Does biopolymers composition in seeds contribute to the flax resistance against the Fusarium infection?

Over the last decades, the cultivation of fibrous flax declined heavily. There are number of reasons for that fact; one of them is flax susceptibility to the pathogen infection. Damages caused mainly by fungi from genus Fusarium lead to the significant losses when cultivating flax, which in turn discourage farmers to grow flax. Therefore, to launch the new products from flax with attractive properties there is a need to obtain new flax varieties with increased resistance to pathogens. In order to obtain the better quality of flax fiber, we previously generated flax with reduced pectin or lignin level (cell wall polymers). The modifications altered also plants' resistance to the Fusarium infection. Undoubtedly, the plant defense system is complex, however, in this article we aimed to investigate the composition of modified flax seeds and to correlate it with the observed changes in the flax resistance to the pathogen attack. In particular, we evaluated the content and composition of carbohydrates (cell wall polymers: pectin, cellulose, hemicelluloses and mucilage), and phenylpropanoid compounds (lignin, lignans, phenolics). From the obtained results we concluded that the observed changes in the vulnerability to pathogens putatively correlate with the antioxidant potential of phenylpropanoids accumulated in seeds, seco‐isolariciresinol and coumaric acid diglycosides in particular, and with pectin level as a carbon source for pathogens. Surprisingly, relatively less important for the resistance was the physical barrier, including lignin and cellulose amount and cellulose structure. Certainly, the hypothesis should be verified on a larger number of genotypes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:992–1004, 2014     

Biochemical,mechanical, and spectroscopic analyses of genetically engineered flax fibers producing bioplastic (poly‐β‐hydroxybutyrate)

The interest in biofibers has grown in recent years due to their expanding range of applications in fields as diverse as biomedical science and the automotive industry. Their low production costs, biodegradability, physical properties, and perceived eco‐friendliness allow for their extensive use as composite components, a role in which they could replace petroleum‐based synthetic polymers. We performed biochemical, mechanical, and structural analyses of flax stems and fibers derived from field‐grown transgenic flax enriched with PHB (poly‐β‐hydroxybutyrate). The analyses of the plant stems revealed an increase in the cellulose content and a decrease in the lignin and pectin contents relative to the control plants. However, the contents of the fibers' major components (cellulose, lignin, pectin) remain unchanged. An FT‐IR study confirmed the results of the biochemical analyses of the flax fibers. However, the arrangement of the cellulose polymer in the transgenic fibers differed from that in the control, and a significant increase in the number of hydrogen bonds was detected. The mechanical properties of the transgenic flax stems were significantly improved, reflecting the cellulose content increase. However, the mechanical properties of the fibers did not change in comparison with the control, with the exception of the fibers from transgenic line M13. The generated transgenic flax plants, which produce both components of the flax/PHB composites (i.e., fibers and thermoplastic matrix in the same plant organ) are a source of an attractive and ecologically safe material for industry and medicine. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The changes in pectin metabolism in flax infected with Fusarium

Fusarium culmorum and Fusarium oxysporum are the most common fungal pathogens of flax (Linum usitatissimum L.), thus leading to the greatest losses in crop yield. A subtractive cDNA library was constructed from flax seedlings exposed for two days to F. oxysporum. This revealed a set of genes that are potentially involved in the flax defense responses. Two of those genes directly participate in cell wall sugar polymer metabolism: UDP-d-glucuronate 4-epimerase (GAE; EC 5.1.3.6) and formate dehydrogenase (FDH; EC 1.2.1.2). GAE delivers the main substrate for pectin biosynthesis, and decreases were detected in its mRNA level after Fusarium infection. FDH participates in the metabolism of formic acid, and the expression level of its gene increased after Fusarium infection. However, metabolite profiling analysis disclosed that the pectin content in the infected plants remained unchanged, but that there were reductions in both the levels of the soluble sugars that serve as pectin precursors, and in the level of formic acid. Since formic acid is the product of pectin demethylesterification, the level of mRNAs coding for pectin methylesterase (EC 3.1.1.11) in the infected flax was measured, revealing a decrease in its expression upon plant infection. Transgenic flax plants overexpressing fungal polygalacturonase (EC 3.2.1.15) and rhamnogalacturonase (EC 3.2.1.-) showed a decrease in the pectin content and an elevated level of formic acid, but the level of expression of the FDH gene remained unchanged. It is suspected that the expression of the formate dehydrogenase gene is directly controlled by the pathogen in the early stage of infection, and additionally by pectin degradation in the later stages.     

