onset of mitosis. this observation might be related to the determination of the basic symmetry of a m. denticulata cell before mitosis, indicated by the development of a three-lobed semicell after removal of the nucleus. in contrast, the snare cycle members were highly expressed in cluster c pointing to a role in further differentiation during the lobe stages for md and md. two tdfs were identified encoding putative glycophosphatidylinositol anchors: md and md belonging to clusters c1a, and c respectively. among other properties, the function of a gpi anchor might be its dominant targeting to a specific membrane domain, possibly establishing a membrane template for morphogenesis. md turned out to be a 179-amino-acid protein containing a signal peptide and a fasciclin domain, with similarity to a fasciclin-like and an agp-like protein from brachypodium sylvaticum and arabidopsis thaliana, respectively. md, similar to a very-long-chain fatty acid-condensing enzyme, might be involved in morphogenesis in accordance to the essential role in cell expansion during plant morphogenesis of arabidopsis. genes involved in cell wall metabolism a total of cell wall-related genes were identified. six tdfs operating in the monosaccharide metabolism, evenly distributed over c and c could be identified as udp-pyrophosphorylases, a phosphoglucomutase, a rhamnose synthase, and a gdp-mannose -epimerase. nine polysaccharide synthesis enzymes all nearly clustered in c among which two cellulose synthases, md and md and one cellulose synthase-like gene of the cslc family, md the exostosin family glycosyltransferases md md md and the glycosyltransferase md might synthesize the hemicellulosic or pectinous part of the cell wall and mucilage as well that is pectic in nature and secreted simultaneously with cell wall material during cell growth. md was the α- -xylosyltransferase, typical of the hemicellulose biosynthetic pathway, whereas md was the xyloglucan endotransglycosylase/hydrolase that is a xyloglucan-modifying enzyme. the open reading frame of md encoded a 277-amino-acid protein with a signal peptide and a gh16-xet domain and therefore designated mdxth the catalytic site deidfeflg, conserved among gh family members and most seed plant xths was present in mdxth as xexdxefxg and immediately followed by a potential n-glycosylation site nxt/s. the other identified tdfs were involved in wall assembly, reorganization, and selective degradation. four of them gave significant hits with expansins: mdexp, mdexp, mdexp, and mdexp. whereas mdexp and mdexp were expressed during the early morphogenetic stages, mdexp and mdexp were up-regulated during later stages. changes in the internal structure of the cell walls, required for cell expansion, might be achieved by the release of hydroxyl radicals mediated by the class-iii peroxidases md and md peroxidase-generated hydroxyl radicals could cause non-enzymatic wall loosening by cleavage of various polysaccharides. the orf of md contained a secretion signal peptide and a pfam peroxidase domain. the h2o substrate for the peroxidase activity was probably generated by the glyoxal oxidases md md and md hydrolytic enzymes included the pectinesterase md the endo-β- -galactanase md and two members of cluster c5: the polygalacturonidase md and the β-glucosidase md possibly involved in degradation of a connecting zone between the primary and the secondary cell wall, thereby enabling shedding of the primary cell wall. phylogenetic relationship of m. denticulata expansin-resembling proteins as the involvement of expansins in cell growth of green algae had not been examined previously, we concentrated the experiments on this class of proteins. the full length characteristics of the m. denticulata expansin-resembling proteins are given in additional file mdexp and mdexp exhibited the highest sequence similarity. phylogenetic analysis of the first dataset revealed that all mdexps were recovered as a monophyletic group with high support. the micrasterias and spirogyra sequences fell within the plant expansins and were most closely related to the expa family, with which they formed a well supported clade. the mdexps are recovered sister to the expa clade and the spirogyra sequences form a paraphyletic assemblage, but the relationships between the micrasterias and spirogyra expansins and the expa clade are poorly supported. the high sequence divergence of expansins within and among micrasterias and spirogyra is shown by the relatively longer branches than those within the expa clade. in the second dataset, the putative expansin sequences of chlorophyta formed a highly divergent clade, separated from the plant expansins by a very long branch. although the relationships between the chlorophyta clade, the dictyostelium clade and the plant expansin families were poorly resolved, the phylogenetic position of the micrasterias clade, closely allied to the expa family, was well supported. domain organization of the m. denticulata expansin-resembling proteins the structural domain organization of the different mdexps was compared with the characteristic structural features of plant expansins. a