Transcriptome profiling of celery petiole tissues reveals peculiarities of the collenchyma cell wall formation.

MAIN CONCLUSION: Transcriptome and biochemical analyses are applied to individual plant cell types to reveal potential players involved in the molecular machinery of cell wall formation in specialized cells such as collenchyma. Plant collenchyma is a mechanical tissue characterized by an irregular, thickened cell wall and the ability to support cell elongation. The composition of the collenchyma cell wall resembles that of the primary cell wall and includes cellulose, xyloglucan, and pectin; lignin is absent. Thus, the processes associated with the formation of the primary cell wall in the collenchyma can be more pronounced compared to other tissues due to its thickening. Primary cell walls intrinsic to different tissues may differ in structure and composition, which should be reflected at the transcriptomic level. For the first time, we conducted transcriptome profiling of collenchyma strands isolated from young celery petioles and compared them with other tissues, such as parenchyma and vascular bundles. Genes encoding proteins involved in the primary cell wall formation during cell elongation, such as xyloglucan endotransglucosylase/hydrolases, expansins, and leucine-rich repeat proteins, were significantly activated in the collenchyma. As the key players in the transcriptome orchestra of collenchyma, xyloglucan endotransglucosylase/hydrolase transcripts were characterized in more detail, including phylogeny and expression patterns. The comprehensive approach that included transcriptome and biochemical analyses allowed us to reveal peculiarities of collenchyma cell wall formation and modification, matching the abundance of upregulated transcripts and their potential substrates for revealed gene products. As a result, specific isoforms of multigene families were determined for further functional investigation.

Using FIBexDB for In-Depth Analysis of Flax Lectin Gene Expression in Response to Fusarium oxysporum Infection.

Plant proteins with lectin domains play an essential role in plant immunity modulation, but among a plurality of lectins recruited by plants, only a few members have been functionally characterized. For the analysis of flax lectin gene expression, we used FIBexDB, which includes an efficient algorithm for flax gene expression analysis combining gene clustering and coexpression network analysis. We analyzed the lectin gene expression in various flax tissues, including root tips infected with Fusarium oxysporum. Two pools of lectin genes were revealed: downregulated and upregulated during the infection. Lectins with suppressed gene expression are associated with protein biosynthesis (Calreticulin family), cell wall biosynthesis (galactose-binding lectin family) and cytoskeleton functioning (Malectin family). Among the upregulated lectin genes were those encoding lectins from the Hevein, Nictaba, and GNA families. The main participants from each group are discussed. A list of lectin genes, the expression of which can determine the resistance of flax, is proposed, for example, the genes encoding amaranthins. We demonstrate that FIBexDB is an efficient tool both for the visualization of data, and for searching for the general patterns of lectin genes that may play an essential role in normal plant development and defense.

Gene Expression Patterns for Proteins With Lectin Domains in Flax Stem Tissues Are Related to Deposition of Distinct Cell Wall Types.

The genomes of higher plants encode a variety of proteins with lectin domains that are able to specifically recognize certain carbohydrates. Plants are enriched in a variety of potentially complementary glycans, many of which are located in the cell wall. We performed a genome-wide search for flax proteins with lectin domains and compared the expression of the encoding genes in different stem tissues that have distinct cell wall types with different sets of major polysaccharides. Over 400 genes encoding proteins with lectin domains that belong to different families were revealed in the flax genome; three quarters of these genes were expressed in stem tissues. Hierarchical clustering of the data for all expressed lectins grouped the analyzed samples according to their characteristic cell wall type. Most lectins differentially expressed in tissues with primary, secondary, and tertiary cell walls were predicted to localize at the plasma membrane or cell wall. These lectins were from different families and had various architectural types. Three out of four flax genes for proteins with jacalin-like domains were highly upregulated in bast fibers at the stage of tertiary cell wall deposition. The dynamic changes in transcript level of many genes for lectins from various families were detected in stem tissue over the course of gravitropic response induced by plant gravistimulation. The data obtained in this study indicate a large number of lectin-mediated events in plants and provide insight into the proteins that take part in tissue specialization and reaction to abiotic stress.

The Toolbox for Fiber Flax Breeding: A Pipeline From Gene Expression to Fiber Quality.