Lignin deficiency in transgenic flax resulted in plants with improved mechanical properties

Flax (Linum usitatissimum L.) is a very important source of natural fibres used by the textile industry. Flax fibres are called lignocellulosic, because they contain mainly cellulose (about 70%), with hemicellulose, pectin and lignin. Lignin is a three-dimensional polymer with a high molecular weight, and it gives rigidity and mechanical resistance to the fibre and plant. Its presence means the fibres have worse elastic properties than non-lignocellulosic fibres, e.g. cotton fibres, which contain no lignin. The main aim of this study was to produce low-lignin flax plants with fibres with modified elastic properties. An improvement in the mechanical properties was expected. The used strategy for CAD down-regulation was based on gene silencing RNAi technology. Manipulation of the CAD gene caused changes in enzyme activity, lignin content and in the composition of the cell wall in the transgenic plants. The detected reduction in the lignin level in the CAD-deficient plants resulted in improved mechanical properties. Young's modulus was up to 75% higher in the generated transgenic plants (CAD33) relative to the control plants. A significant increase in the lignin precursor contents and a reduction in the pectin and hemicellulose constituents was also detected. A decrease in pectin and hemicellulose, as well as a lower lignin content, might lead to improved extractability of the fibres. However, the resistance of the transgenic lines to Fusarium oxysporum was over two-fold lower than for the non-transformed plants. Since Fusarium species are used as retting organisms and had been isolated from retted flax, the increased sensitivity of the CAD-deficient plant to F. oxysporum infection might lead to improved flax retting.     

间歇低温胁迫对桃果实细胞壁代谢的影响

桃果实在成熟过程中细胞壁干物质不断减少,随着共价结合果胶质和离子结合果胶质减少,水溶性果胶质明显增加,纤维素也逐渐减少,但半纤维素含量变化较小.低温胁迫造成果胶质和纤维素的降解过程受阻,从而造成较高分子量果胶质的积累,果汁粘度升高.中途加温则能促进果胶质和纤维素的增溶和解聚,引导细胞进行与果实成熟有关的细胞壁代谢.14C-蔗糖标记试验表明,在细胞壁不断降解的同时,也进行着合成.在果实成熟的启动阶段,细胞壁的合成能力加强.果实衰老过程与细胞壁合成减少有着直接的联系.受到低温伤害的果实细胞壁物质含量高于正常果实的原因,并不是其合成水平的升高,而是其降解的减慢.     

Arabidopsis fragile fiber8, which encodes a putative glucuronyltransferase, is essential for normal secondary wall synthesis

Secondary walls in vessels and fibers of dicotyledonous plants are mainly composed of cellulose, xylan, and lignin. Although genes involved in biosynthesis of cellulose and lignin have been intensively studied, little is known about genes participating in xylan synthesis. We found that Arabidopsis thaliana fragile fiber8 (fra8) is defective in xylan synthesis. The fra8 mutation caused a dramatic reduction in fiber wall thickness and a decrease in stem strength. FRA8 was found to encode a member of glycosyltransferase family 47 and exhibits high sequence similarity to tobacco (Nicotiana plumbaginifolia) pectin glucuronyltransferase. FRA8 is expressed specifically in developing vessels and fiber cells, and FRA8 is targeted to Golgi. Comparative analyses of cell wall polysaccharide fractions from fra8 and wild-type stems showed that the xylan and cellulose contents are drastically reduced in fra8, whereas xyloglucan and pectin are elevated. Further structural analysis of cell walls revealed that although wild-type xylans contain both glucuronic acid and 4-O-methylglucuronic acid residues, xylans from fra8 retain only 4-O-methylglucuronic acid, indicating that the fra8 mutation results in a specific defect in the addition of glucuronic acid residues onto xylans. These findings suggest that FRA8 is a glucuronyltransferase involved in the biosynthesis of glucuronoxylan during secondary wall formation.     

Effect of pectin methylesterase gene expression on pea root development.

Expression of an inducible gene with sequences common to genes encoding pectin methylesterase (PME) was found to be tightly correlated, both spatially and temporally, with border cell separation in pea root caps. Partial inhibition of the gene's expression by antisense mRNA in transgenic pea hairy roots prevented the normal separation of root border cells from the root tip into the external environment. This phenotype was correlated with an increase in extracellular pH, reduced root elongation, and altered cellular morphology. The translation product of the gene exhibited PME activity in vitro. These results are consistent with the long-standing hypothesis that the demethylation of pectin by PME plays a key role in cell wall metabolism.     