secretion signal peptide was present in all of them. while the pollen-allerg- domain occurred in all proteins, except mdexp the gh domain was found in mdexp and mdexp only, albeit with insignificant e-values. nevertheless, in all sequences, a dpbb- domain was present, a rare lipoprotein a-like double-psi beta-barrel, to which gh belongs, and even twice in mdexp. the eight cystenyl residues forming disulfide bridges in fungal gh enzymes and maintaining their folded structure were conserved in the expansin domain of some of the plant expansin groups and also in the mdexps. in m. denticulata, the ggacgy motif was present as ggscgy/f, whereas the gxxcgxcf/y motif in the same expansin domain was fully conserved. a third motif characteristic for this domain, the y/frrvpc motif, varied among the mdexps. the key residues of the gh catalytic site, conserved among expa and expb proteins, were absent. in land plant expansins, the pollen-allergen domain contains four conserved tryptophan residues that form part of the hydrophobic core of this domain. in the mdexps up to two of these residues occurred and were fully conserved, when the structurally related amino acids phenylalanine and tyrosine are taken into account. although the highly conserved hatfyg motif near the n-terminus is characteristic of expa proteins, this motif could not be found in the mdexps. the expa and expb proteins were distinguished by the presence or absence of short stretches of amino acids at conserved positions at either side of the hfdl motif in the gh active site. according to the phylogeny, the mdexps contained an α-insertion characteristic of expas, but they lacked the four highly conserved n-terminal residues'gwcn' found in other expas. of the hfdl motif, only the leucine residue was conserved. however, the long c-terminal extension of mdexp was typical for exla proteins. although mdexps were heterogeneous and divergent, they clearly shared several characteristics of the expa protein domains, supporting our phylogenetic results. when a domain is present, its position is given. a, unique characteristic of the expa family; b, unique characteristic of the expb family; la, unique characteristic of the exla family; lb, unique characteristic of the exlb family subcellular localization of the expansin-resembling mdexp and phenotypic changes due to its overexpression the orf of the m. denticulata expansin-resembling protein with the highest mrna levels during cell growth, namely mdexp was cloned into an overexpression vector to allow c-terminal fusions to the green fluorescence protein. as observed by confocal laser scanning microscopy of transiently mdexp2-gfp-overexpressing interphase cells, the mdexp2-gfp fluorescence occurred as motile cytoplasmic dots but could not be observed in the secondary cell wall itself, probably because of quenching due to a low apoplast ph. therefore, mdexp2-gfp-overexpressing interphase cells were processed for transmission electron microscopy and stained with gfp antibodies and protein a-gold to investigate whether the mdexp2-gfp protein localizes into the secondary cell wall. indeed, a positive signal was observed in the secondary cell wall, albeit not abundantly, probably due to the instability of the gfp protein in this acid compartment. in addition, mucilage vesicles still attached to distal golgi cisternae and some released from the dictyosome were stained. this immunogold labelling indicated that the punctate pattern of the gfp fluorescence could correspond to golgi-derived mucilage vesicles and that the fusion protein was directed to the wall via the endoplasmic reticulum-golgi secretory pathway. no staining was observed in experiments for specificity control consisting of sections treated with protein a-gold alone. in control sections of transgenic cells producing the free gfp, labelling occurred in the cytoplasm and was absent from the cell wall and cell organelles. next, independent transient transgenic cells were isolated and further analysed. a group of cells lost the gfp-fluorescence within a few days and divided, resulting in normal daughter cells, while the majority of the cells died, possibly because of strong mdexp overexpression as indicated by their bright gfp fluorescence. however, in eight independent cell lines, a range of phenotypes related to mdexp overexpression during cell division and growth could be observed. line exhibited strong lobe elongation without loss of growth polarity after the first cell division. the lobes were stretched and rounded instead of flattened at their tips. after the second cell division of line and in all other cases, the growth polarity was altered. line lost its planarity upon cell division and, thus, had the most severe phenotype. new semicells, without the characteristically lobed morphology, but almost without indentations, grew out three-dimensionally. upon a new cell division of one of the daughter cells, the same phenotype was observed, whereas the newly formed semicells were also fused with each other. in lines, and axial but not radial elongation was impaired, resulting in semicells with a stunted polar lobe and fused lateral