Why do cells elongate

Xyloglucans and homogalacturonans are synthesized there actively together with mixed-linkage glucans and glucuronoarabinoxylans. Rhamnogalacturonans-I with the side-chains of branched 1,4-galactan and arabinan persisted in cell walls throughout the development. Thus, the machinery to generate the type I primary cell wall constituents is completely established and operates. The expression of glycosyltransferases responsible for mixed-linkage glucan and glucuronoarabinoxylan synthesis peaks at active or late elongation.

These findings widen the number of jigsaw pieces which should be put together to solve the puzzle of grass cell growth. The ability to expand or to elongate many times compared to the initial size is a vital property of plant cells. Cells which are capable to grow are surrounded by a thin primary cell wall PCW. The enlargement of plant cells occurs under the action of turgor pressure and is controlled by the mechanical properties of their cell walls.

Mechanical properties, in turn, depend on the cell wall composition and architecture. The mechanisms underlying the growth of plant cells have mainly been studied in dicotyledonous species and non-commelinoid monocots with type I primary cell walls Fig.

Cellulose in the form of microfibrils is present in plant cell walls of all types. Type I cell walls also have pectins and xyloglucans XyGs as the basic constituents 1. Hydrated pectin matrix fills the spaces between cellulose microfibrils. The major part of XyGs also exists between microfibrils in a coiled conformation or interacts with them in an extended conformation.

However, minor portion of XyGs is entrapped between cellulose strands 2. These local interactions of XyGs with cellulose named "biomechanical hotspots" were proposed to form microfibril junctions and integrate them into one load-bearing network 3. Alterations in the pectin structure are also considered a potential mechanism regulating wall expansion. Changes in cell wall hydration, the degree of cross-linking or accessibility of individual molecules to degrading enzymes are supposed to be a mechanism underlying the modulation of cell wall mechanics by pectin modifications 4 , 5.

Different types of plant cell wall. Models may not be to scale. Based on chronological order Buckeridge et al. The Poaceae family, which includes cereals, is characterized by type II primary cell walls Fig.

MLGs, together with low substituted GAXs are thought to cover cellulose, while high substituted GAXs are localized in a space between microfibrils 6 , 7 , 8. However, in vitro studies have shown that neither arabinoxylan AX nor MLG serve as equivalents of either XyG or pectin in interactions with cellulose 9 , These observations discourage direct extrapolations of recent findings in the field of plant cell growth mechanisms obtained from dicots onto grasses.

Many specialized cells, like tracheids, vessels, fibers and sclereids, deposit thickened secondary cell wall SCW inside the primary cell wall. This provides additional mechanical strength to plant tissues. Usually, it happens after cessation of expansion growth because solid SCW is not extensible. However, when secondary thickenings have a spiral or annular character of deposing they do not hinder cell elongation. SCWs Fig. Interaction of these three polymers occurs mainly through xylans The pattern of xylan backbone decoration determines the conformation of this polysaccharide and provides the conditions to interact either with cellulose or with lignin 13 , Grasses have a more complex structure of both lignin and xylans However, the general architecture of SCW is similar in grasses and dicots Fig.

The importance of the set and structural nuances of cell wall polysaccharides for plant development is getting more and more recognized. However, the molecular details of the processes in elongating cells of grasses remain elusive and need a combination of approaches and adequate model systems for elucidation. The transcriptomic analysis is a powerful tool for identifying genes that modulate biological processes in a living cell.

Over the past decade, numerous RNA-Seq and microarray analyses have been conducted on different grass organ and tissue samples to identify key participants in elongation growth. Maize internodes were among the most popular objects of such studies 16 , 17 , However, the comparison between elongating and non-elongating internodes shifts the focus to genes involved in growth cessation and secondary cell wall formation.