The goal of any plant breeding program is to improve quality of a target crop. Crop quality is a comprehensive feature largely determined by biological background. To improve the quality parameters of crops grown for the production of fiber, a functional approach was used to search for genes suitable for the effective manipulation of technical fiber quality. A key step was to identify genes with tissue and stage-specific pattern of expression in the developing fibers. In the current study, we investigated the relationship between gene expression evaluated in bast fibers of developing flax plants and the quality parameters of technical fibers measured after plant harvesting. Based on previously published transcriptomic data, two sets of genes that are upregulated in fibers during intrusive growth and tertiary cell wall deposition were selected. The expression level of the selected genes and fiber quality parameters were measured in fiber flax, linseed (oil flax) cultivars, and wild species that differ in type of yield and fiber quality parameters. Based on gene expression data, linear regression models for technical stem length, fiber tensile strength, and fiber flexibility were constructed, resulting in the identification of genes that have high potential for manipulating fiber quality. Chromosomal localization and single nucleotide polymorphism distribution in the selected genes were characterized for the efficacy of their use in conventional breeding and genome editing programs. Transcriptome-based selection is a highly targeted functional approach that could be used during the development of new cultivars of various crops.

Rearrangement of the Cellulose-Enriched Cell Wall in Flax Phloem Fibers over the Course of the Gravitropic Reaction.

The plant cell wall is a complex structure consisting of a polysaccharide network. The rearrangements of the cell wall during the various physiological reactions of plants, however, are still not fully characterized. Profound changes in cell wall organization are detected by microscopy in the phloem fibers of flax (Linum usitatissimum) during the restoration of the vertical position of the inclined stems. To characterize the underlying biochemical and structural changes in the major cell wall polysaccharides, we compared the fiber cell walls of non-inclined and gravistimulated plants by focusing mainly on differences in non-cellulosic polysaccharides and the fine cellulose structure. Biochemical analysis revealed a slight increase in the content of pectins in the fiber cell walls of gravistimulated plants as well as an increase in accessibility for labeling non-cellulosic polysaccharides. The presence of galactosylated xyloglucan in the gelatinous cell wall layer of flax fibers was demonstrated, and its labeling was more pronounced in the gravistimulated plants. Using solid state NMR, an increase in the crystallinity of the cellulose in gravistimulated plants, along with a decrease in cellulose mobility, was demonstrated. Thus, gravistimulation may affect the rearrangement of the cell wall, which can enable restoration in a vertical position of the plant stem.

Stimulation of adventitious root formation by the oligosaccharin OSRG at the transcriptome level.

Oligosaccharins, which are biologically active oligosaccharide fragments of cell wall polysaccharides, may regulate the processes of growth and development as well as the response to stress factors. We characterized the effect of the oligosaccharin that stimulates rhizogenesis (OSRG) on the gene expression profile in the course of IAA-induced formation of adventitious roots in hypocotyl explants of buckwheat (Fagopyrum esculentum Moench.). The transcriptomes at two stages of IAA-induced root primordium formation (6 h and 24 h after induction) were compared after either treatment with auxin alone or joint treatment with auxin and OSRG. The set of differentially expressed genes indicated the special importance of oligosaccharin at the early stage of auxin-induced adventitious root formation. The list of genes with altered mRNA abundance in the presence of oligosaccharin included those, which Arabidopsis homologs encode proteins directly involved in the response to auxin as well as proteins that contribute to redox regulation, detoxification of various compounds, vesicle trafficking, and cell wall modification. The obtained results contribute to understanding the mechanism of adventitious root formation and demonstrate that OSRG is involved in fine-tuning of ROS and auxin regulatory modes involved in root development.

Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles.

