Periodic changes in chain lengths distribution parameters of wheat starch during endosperm development.

This study analyzed the molecular structure of developing wheat endosperm starch at different stages after anthesis (DAA) using chain length distribution analysis by size exclusion chromatography (SEC) and fluorophore-assisted carbohydrate electrophoresis. Our results revealed periodic changes in the content of both amylose and amylopectin fractions. Specifically, the content of amylose chains with a degree of polymerization (DP) > 100 significantly decreased from 5 to 10 DAA (28% to 21%) and from 15 to 20 DAA (29% to 26%), but increased between 10 and 15 DAA (21% to 29%) and 20 to 25 DAA (30.0% to 33%). Conversely, the content of short amylopectin chains with DP ≤ 32 showed the opposite trend. Interestingly, mRNA expression levels of key starch biosynthesis genes did not exhibit periodic changes. These findings contribute to our understanding of starch biosynthesis and provide important insights for the development of starch-based products.

Bricks out of the wall: polysaccharide extramural functions.

Plant polysaccharides are components of plant cell walls and/or store energy. However, this oversimplified classification neglects the fact that some cell wall polysaccharides and glycoproteins can localize outside the relatively sharp boundaries of the apoplastic moiety, where they adopt functions not directly related to the cell wall. Such polysaccharide multifunctionality (or 'moonlighting') is overlooked in current research, and in most cases the underlying mechanisms that give rise to unconventional ex muro trafficking, targeting, and functions of polysaccharides and glycoproteins remain elusive. This review highlights major examples of the extramural occurrence of various glycan cell wall components, discusses the possible significance and implications of these phenomena for plant physiology, and lists exciting open questions to be addressed by future research.

An aptamer highly specific to cellulose enables the analysis of the association of cellulose with matrix cell wall polymers in vitro and in muro.

The current toolbox of cell wall-directed molecular probes has been pivotal for advancing basic and application-oriented plant carbohydrate research; however, it still exhibits limitations regarding target diversity and specificity. Scarcity of probes targeting intramolecular associations between cell wall polymers particularly hinders our understanding of the cell wall microstructure and affects the development of effective means for its efficient deconstruction for bioconversion. Here we report a detailed characterization of a cellulose-binding DNA aptamer CELAPT MINI using a combination of various in vitro biochemical, biophysical, and molecular biology techniques. Our results show evidence for its high specificity towards long non-substituted ß-(1-4)-glucan chains in both crystalline and amorphous forms. Fluorescent conjugates of CELAPT MINI are applicable as in situ cellulose probes and are well suited for various microscopy techniques, including super-resolution imaging. Compatibility of fluorescent CELAPT MINI variants with immunodetection of cell wall matrix polymers enabled them simultaneously to resolve the fibrillar organization of complex cellulose-enriched pulp material and to quantify the level of cellulose masking by xyloglucan and xylan. Using enzymatically, chemically, or genetically modulated Brachypodium internode sections we showed the diversity in cell wall packing among various cell types and even cell wall microdomains. We showed that xylan is the most prominent, but not the only, cellulose-masking agent in Brachypodium internode tissues. These results collectively highlight the hitherto unexplored potential to expand the cell wall probing toolbox with highly specific and versatile in vitro generated polynucleotide probes.

Current and future advances in fluorescence-based visualization of plant cell wall components and cell wall biosynthetic machineries.

Plant cell wall-derived biomass serves as a renewable source of energy and materials with increasing importance. The cell walls are biomacromolecular assemblies defined by a fine arrangement of different classes of polysaccharides, proteoglycans, and aromatic polymers and are one of the most complex structures in Nature. One of the most challenging tasks of cell biology and biomass biotechnology research is to image the structure and organization of this complex matrix, as well as to visualize the compartmentalized, multiplayer biosynthetic machineries that build the elaborate cell wall architecture. Better knowledge of the plant cells, cell walls, and whole tissue is essential for bioengineering efforts and for designing efficient strategies of industrial deconstruction of the cell wall-derived biomass and its saccharification. Cell wall-directed molecular probes and analysis by light microscopy, which is capable of imaging with a high level of specificity, little sample processing, and often in real time, are important tools to understand cell wall assemblies. This review provides a comprehensive overview about the possibilities for fluorescence label-based imaging techniques and a variety of probing methods, discussing both well-established and emerging tools. Examples of applications of these tools are provided. We also list and discuss the advantages and limitations of the methods. Specifically, we elaborate on what are the most important considerations when applying a particular technique for plants, the potential for future development, and how the plant cell wall field might be inspired by advances in the biomedical and general cell biology fields.

The source of inorganic nitrogen has distinct effects on cell wall composition in Brachypodium distachyon.

Plants have evolved different strategies to utilize various forms of nitrogen (N) from the environment. While regulation of plant growth and development in response to application of inorganic N forms has been characterized, our knowledge about the effect on cell wall structure and composition is quite limited. In this study, we analysed cell walls of Brachypodium distachyon supplied with three types of inorganic N (NH4NO3, NO3-, or NH4+). Cell wall profiles showed distinct alterations in both the quantity and structures of individual polymers. Nitrate stimulated cellulose, but inhibited lignin deposition at the heading growth stage. On the other hand, ammonium supply resulted in higher concentration of mixed linkage glucans. In addition, the chemical structure of pectins and hemicelluloses was strongly influenced by the form of N. Supply of only NO3- led to alteration in xylan substitution and to lower esterification of homogalacturonan. We conclude that the physiological response to absorption of different inorganic N forms includes pleotropic remodelling of type II cell walls.