Additionally, actively growing intercalary meristems contain vascular tissues; otherwise, the meristematic regions would interrupt the transport continuity and mechanically weaken the stem Thus, even in its base, the internode represents a mixture of dividing and differentiated cells, which complicates the analysis. The primary root is a more convenient model system to study elongation growth. Several zones containing cells at different stages of development can be separated from each other based on the distance from the root apex This unique characteristic was partially employed in the large-scale analysis of transcriptomes 21 and proteomes 22 in various parts of the maize root system.

Relatively large root fragments that combined several zones were used in these studies, increasing the difficulty of distinguishing the cell division and cell elongation stages, and identifying the important processes occurring at the transition between these stages. We have applied an RNA-Seq analysis to five zones in the apical part of maize root before root hair emergence to reveal key participants involved in the initiation, realization and cessation of coordinated elongation growth in a plant with type II cell walls Fig.

Clustering and co-expression approaches used for genes encoding numerous glycosyltransferases GTs involved in the biosynthesis of cell wall polysaccharides, coupled with extensive immunohistochemical analysis to determine the distribution and dynamics of particular polysaccharide motifs, revealed several patterns of cell wall polysaccharide deposition that corresponded to important stages in cell development.

Schematic representation of the collection of samples from maize root in the current study A and for the proteome analysis reported by Marcon et al. Ten individual libraries of mRNAs from two biological replicates of five different samples of maize primary root were analyzed using Illumina sequencing technology. Three hundred thirty million cleaned and filtered 60 bp single-end Illumina reads with quality scores greater than Q30 were used in further analyses.

Notably, The annotation of the Z. Two hundred sixty-four genes belonging to 12 GT families and one methyl-transferase family were expressed in maize root. Their expression patterns were analyzed using a clustering analysis, and 6 clusters were identified Table S1. The phylogenetic analysis of GTs and the comparison with known members of the same GT families in rice and Arabidopsis were performed to further characterize the genes and determine the clade of the family Fig.

S1 — S The biosynthesis of the backbones for several cell wall polysaccharides is mediated by the enzymes encoded by members of the cellulose synthase CesA gene superfamily. The phylogenetic tree was built with known members of CesA superfamily in Arabidopsis and rice Fig. Among the three examined species, the CslB clade was represented only by Arabidopsis sequences, while the CslF and CslH clades included only rice and maize genes.

Two recent studies reported 20 members of this clade in maize 18 , 23 however, both studies used older versions of the Zea mays genome. Heat map color coding is applied separately to each gene subgroup. The underlined gene names indicate the baits for co-expression analysis. The genes co-expressed with maize primary cell wall CesAs are labelled in blue, and genes co-expressed with secondary cell wall CesAs are labelled in red.

Annotations are based on the study by Penning et al. The annotations shown in blue and in red are CesAs assigned to primary and secondary cell wall formation, respectively, by Penning et al. Cap—root cap, Mer—meristem, eElong—early elongation zone, Elong—zone of active elongation, lElong—zone of late elongation before root hair initiation, and RH—root hair zone. No data, i. Penning et al.

Transcripts of these genes were relatively abundant in the meristem zone. Four- to five-fold up-regulation was characteristic of these genes in the early elongation zone, with further increase in the elongation zone and two-fold down-regulation at the late elongation stage.

According to the proteomic study performed by Marcon et al. Both stele and cortex tissues in the root hair region of young maize root were characterized by high levels of these cellulose synthases These features of the transcription and translation of particular cellulose synthase genes probably reflect the high demand for new cell wall material by rapidly elongating cells. The six mentioned cellulose synthases were co-expressed with each other, with correlation coefficients greater than 0.

The ZmCesA genes were expressed at low levels in the root cap, meristem and early elongation zone. Significant increases in the levels of their transcripts occurred in the elongation zone, with further increases in the TGR values in the late elongation zone.

High levels of the corresponding proteins were detected in the stele of the root hair region of maize root A virtual absence of certain transcripts and proteins at the earlier stages of cell development, transcriptional up-regulation in the elongation zone that peaked at the stage of late elongation, and the high levels of proteins in the stele of the root hair region corresponded to the development of the vascular system and secondary cell wall thickening in maize root.