Phloem fibers are important elements of plant architecture and the target product of many fiber crops. A key stage in fiber development is intrusive elongation, the mechanisms of which are largely unknown. Integrated analysis of miRNA and mRNA expression profiles in intrusivelygrowing fibers obtained by laser microdissection from flax (Linum usitatissimum L.) stem revealed all 124 known flax miRNA from 23 gene families and the potential targets of differentially expressed miRNAs. A comparison of the expression between phloem fibers at different developmental stages, and parenchyma and xylem tissues demonstrated that members of miR159, miR166, miR167, miR319, miR396 families were down-regulated in intrusively growing fibers. Some putative target genes of these miRNA families, such as those putatively encoding growth-regulating factors, an argonaute family protein, and a homeobox-leucine zipper family protein were up-regulated in elongating fibers. miR160, miR169, miR390, and miR394 showed increased expression. Changes in the expression levels of miRNAs and their target genes did not match expectations for the majority of predicted target genes. Taken together, poorly understood intrusive fiber elongation, the key process of phloem fiber development, was characterized from a miRNA-target point of view, giving new insights into its regulation.

Flax rhamnogalacturonan lyases: phylogeny, differential expression and modeling of protein structure.

Rhamnogalacturonan lyases (RGLs; EC 4.2.2.23) degrade the rhamnogalacturonan I (RG-I) backbone of pectins present in the plant cell wall. These enzymes belong to polysaccharide lyase family 4, members of which are mainly from plants and plant pathogens. RGLs are investigated, as a rule, as pathogen 'weapons' for plant cell wall degradation and subsequent infection. Despite the presence of genes annotated as RGLs in plant genomes and the presence of substrates for enzyme activity in plant cells, evidence supporting the involvement of this enzyme in certain processes is limited. The differential expression of some RGL genes in flax (Linum usitatissimum L.) tissues, revealed in our previous work, prompted us to carry out a total revision (phylogenetic analysis, analysis of expression and protein structure modeling) of all the sequences of flax predicted as coding for RGLs. Comparison of the expressions of LusRGL in various tissues of flax stem revealed that LusRGLs belong to distinct phylogenetic clades, which correspond to two co-expression groups. One of these groups comprised LusRGL6-A and LusRGL6-B genes and was specifically upregulated in flax fibers during deposition of the tertiary cell wall, which has complex RG-I as a key noncellulosic component. The results of homology modeling and docking demonstrated that the topology of the LusRGL6-A catalytic site allowed binding to the RG-I ligand. These findings lead us to suggest the presence of RGL activity in planta and the involvement of special isoforms of RGLs in the modification of RG-I of the tertiary cell wall in plant fibers.

Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection.

The intrusive growth, a type of plant cell elongation occurring in the depths of plant tissues, is characterized by the invasion of a growing cell between its neighbours due to a higher rate of elongation. In order to reveal the largely unknown molecular mechanisms of intrusive growth, we isolated primary flax phloem fibers specifically at the stage of intrusive growth by laser microdissection. The comparison of the RNA-Seq data from several flax stem parts enabled the characterization of those processes occurring specifically during the fiber intrusive elongation. The revealed molecular players are summarized as those involved in the supply of assimilates and support of turgor pressure, cell wall enlargement and modification, regulation by transcription factors and hormones, and responses to abiotic stress factors. The data obtained in this study provide a solid basis for developing approaches to manipulate fiber intrusive elongation, which is of importance both for plant biology and the yield of fiber crops.

Plant 'muscles': fibers with a tertiary cell wall.

Plants, although sessile organisms, are nonetheless able to move their body parts; for example, during root contraction of geophytes or in the gravitropic reaction by woody stems. One of the major mechanisms enabling these movements is the development of specialized structures that possess contractile properties. Quite unlike animal muscles, for which the action is driven by protein-protein interactions in the protoplasma, the action of plant 'muscles' is polysaccharide-based and located in the uniquely designed, highly cellulosic cell wall that is deposited specifically in fibers. This review describes the development of such cell walls as a widespread phenomenon in the plant kingdom, gives reasons why it should be considered as a tertiary cell wall, and discusses the mechanism of action of the 'muscles'. The origin of the contractile properties lies in the tension of the axially oriented cellulose microfibrils due to entrapment of rhamnogalacturonan-I aggregates that limits the lateral interaction of microfibrils. Long side chains of the nascent rhamnogalacturonan-I are trimmed off during cell wall maturation leading to tension development. Similarities in the tertiary cell wall design in fibers of different plant origin indicate that the basic principles of tension creation may be universal in various ecophysiological situations.

Phloem fibres as motors of gravitropic behaviour of flax plants: level of transcriptome.