Improving retting of fibre through genetic modification of flax to express pectinases

Flax (Linum usitatissimum L.) is a raw material used for important industrial products. Linen has very high quality textile properties, such as its strength, water absorption, comfort and feel. However, it occupies less than 1% of the total textile market. The major reason for this is the long and difficult retting process by which linen fibres are obtained. In retting, bast fibre bundles are separated from the core, the epidermis and the cuticle. This is accomplished by the cleavage of pectins and hemicellulose in the flax cell wall, a process mainly carried out by plant pathogens like filamentous fungi. The remaining bast fibres are mainly composed of cellulose and lignin. The aim of this study was to generate plants that could be retted more efficiently. To accomplish this, we employed the novel approach of transgenic flax plant generation with increased polygalacturonase (PGI ) and rhamnogalacturonase (RHA) activities. The constitutive expression of Aspergillus aculeatus genes resulted in a significant reduction in the pectin content in tissue-cultured and field-grown plants. This pectin content reduction was accompanied by a significantly higher (more than 2-fold) retting efficiency of the transgenic plant fibres as measured by a modified Fried’s test. No alteration in the lignin or cellulose content was observed in the transgenic plants relative to the control. This indicates that the over-expression of the two enzymes does not affect flax fibre composition. The growth rate and soluble sugar and starch contents were in the range of the control levels. It is interesting to note that the RHA and PGI plants showed higher resistance to Fusarium culmorum and F. oxysporum attack, which correlates with the increased phenolic acid level. In this report, we demonstrate for the first time that over-expression of the A. aculeatus genes results in flax plants more readily usable for fibre production. The biochemical parameters of the cell wall components indicated that the fibre quality remains similar to that of wild-type plants, which is an important pre-requisite for industrial applications. Magdalena Musialak and Magdalena Wróbel-Kwiatkowska participated equally in the preparation of this paper     

Effects of hypergravity on growth and cell wall properties of cress hypocotyls

Elongation growth of etiolated hypocotyls of cress (Lepidium sativum L.) was suppressed when they were exposed to basipetal hypergravity at 35 x g and above. Acceleration at 135 x g caused a decrease in the mechanical extensibility and an increase in the minimum stress-relaxation time of the cell wall. Such changes in the mechanical properties of the cell wall were prominent in the lower regions of hypocotyls. The amounts of cell wall polysaccharides per unit length of hypocotyls increased under the hypergravity condition and, in particular, the increase in the amount of cellulose in the lower regions was conspicuous. Hypergravity did not influence the neutral sugar composition of either the pectin or the hemicellulose fraction. The amount of lignin was also increased by hypergravity treatment, although the level was low. The data suggest that hypergravity modifies the metabolism of cell wall components and thus makes the cell wall thick and rigid, thereby inhibiting elongation growth of cress hypocotyls. These changes may contribute to the plants' ability to sustain their structures against hypergravity.     

Differential expression and activity of matrix metalloproteinase-2 and -9 in the calreticulin deficient cells.

Calreticulin is an endoplasmic reticulum protein important in cardiovascular development. Deletion of the calreticulin gene leads to defects in the heart and the formation of omphaloceal. These defects could both be due to changes in the extracellular matrix composition. Matrix metalloproteinases (MMP)-2 and MMP-9 are two of the MMPs which are essential for cardiovascular remodelling and development. Here, we tested the hypothesis that the defects observed in the heart and body wall of the calreticulin null embryos are due to alterations in MMP-2 and MMP-9 activity. Our results demonstrate that there is a significant decrease in the MMP-9 and increase in the MMP-2 activity and expression in the calreticulin deficient cells. We also showed that there is a significant increase in the expression level of membrane type-1 matrix metalloproteinase (MT1-MMP). In contrast, there was no change in the tissue inhibitor of matrix metalloproteinase (TIMP)-1 or -2 in the calreticulin deficient cells as compared to the wild type cells. Interestingly, the inhibition of the MEK kinase pathway using PD98059 attenuated the decrease in the MMP-9 mRNA with no effect on the MMP-2 mRNA level in the calreticulin deficient cells. Furthermore, PI3 kinase inhibitor decreased the expression of both the MMP-2 and MMP-9. This study is the first report on the role of calreticulin in regulating MMP activity.     