lobes. sometimes, the second division gave rise to a similar morphology, but in most cases the phenotype was lost over one to two subsequent generations. that all phenotypes still had the gfp signal and none of them resulted from control experiments with transgenic cells expressing only the gfp suggests that they were related to the expression of the transgene. discussion genome-wide expression analysis revealed a role for rab and snare cycles in membrane fusions and for agp-like proteins in cell pattern establishment. agps, differing in composition from land plants, had recently been found to be present in the growing primary cell wall of micrasterias. our analysis further suggests an involvement of class-iii peroxidases, xth and expansins in cell wall growth. class-iii peroxidases had been considered absent in green algae, although a mrna occurred in the desmid closterium. here, a full length algal class-iii peroxidase is linked to cell growth. furthermore, despite their supposed lack of xyloglucans, xet activity was found in the streptophyte chara and the chlorophyte ulva. recently, the -β-glucan has been determined as the main constituent of the secondary cell wall of micrasterias and in this study, the first algal xth was identified. the green algae valonia and nitella exhibit acid-induced wall extension, but this response is seemingly not mediated by proteins. contrary to the assumption of a land plant specific mechanism, four genes with significant similarity to expansins were up-regulated during cell growth of micrasterias, in agreement with a presumed ancient evolutionary origin. based on significant blast similarities with the expansin domains, global pairwise alignment and phylogeny, and structural features like the presence of a secretion signal, mdexp mdexp and mdexp are considered expansins, but considerably diverge in gene architecture from embryophytic expansins, as indicated by both domain analysis and phylogeny. these results add to the evidence that expansins are not strongly conserved through evolution. the key residues of the gh domain catalytic site and the hfdl motif, which are present in land plants and spirogyra, do not occur in micrasterias. the hfdl motif is present in most groups of plant expansins, but is absent in a few plant expa and expb proteins. the eight n-terminal cysteines required for protein folding and the four c-terminal tryptophans or related residues involved in cellulose binding are conserved between micrasterias, spirogyra and land plants and can be considered as key characteristics of plant expansins. the ggxcgy/f and the gxxcgxcf/y motifs in the gh domain are conserved as well. the only constant difference in the conserved amino acid residues in micrasterias when compared to land plants is the occurrence of a serine residue instead of an alanine residue in the ggacgy motif of the gh domain. as expansins disrupt noncovalent bonding between cellulose microfibrils and matrix glucans that stick to the microfibril, we hypothesize that the characteristics of the mdexps might be related to the dominant mlg in the secondary cell walls of micrasterias instead of the -β-glucan backbone present in dicotyledonous plants. the occurrence of mlg in lichens, fungi, green algae, horsetails, and poales has been suggested to result from convergent evolution, whereas the occurrence of distinct mdexps might be connected to two different cell wall types, implied by their different temporal expression patterns. based on the present expansin phylogeny, combined with current hypotheses on the evolution of the closest relatives to land plants, expansins can fairly be assumed to have evolved before the origin of land plants. however, the unresolved relationships between embryophytes and the streptophyte lineages zygnematophyceae, coleochaetophyceae and charophyceae hamper a solid reconstruction of expansin gene history. assuming that the zygnematophyceae form the closest living relatives to land plants, a possible scenario would be that expansins evolved into two lineages in a common ancestor of embryophytes and zygnematophyceae. the apparent lack of expb and exl in micrasterias and spirogyra might be due to gene loss, early in the evolution of the zygnematophyceae. it should be emphasized however, that the ancient relationships among expansin families are difficult to resolve. therefore the phylogenetic positions of the green algal expansin-resembling genes should be interpreted with care, hinting at a complete divergence of the plant expansin families within the embryophytic lineage. distinct differences in gene architecture between micrasterias and embryophytic expansins have raised the question whether the biochemical functions of mdexps and embryophytic expansins are similar. to this end, we studied functionally mdexp the mdexp with the highest expression levels during growth, through localization and overexpression. a gfp antibody detecting the mdexp2-gfp fusion protein was used, because the sequence conservation was too low for the available plant expansin antibodies. unfortunately, currently, because only transient genetic transformation of micrasterias