Restoration of stem vertical position after plant inclination is a widely spread version of plant orientation in accordance with gravity vector direction. Gravitropic behaviour of flax plants involves the formation of curvature in stem region that has ceased elongation long in advance of stem inclination. The important participants of such behaviour are phloem fibres with constitutively formed tertiary cell wall (G-layer). We performed the large-scale transcriptome profiling of phloem fibres isolated from pulling and opposite sides of gravitropic curvature and compared with control plant fibres. Significant changes in transcript abundance take place for genes encoding proteins of several ion channels, transcription factors and other regulating elements. The largest number of upregulated genes belonged to the cell wall category; many of those were specifically upregulated in fibres of pulling stem side. The obtained data permit to suggest the mechanism of fibre participation in gravitropic reaction that involves the increase of turgor pressure and the rearrangements of cell wall structure in order to improve contractile properties, and to identify the regulatory elements that operate specifically in the fibres of the pulling stem side making gelatinous phloem fibres an important element of gravitropic response in herbaceous plants.

Cellulosic fibres of flax recruit both primary and secondary cell wall cellulose synthases during deposition of thick tertiary cell walls and in the course of graviresponse.

Cellulose synthesising complex consists of cellulose synthase (CESA) subunits encoded by a multigene family; different sets of CESA genes are known to be expressed during primary and secondary cell wall formation. We examined the expression of LusCESAs in flax (Linum usitatissimum L.) cellulosic fibres at various stages of development and in the course of graviresponse by means of RNA-Seq and quantitative PCR. Transcripts for both primary and secondary cell wall-related CESAs were abundant in fibres depositing highly cellulosic tertiary cell walls. Gravistimulation of flax plants temporally increased the abundance of CESA transcripts, specifically in phloem fibres located at the pulling stem side. Construction of coexpression networks for LusCESAs revealed that both primary and secondary cell wall-related CESAs were involved in the joint coexpression group in fibres depositing tertiary cell walls, as distinct from other tissues, where these genes were within separate groups. The obtained data suggest that fibres depositing tertiary cell walls have a specific mechanism of cellulose biosynthesis and a specific way of its regulation.

Transcriptome portrait of cellulose-enriched flax fibres at advanced stage of specialization.

Functional specialization of cells is among the most fundamental processes of higher organism ontogenesis. The major obstacle to studying this phenomenon in plants is the difficulty of isolating certain types of cells at defined stages of in planta development for in-depth analysis. A rare opportunity is given by the developed model system of flax (Linum usitatissimum L.) phloem fibres that can be purified from the surrounding tissues at the stage of the tertiary cell wall deposition. The performed comparison of the whole transcriptome profile in isolated fibres and other portions of the flax stem, together with fibre metabolism characterization, helped to elucidate the general picture of the advanced stage of plant cell specialization and to reveal novel participants potentially involved in fibre metabolism regulation and cell wall formation. Down-regulation of all genes encoding proteins involved in xylan and lignin synthesis and up-regulation of genes for the specific set of transcription factors transcribed during tertiary cell wall formation were revealed. The increased abundance of transcripts for several glycosyltransferases indicated the enzymes that may be involved in synthesis of fibre-specific version of rhamnogalacturonan I.

Aspen Tension Wood Fibers Contain ß-(1---> 4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls.

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). ß-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. ß-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, ß-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high ß-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.

Differential expression of α-L-arabinofuranosidases during maize (Zea mays L.) root elongation.

MAIN CONCLUSION: Specific α- l -arabinofuranosidases are involved in the realisation of elongation growth process in cells with type II cell walls. Elongation growth in a plant cell is largely based on modification of the cell wall. In type II cell walls, the Ara/Xyl ratio is known to decrease during elongation due to the partial removal of Ara residues from glucuronoarabinoxylan. We searched within the maize genome for the genes of all predicted α-L-arabinofuranosidases that may be responsible for such a process and related their expression to the activity of the enzyme and the amount of free arabinose measured in six zones of a growing maize root. Eight genes of the GH51 family (ZmaABFs) and one gene of the GH3 family (ZmaARA-I) were identified. The abundance of ZmaABF1 and 3-6 transcripts was highly correlated with the measured enzymatic activity and free arabinose content that significantly increased during elongation. The transcript abundances also coincided with the pattern of changes in the Ara/Xyl ratio of the xylanase-extractable glucuronoarabinoxylan described in previous studies. The expression of ZmaABF3, 5 and 6 was especially up-regulated during elongation although corresponding proteins are devoid of the catalytic glutamate at the proper position. ZmaABF2 transcripts were specifically enriched in the root cap and meristem. A single ZmaARA-I gene was not expressed as a whole gene but instead as splice variants that encode the C-terminal end of the protein. Changes in the ZmaARA-I transcript level were rather moderate and had no significant correlation with free arabinose content. Thus, elongation growth of cells with type II cell walls is accompanied by the up-regulation of specific and predicted α-L-arabinofuranosidase genes, and the corresponding activity is indeed pronounced and is important for the modification of glucuronoarabinoxylan, which plays a key role in the modification of the cell wall supramolecular organisation.