Expression level TGR, red-blue heat map of genes potentially involved in GAX backbone decoration in maize root and relative levels of the corresponding proteins averaged and normalized total spectral counts 22 , red-green heat map. Expression level TGR, red-blue heat map and relative protein abundance averaged and normalized total spectral counts 22 , red-green heat map of genes potentially involved in XyG synthesis in maize root.

The gene co-expressed with maize primary cell wall CesAs is labelled in blue. Green indicates genes co-expressed with ZmCslC5c underlined and displaying a correlation coefficient greater than 0. Expression level TGR, red-blue heat map and relative protein abundance averaged and normalized total spectral counts 22 , red-green heat map of genes potentially involved in HG and RG-I biosynthesis in maize root. Green indicates genes co-expressed with ZmCslC5c presenting correlation coefficients greater than 0.

Members of the cellulose synthase-like H, F and J clades of the CesA superfamily mediate MLG synthesis in grasses and heterologous expression systems According to the phylogenetic tree, seven sequences of maize genes were grouped with rice CslFs, and one with rice CslHs genes Fig.

No representatives of CslJ subfamily were found. Two maize orthologues of the rice CslF6 gene Zmd and Zmd exhibited the highest TGRs in the active elongation zone and were co-expressed ZmCesAs, which are predicted to be involved in primary cell wall formation Fig.

Another member of the CslF clade Zmd, the orthologue of rice and barley CslF3 was up-regulated in the meristem and early elongation zone. This GT was recently shown to possess novel activity, mediating the synthesis of glucoxylan Protein products of this gene were detected in maize root segments before, but not after, root hair initiation GAXs are the major noncellulosic polysaccharides present in the type II primary walls.

Xylosyl-transferase activity in vitro has only been observed for IRX10 26 , IRX9 and IRX14 are believed to function as structural components of the xylan synthase complex with no catalytic activity per se Four of them were shorter than amino acids and were excluded from further analysis. All were expressed in maize root, with the highest TGR values observed in the zones of active or late elongation Fig. The GT43 family is represented in the maize genome by 15 members encoding proteins containing the PF domain Fig.

All these genes were expressed Fig. Ten belonged to either primary or secondary cell wall-related co-expression networks. The GAX backbone can be decorated with arabinose, glucuronic acid, xylose or oligosaccharide chains.

The attachment of arabinosyl and xylosyl residues to the GAX molecule is mediated by GTs of the GT61 family 28 , 29 , while GT8 members are required for the glucuronosyl substitution of the xylan backbone The GT61 family has been subdivided into several clades with specific characterized members Fig.

Zmd had highest TGR value among all genes belonging to this subclade. Two other genes on the same branch Zmd and Zmd were co-expressed with primary or secondary cell wall-related CesAs, respectively Fig. The maize orthologue of OsXYXT1 Zmd had highest TGR values in the early elongation and elongation zones, and was significantly down-regulated in the late elongation zone of maize root Fig. Three maize members of the GT61 clade B were predominantly expressed in the elongation zone, while two others were expressed in the late elongation zone of maize root.

Five of these genes were expressed in maize primary root, with the maximum of TGR values in either the active elongation or late elongation stages. Thus, two different sets of GTs, both of which are sufficient for the production of highly substituted xylans, are expressed in maize primary root. Genes and proteins of these enzymes appear along the root length in a manner corresponding to primary and secondary cell wall biosynthesis.

Further substitution of xylose with galactose or fucosyl-galactose may occur in cell walls of Poales 35 , CslC4 of Arabidopsis induced XyG backbone synthesis in heterologous systems The maize genome contains eight members of the CslC clade Fig. S1 ; all eight genes were expressed in maize root Fig. Two orthologues of the Arabidopsis CslC5 gene Zmd and Zmd were expressed at high levels, with maximum TGR values observed in the root cap and meristem Fig.