is possible, immunoelectron microscopic detection in the growing cell walls is unfeasible. nevertheless, the ectopically produced protein was targeted to the fully-grown secondary cell wall. in addition, the phenotypic results obtained from its overexpression suggest that mdexp can alter the cell wall shape, but this effect on growth cannot be excluded to result from saturation or blockage of the membrane trafficking of other essential proteins. the phenotypes were remarkably variable, whereby the phenotype severity did not seem to directly correlate with expansin abundance, as reported previously. although a phenotype could be observed corresponding to the expected enhanced wall extensibility due to increased expansin levels, the elongation growth impaired in most cases, but not the lateral expansion, resulting in the fusion of the lateral lobes. a number of factors might explain the reduced growth of tomato overexpressing an expansin. all together, the growth phase-specific expression, the accumulation in the cell walls, and its overexpression phenotype, allow us to to hypothesize that mdexp might have a biochemical function related to that of land plant expansins.  our study provides novel data on gene expression during morphogenesis and cell growth in the desmid micrasterias denticulata and adds to our understanding of the evolution of genes involved in cell wall formation in green algae and land plants. cell walls have played crucial roles in the colonization of land by plants. for a detailed understanding of how cell walls have evolved, cell wall components and cell wall-related genes in land plants and their closest relatives, the streptophyte green algae need to be analyzed comprehensively. although some cell wall components appear to be adaptations of land plants, cell wall evolution after the colonization of land is seemingly characterized by the elaboration of a pre-existing set of genes and polysaccharides rather than by substantial innovations. the data add to the growing body of evidence that the evolutionary origins of many cell wall components and regulating genes in embryophytes antedate the colonization of land. 
overexpression phenotypes ranged from lobe elongation to loss of growth polarity and planarity. the multilobed cell wall of micrasterias denticulata that rebuilds symmetrically after cell division and consists of pectin and cellulose, makes this unicellular streptophyte alga an interesting model system to study the molecular controls on cell shape and cell wall formation in green plants. four transcripts showed high similarity to expansins that had not been examined previously in green algae. streptophyte green algae share several characteristics of cell growth and cell wall formation with their relatives, the embryophytic land plants. our study demonstrates the potential of m. denticulata as a unicellular model system, in which cell growth mechanisms have been discovered similar to those in land plants. these results indicate that mdexp can alter the cell wall structure and, thus, might have a function related to that of land plant expansins during cell morphogenesis. phylogenetic analysis suggests that these genes are most closely related to the plant expansin a family, although their domain organization is very divergent. additionally, evidence is provided that the evolutionary origins of many cell wall components and regulatory genes in embryophytes precede the colonization of land. a gfp-tagged version of the expansin-resembling protein mdexp localized to the cell wall and in golgi-derived vesicles.
 streptophyte green algae share several characteristics of cell growth and cell wall formation with their relatives, the embryophytic land plants. the multilobed cell wall of micrasterias denticulata that rebuilds symmetrically after cell division and consists of pectin and cellulose, makes this unicellular streptophyte alga an interesting model system to study the molecular controls on cell shape and cell wall formation in green plants.  genome-wide transcript expression profiling of synchronously growing cells identified genes of which the expression correlated with the growth phase. four transcripts showed high similarity to expansins that had not been examined previously in green algae. phylogenetic analysis suggests that these genes are most closely related to the plant expansin a family, although their domain organization is very divergent. a gfp-tagged version of the expansin-resembling protein mdexp localized to the cell wall and in golgi-derived vesicles. overexpression phenotypes ranged from lobe elongation to loss of growth polarity and planarity. these results indicate that mdexp can alter the cell wall structure and, thus, might have a function related to that of land plant expansins during cell morphogenesis.  our study demonstrates the potential of m. denticulata as a unicellular model system, in which cell growth mechanisms have been discovered similar to those in land plants. additionally, evidence is provided that the evolutionary origins of many cell wall components and regulatory genes in embryophytes precede the colonization of land. 