Chitinase-like (CTL) and cellulose synthase (CESA) gene expression in gelatinous-type cellulosic walls of flax (Linum usitatissimum L.) bast fibers.

Plant chitinases (EC 3.2.1.14) and chitinase-like (CTL) proteins have diverse functions including cell wall biosynthesis and disease resistance. We analyzed the expression of 34 chitinase and chitinase-like genes of flax (collectively referred to as LusCTLs), belonging to glycoside hydrolase family 19 (GH19). Analysis of the transcript expression patterns of LusCTLs in the stem and other tissues identified three transcripts (LusCTL19, LusCTL20, LusCTL21) that were highly enriched in developing bast fibers, which form cellulose-rich gelatinous-type cell walls. The same three genes had low relative expression in tissues with primary cell walls and in xylem, which forms a xylan type of secondary cell wall. Phylogenetic analysis of the LusCTLs identified a flax-specific sub-group that was not represented in any of other genomes queried. To provide further context for the gene expression analysis, we also conducted phylogenetic and expression analysis of the cellulose synthase (CESA) family genes of flax, and found that expression of secondary wall-type LusCESAs (LusCESA4, LusCESA7 and LusCESA8) was correlated with the expression of two LusCTLs (LusCTL1, LusCTL2) that were the most highly enriched in xylem. The expression of LusCTL19, LusCTL20, and LusCTL21 was not correlated with that of any CESA subgroup. These results defined a distinct type of CTLs that may have novel functions specific to the development of the gelatinous (G-type) cellulosic walls.

Development of cellulosic secondary walls in flax fibers requires beta-galactosidase.

Bast (phloem) fibers, tension wood fibers, and other cells with gelatinous-type secondary walls are rich in crystalline cellulose. In developing bast fibers of flax (Linum usitatissimum), a galactan-enriched matrix (Gn-layer) is gradually modified into a mature cellulosic gelatinous-layer (G-layer), which ultimately comprises most of the secondary cell wall. Previous studies have correlated this maturation process with expression of a putative ß-galactosidase. Here, we demonstrate that ß-galactosidase activity is in fact necessary for the dynamic remodeling of polysaccharides that occurs during normal secondary wall development in flax fibers. We found that developing stems of transgenic (LuBGAL-RNAi) flax with reduced ß-galactosidase activity had lower concentrations of free Gal and had significant reductions in the thickness of mature cellulosic G-layers compared with controls. Conversely, Gn-layers, labeled intensively by the galactan-specific LM5 antibody, were greatly expanded in LuBGAL-RNAi transgenic plants. Gross morphology and stem anatomy, including the thickness of bast fiber walls, were otherwise unaffected by silencing of ß-galactosidase transcripts. These results demonstrate a specific requirement for ß-galactosidase in hydrolysis of galactans during formation of cellulosic G-layers. Transgenic lines with reduced ß-galactosidase activity also had biochemical and spectroscopic properties consistent with a reduction in cellulose crystallinity. We further demonstrated that the tensile strength of normal flax stems is dependent on ß-galactosidase-mediated development of the phloem fiber G-layer. Thus, the mechanical strength that typifies flax stems is dependent on a thick, cellulosic G-layer, which itself depends on ß-galactosidase activity within the precursor Gn-layer. These observations demonstrate a novel role for matrix polysaccharides in cellulose deposition; the relevance of these observations to the development of cell walls in other species is also discussed.