Increased Expression of Retinal TIMP3 mRNA in Simplex Retinitis Pigmentosa Is Localized to Photoreceptor-Retaining Regions

Abstract: The human tissue inhibitor of metalloproteinases-3 (TIMP3) gene is the most recently characterized member of a family of genes whose products are implicated in extracellular matrix (ECM) remodelling. We previously described an increase in expression of TIMP3 mRNA in retinas affected by the progressive photoreceptor degenerative disease, simplex retinitis pigmentosa (RP). To gain further insight into the association between TIMP3 overexpression and retinal degeneration, we have analyzed the cellular localization of TIMP3 mRNA in control and simplex RP retinas using in situ hybridization. No TIMP3 mRNA expression was detectable in control neural retina. In RP-affected retinas, overexpression of TIMP3 mRNA was observed in photoreceptor inner segments and in the ganglion cell layer only in those regions retaining relatively nondystrophic retinal architecture. Modulation of TIMP3 expression in these regions, possibly in association with matrix metalloproteinases, may reflect remodelling of the retinal ECM and concomitant reorganization of neuronal connectivity.     

Effect of water deficit on the cell wall of the date palm (Phoenix dactylifera ‘Deglet nour’, Arecales) fruit during development

Date palm (Phoenix dactylifera) is an important crop providing a valuable nutrition source for people in many countries including the Middle East and North Africa. In recent years, the amount of rain in North Africa and especially in the Tunisian palm grove areas has dropped significantly. We investigated the growth and cell wall remodelling of fruits harvested at three key development stages from trees grown with or without water supply. During development, cell wall solubilization and remodelling was characterized by a decrease of the degree of methylesterification of pectin, an important loss of galactose content and a reduction of the branching of xylan by arabinose in irrigated condition. Water deficit had a profound effect on fruit size, pulp content, cell wall composition and remodelling. Loss of galactose content was not as important, arabinose content was significantly higher in the pectin‐enriched extracts from non‐irrigated condition, and the levels of methylesterification of pectin and O‐acetylation of xyloglucan were lower than in irrigated condition. The lower levels of hydrophobic groups (methylester and O‐acetyl) and the less intensive degradation of the hydrophilic galactan, arabinan and arabinogalactan in the cell wall may be implicated in maintaining the hydration status of the cells under water deficit.     

Mechanical properties of primary plant cell wall analogues

Mechanical effects of turgor pressure on cell walls were simulated by deforming cell wall analogues based on Acetobacter xylinus cellulose under equi-biaxial tension. This experimental set-up, with associated modelling, allowed quantitative information to be obtained on cellulose alone and in composites with pectin and/or xyloglucan. Cellulose was the main load-bearing component, pectin and xyloglucan leading to a decrease in modulus when incorporated. The cellulose-only system could be regarded as an essentially linear elastic material with a modulus ranging from 200 to 500 MPa. Pectin incorporation modified extensibility properties of the system by topology/architecture changes of cellulose fibril assemblies, but the cellulose/pectin composites could still be described as a linear elastic material with a modulus ranging from 120 to 250 MPa. The xyloglucan/cellulose composite could not be modelled as a linear elastic material. Introducing xyloglucan into a cellulose network or a cellulose/pectin composite led to very compliant materials characterised by time-dependent creep behaviour. Modulus values obtained for the composite materials were compared with mechanical data found for plant-derived systems. After comparing bi-axial and uni-axial behaviour of the different composites, structural models were proposed to explain the role of each polysaccharide in determining the mechanical properties of these plant primary cell wall analogues.     

Cadmium-induced alterations of the structural features of pectins in flax hypocotyl

In the course of our studies on the putative role of pectins in the control of cell growth, we have investigated the effect of cadmium on their composition, remodelling and distribution within the epidermis and fibre tissues of flax hypocotyl (Linum usitatissimum L.). Cadmium-stressed seedlings showed a significant inhibition of growth whereas the hypocotyl volume did not significantly change, due to the swelling of most tissues. The structural alterations consisted of significant increase of the thickness of all cell walls and the marked collapse of the sub-epidermal layer. The pectic epitopes recognized by the anti-PGA/RGI and JIM5 antibodies increased in the outer parts of the epidermis (external tangential wall and junctions) and fibres (primary wall and junctions). Concomitantly, there was a remarkable decrease of JIM7 antibody labelling and consequently an increase of the ratio JIM5/JIM7. Conversely, the ratio JIM7/JIM5 increased in the wall domains closest to the plasmalemma, which would expel the cadmium ions from the cytoplasm. The hydrolysis of cell walls revealed a cadmium-induced increase of uronic acid in the pectic matrix. Sequential extractions showed a remodelling of both homogalacturonan and rhamnogalacturonan I. In fractions enriched in primary walls, the main part of the pectins became cross-linked and could be extracted only with alkali. In fractions enriched in secondary walls, the homogalacturonan moieties were found more abundantly in the calcium-chelator extract while the rhamnogacturonan level increased in the boiling water extract.