The impact of silicon on cell wall composition and enzymatic saccharification of Brachypodium distachyon.

BACKGROUND: Plants and in particular grasses benefit from a high uptake of silicon (Si) which improves their growth and productivity by alleviating adverse effects of biotic and abiotic stress. However, the silicon present in plant tissues may have a negative impact on the processing and degradation of lignocellulosic biomass. Solutions to reduce the silicon content either by biomass engineering or development of downstream separation methods are therefore targeted. Different cell wall components have been proposed to interact with the silica pool in plant shoots, but the understanding of the underlying processes is still limited. RESULTS: In the present study, we have characterized silicon deposition and cell wall composition in Brachypodium distachyon wild-type and low-silicon 1 (Bdlsi1-1) mutant plants. Our analyses included different organs and plant developmental stages. In the mutant defective in silicon uptake, low silicon availability favoured deposition of this element in the amorphous form or bound to cell wall polymers rather than as silicified structures. Several alterations in non-cellulosic polysaccharides and lignin were recorded in the mutant plants, indicating differences in the types of linkages and in the three-dimensional organization of the cell wall network. Enzymatic saccharification assays showed that straw from mutant plants was marginally more degradable following a 190 °C hydrothermal pretreatment, while there were no differences without or after a 120 °C hydrothermal pretreatment. CONCLUSIONS: We conclude that silicon affects the composition of plant cell walls, mostly by altering linkages of non-cellulosic polymers and lignin. The modifications of the cell wall network and the reduced silicon concentration appear to have little or no implications on biomass recalcitrance to enzymatic saccharification.

Silicon affects seed development and leaf macrohair formation in Brachypodium distachyon.

Silicon (Si) has many beneficial effects in plants, especially for the survival from biotic and abiotic stresses. However, Si may negatively affect the quality of lignocellulosic biomass for bioenergy purposes. Despite many studies, the regulation of Si distribution and deposition in plants remains to be fully understood. Here, we have identified the Brachypodium distachyon mutant low-silicon 1 (Bdlsi1-1), with impaired channeling function of the Si influx transporter BdLSI1, resulting in a substantial reduction of Si in shoots. Bioimaging by laser ablation-inductively coupled plasma-mass spectrometry showed that the wild-type plants deposited Si mainly in the bracts, awns and leaf macrohairs. The Bdlsi1-1 mutants showed substantial (>90%) reduction of Si in the mature shoots. The Bdlsi1-1 leaves had fewer, shorter macrohairs, but the overall pattern of Si distribution in bracts and leaf tissues was similar to that in the wild-type. The Bdlsi1-1 plants supplied with Si had significantly lower seed weights, compared to the wild-type. In low-Si media, the seed weight of wild-type plants was similar to that of Bdlsi1-1 mutants supplied with Si, while the Bdlsi1-1 seed weight decreased further. We conclude that Si deficiency results in widespread alterations in leaf surface morphology and seed formation in Brachypodium, showing the importance of Si for successful development in grasses.

DNA barcoding and isolation of vertically transmitted ascomycetes in sorghum from Burkina Faso: Epicoccum sorghinum is dominant in seedlings and appears as a common root pathogen.

Molecular identification of fungal taxa commonly transmitted through seeds of sorghum in Western Africa is lacking. In the present study, farm-saved seeds, collected from four villages in Northern Burkina Faso, were surface sterilized and the distribution of fungal DNA in seeds and seven-day-old seedlings was analyzed by 18S ribosomal DNA (rDNA) amplicon sequencing. More than 99% of the fungal rDNA was found to originate from ascomycetes. The distribution of ascomycetes at species level was subsequently analyzed by barcoding of ITS2 rDNA. Eighteen Operational Taxonomic Units (OTUs) were identified from seedlings, compared to 29 OTUs from seeds. The top-eight most abundant ascomycete OTUs from seedlings were annotated as: Epicoccum sorghinum, Fusarium thapsinum, four different Curvularia spp., Exserohilum rostratum and Alternaria longissima. These OTUs were also present in amplicons from seed samples collected in Central Burkina Faso confirming a common occurrence. E. sorghinum was highly predominant in seedlings both measured by DNA analysis and by isolation. The dominance of E. sorghinum was particularly strong in roots from poorly growing seedlings. Pathogenicity of E. sorghinum isolates was compared to F. thapsinum by inoculation to seeds in vitro. Both fungal species caused significant inhibition of seedling growth (P<0.001) and Koch's postulates were fulfilled. Extensive, dark necrosis in roots was a typical symptom of E. sorghinum, whereas wilting of leaves was caused primarily by F. thapsinum. This study provides the first molecular approach to characterize the seedling mycoflora of sorghum in Western Africa and suggests E. sorghinum as a common root pathogen.