Natalia Mokshina

Development of cellulosic secondary walls in flax fibers requires β-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 β-galactosidase. Here, we demonstrate that β-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 β-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 β-galactosidase transcripts. These results demonstrate a specific requirement for β-galactosidase in hydrolysis of galactans during formation of cellulosic G-layers. Transgenic lines with reduced β-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 β-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 β-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.

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.

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

Entrapment of pectic galactan and acidic arabinogalactan II within cellulose fibrils is a distinctive feature of aspen tension wood gelatinous fibers with contractile properties. 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). β-(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. β-(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, β-(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 β-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.

Cellulosic Fibers: Role of Matrix Polysaccharides in Structure and Function

Cellulose, being the major cell wall component and the most abundant organic matter, produced by living organisms, is not uniformly distributed within plant tissues. There are numerous cells, like the parenchyma ones, which even at maturity have thin cell wall. Thick cell walls are characteristic for the tissues with mechanical function. Among those, there are cell walls, which contain several major components, and those, the predominant component of which is cellulose. The most pure natural cellulose is considered to be present in cotton seed hairs (sometimes erroneously called “cotton fibers”) – over 90% of cell wall [1]. Very close to this value is a special group of plant fibers – cellulosic or gelatinous fibers, the proportion of cellulose in which amounts for 85-90% [2,3]. The cell wall thickness in such fibers may reach 15 uf06dm, as compared to 0.2 uf06dm in cells with thin cell wall. So, the very significant portion of total plant cellulose may be concentrated within the gelatinous fibers, making them the important source for production of biofuels and bio-based products. An additional attractiveness of cellulosic fibers for such applications comes from the fact that gelatinous cell wall layers are devoid of lignin – the major hurdle in using plant biomass [1].

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.

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.

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.

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.

Pretreatment of Sugar Beet Pulp with Dilute Sulfurous Acid is Effective for Multipurpose Usage of Carbohydrates

Sulfurous acid was used for pretreatment of sugar beet pulp (SBP) in order to achieve high efficiency of both extraction of carbohydrates and subsequent enzymatic hydrolysis of the remaining solids. The main advantage of sulfurous acid usage as pretreatment agent is the possibility of its regeneration. Application of sulfurous acid as hydrolyzing agent in relatively low concentrations (0.6–1.0xa0%) during a short period of time (10–20xa0min) and low solid to liquid ratio (1:3, 1:6) allowed effective extraction of carbohydrates from SBP and provided positive effect on subsequent enzymatic hydrolysis. The highest obtained concentration of reducing substances (RS) in hydrolysates was 8.5xa0%; up to 33.6xa0% of all carbohydrates present in SBP could be extracted. The major obtained monosaccharides were arabinose and glucose (9.4 and 7.3xa0g/l, respectively). Pretreatment of SBP with sulfurous acid increased 4.6 times the yield of glucose during subsequent enzymatic hydrolysis of remaining solids with cellulase cocktail, as compared to the untreated SBP. Total yield of glucose during SBP pretreatment and subsequent enzymatic hydrolysis amounted to 89.4xa0% of the theoretical yield. The approach can be applied directly to the wet SBP. Hydrolysis of sugar beet pulp with sulfurous acid is recommended for obtaining of individual monosaccharides, as well as nutritional media.

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.