A greater than fold decrease in transcript abundance was observed in the zone of active elongation. Proteins encoded by these genes were expressed at the highest levels in the meristematic region of maize root Zmd is also the orthologue of OsCslC3, which is predicted to function as a XyG backbone synthase in rice This gene was chosen as the bait in the co-expression analysis to identify other participants in XyG synthesis in maize Fig.

The XyG backbone is decorated by xylose residues. Twelve representatives of XXT clade Fig. S7 were expressed Fig. The maximum levels of the transcripts of almost all GT34 members were observed in the root cap or meristem zone of maize root. Expression decreased at later stages of cell development.

Five of these genes were co-expressed with ZmCslC5c Fig. Proteins of the GT34 family were mainly detected in the elongating part of maize root, and rarely in the root hair region. Xylose residues attached to the XyG backbone may be further substituted at the O 2 position with galactose.

Representatives of the GT47 family possess various activities, and the family is divided into several clades Fig. The vast majority of these genes were expressed with the highest TGR values in root cap and meristem zones. A recent study of Sorghum revealed two proteins functioning as the XyG galactosyl-transferases The Zmd gene is an orthologue of one of these genes in maize. Fucose-containing side-chains of XyG are barely detectable in Poaceae species However, fucogalactoxyloglucans were recently identified in young tissues of rice XyG fucosylation is catalyzed by members of the GT37 family Fourteen of these genes were expressed in analyzed root zones, and among them, four orthologues of AtFUT1 Zmd, Zmd, Zmd, and Zmd known to be involved in fucosylation of XyGs in Arabidopsis 44 and one orthologue of rice OsFUT1 Zmd were detected.

All five presented the highest TGR values in the root cap zone, and corresponding proteins were observed only in the meristematic part of maize root Fig.

Homogalacturonan HG usually is the most abundant pectic polysaccharide. Forty-seven members of the GT8 family were identified in the fourth version of the maize genome, although three of these genes are too short to encode active enzymes.

Corresponding proteins were not detected at high levels, but were mainly observed in the elongating part of the root in the study by Marcon and colleagues HG is synthesized in a highly methyl-esterified form and is postulated to lose ester groups during the elongation growth of cells 5. AtQUA2 is characterized on the protein level as pectin-methyl-transferase in Arabidopsis 49 , Recently, members of GT RRT clade , which were previously annotated as fucosyl-transferases, were shown to catalyze rhamnose attachment to the RG-I backbone acceptor The RRT clade is represented by eight genes in the maize genome Fig.

All of these genes were expressed in the primary root. Only the protein encoded by Zmd was detected This gene and two other members of this clade belonged to the co-expression network of CesAs genes responsible for primary cell wall formation.

The RG-I backbone can be substituted at the O 2 position of rhamnosyl residues. Three orthologues of this gene are present in the Zea mays genome.

All of these genes were expressed in the apical part of maize root. No corresponding proteins were detected This enzyme belongs to the GT47 family clade C Fig. Eight members of this clade were expressed in the maize primary root. However, corresponding proteins were detected only in the cortex of the root hair region The presence of transcripts and proteins of GTs essential for the synthesis of a particular polysaccharide does not indicate that this polymer is indeed produced.

We performed immunohistochemical analysis with a set of monoclonal antibodies to trace the dynamics of cell wall polysaccharides during the elongation growth of maize root. Calcofluor White stained cell walls in maize root evenly in the meristem, early elongation and elongation zones Fig.

Yellow coloration appeared in the vascular parenchyma in the late elongation zone, indicating its lignification. Brighter staining of the vascular ring corresponded to secondary cell wall thickening Fig. No fluorescence was detected in negative control samples primary antibodies were omitted under the observation conditions. However, their labelling was weaker in meristem region. The rhizodermis was only labelled by the AX1 antibody in the late elongation zone.

The LM27 antibody recognizing grass heteroxylans 56 did not label meristematic cells. At the stage of early elongation, it bound the rhizodermis and outer cell layer of the cortex. A similar pattern was observed in the elongation zone. Labelling was increased centripetally in the late elongation zone and observed in several layers of the cortex Fig. The LM28 antibody specific for glucuronoxylans 56 labelled all tissues except rhizodermis.

The preference for binding to stele tissues was observed in the early elongation, elongation and late elongation zones Fig. The LM11 antibody is often used to detect secondary cell wall xylans. It was raised against xylooligosaccharides 57 and probably requires a less substituted backbone fragment in contrast to other anti-GAX probes used in the current study. The LM11 antibody labelled vascular tissues in the elongation and late elongation zones.

Brighter labelling was also observed in the rhizodermis in the late elongation zone Fig. The epitope of LM25, the antibody specific for galactoxyloglucans 58 , was detected at relatively high levels in the meristematic region of maize root and root cap cells, and in the slime produced by these cells.

The labelling intensity increased in the early elongation zone and then decreased in the elongation and late elongation zones.

However, the root slime in these zones still bound the antibody Fig. A similar pattern of labelling was observed with another xyloglucan-specific antibody, LM15 data not shown. LM20 epitopes were present only in the meristematic region, and the labelling became weaker in subsequent zones. In contrast, LM19 labelling became more intense in the early elongation zone.

Rhizodermis or root slime did not possess the epitopes for the LM19 antibody. Elongation and late elongation zones were characterized by stronger labelling with the LM19 antibody in the root stele. The root cap cells and root slime were labelled by this antibody. Cell walls in cell corners of middle cortex and pith were strongly labelled throughout root development Fig. Three antibodies specific for RG-I side-chains were also used. The early elongation zone possessed lower levels of epitopes for the LM5 antibody, while a gradual increase in the labelling intensity was observed in stele tissues in the elongation and late elongation zones.

Phloem cells were enriched in LM5 epitopes Fig. LM26 labelling was much stronger than LM5 labelling Fig. All tissues analyzed at all of the developmental stages possessed epitopes for the LM26 antibody. The antibody was distributed evenly in all tissues and in all zones of maize root Fig.

Transcriptomic data for all GTs recognized in the maize genome were subjected to a cluster analysis. Six clusters were revealed, four of which were the most populated Fig. Cluster 1 included genes expressed at high levels in the root cap and meristem zone and at lower levels in subsequent zones of maize root.

It was enriched with numerous GTs potentially involved in XyG biosynthesis. According to the data reported by Marcon et al. EMBO J. Differential growth regulation in plants—the acid growth balloon theory. Plant Biol. Evans, M. Responses of Arabidopsis roots to auxin studied with high temporal resolution: comparison of wild type and auxin-response mutants. Planta , — Fendrych, M.

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Cell Mol. Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis. Rayle, D. Enhancement of wall loosening and elongation by Acid solutions. Richardson, S. Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton.

Cell 15, — Scheuring, D. Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression. Vacuolar staining methods in plant cells. Methods Mol. Cell cycle-dependent changes in Golgi stacks, vacuoles, clathrin-coated vesicles and multivesicular bodies in meristematic cells of Arabidopsis thaliana: a quantitative and spatial analysis.

Shih, H. CB 24, — Silady, R. Takemoto, K. Distinct sets of tethering complexes. Veytsman, B. A model of cell wall expansion based on thermodynamics of polymer networks. Viotti, C. The endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuole in Arabidopsis. According to "Biology," plant cell elongation is believed to take place in a manner described by the acid growth hypothesis.

Plant cells are surrounded by a tough but flexible layer of cellulose and other molecules called the cell wall. The cell wall helps to counteract osmotic pressure created by diffusion of water across the plant's cell membrane; it also, however, can restrict cell growth. To elongate, plant cells must temporarily loosen their cell wall.

The acid growth hypothesis posits that plant cells elongate by pumping hydrogen ions across their membrane. Auxin is the plant hormone responsible for cell elongation in shoots.