Enhancing high-fibre cereal food quality through microbial-enzymatic processing: Mechanisms, applications, and prospects.

Miscanthus is a perennial energy grass characterized by a high productivity and resource-use efficiency, making it an ideal biomass feedstock for the production of cellulosic biofuels and a wide range of other biobased value-chains. However, the large-scale commercialization of converting biomass into cellulosic biofuel is hindered by our inability to efficiently deconstruct the plant cell wall. The plant cell wall is a complex and dynamic structure and its components are extensively cross-linked into an unyielding matrix. The production of biofuel depends on the extraction, hydrolysis and fermentation of cell wall polysaccharides, which currently requires energetically and chemically intensive processing operations that negatively affect the economic viability and sustainability of the industry. To address this challenge it is envisioned that the bioenergy feedstocks can be compositionally tailored to increase the accessibility and extractability of cell wall polysaccharides, which would allow a more efficient conversion of biomass into biofuel under milder processing conditions. Extensive phenotypic and genetic diversity in cell wall composition and conversion efficiency was observed in different miscanthus species, including M. sinensis, M. sacchariflorus and interspecific hybrids between these two species. In multiple experiments a twofold increase in the release of fermentable sugars was observed in ‘high quality’ accessions compared to ‘low quality’ accessions. The exhaustive characterization of eight highly diverse M. sinensis genotypes revealed novel and distinct breeding targets for different bioenergy conversion routes. The key traits that contributed favourably to the conversion efficiency of biomass into biofuel were a high content of hemicellulosic polysaccharides, extensive cross-linking of hemicellulosic polysaccharides (revealed by a high content of trans-ferulic acids and a high ratio of arabinose-to-xylose), a low lignin content and extensive incorporation of para-coumaric acid into the lignin polymer. Lignin is widely recognized as one of the key factors conveying recalcitrance against enzymatic deconstruction of the cell wall. The incorporation of para-coumaric acid into the lignin polymer is hypothesized to make lignin more easily degradable during alkaline pretreatment, one of the most widely applied processing methods that is used to pretreat biomass prior to enzymatic hydrolysis. Previous studies have shown that reducing lignin content is often implicated in reduced resistance of plants to lodging. We hypothesize that extensively cross-linked hemicellulosic polysaccharides may fulfil a similar function in supporting cell wall structural rigidity and increasing the content of hemicellulosic polysaccharides may be a way to reduce lignin content without adversely affecting cell wall rigidity. This strategy can be used to improve biomass quality for biobased applications, as hemicellulosic polysaccharides are more easily degradable during industrial processing than lignin. Furthermore, hemicellulosic polysaccharides adhere to cellulose, which negatively affects the level of cellulose crystallinity. Crystalline cellulose is harder to degrade than its more amorphous form. Therefore the reduction of cellulose crystallinity is another mechanism through which increasing the content of hemicellulosic polysaccharides positively contributes to cell wall degradability. These results provided new insights into the traits that may be targeted to improve the quality of lignocellulose feedstocks. However, evaluation of complex biochemical traits for selection purposes is hindered by the fact that their accurate quantification is a costly, lengthy and laborious procedure. To overcome these limitations an accurate and high-throughput method was developed based on near-infrared spectroscopy. Through extensive calibration we developed accurate prediction models for a wide range of biomass quality characteristics, which may be readily implemented as a phenotyping tool for selection purposes. Additionally, progress through breeding may substantially be improved by marker-assisted selection, which will reduce the need for the evaluation of genotype performance in multi-year field trials. To this end, a biparental M. sinensis mapping population of 186 individuals was developed and genotyped using a genotyping-by-sequencing approach. A total of 564 short-sequence markers were used to construct a new M. sinensis genetic map. Cell wall composition and conversion efficiency were observed to be highly heritable and quantitatively inherited properties. This is the first genetic study in miscanthus to map quantitative trait loci (QTLs) for biomass quality properties and is a first step towards the application of marker-assisted selection for biomass quality properties. Through the evaluation of a diverse set of miscanthus genotypes in multiple locations we demonstrated that in addition to genotypic variation, growing conditions may have a substantial influence on cell wall composition and conversion efficiency. While further research is needed to identify which specific environmental parameters are responsible for the observed effects, these results clearly indicate that the environmental influence on biomass quality needs to be taken into account in order to match genotype, location and end-use of miscanthus as a lignocellulose feedstock. Moreover, significant genotype-by-environment interaction effects were observed for cell wall composition and conversion efficiency, indicating variation in environmental sensitivity across genotypes. Although the magnitude of the genotypic differences was small in comparison to genotype and environmental main effects, this affected the ranking of accession across environments. Stability analysis indicated some stable accessions performed relatively across diverse locations. In addition to trialing miscanthus in diverse locations, we also evaluated miscanthus biomass quality under drought conditions for a number of reasons: 1) drought stress is linked to a differential expression of cell wall biosynthesis genes, 2) incidence of drought events is increasing due to climate change, 3) irrigation is likely to be uneconomical during the cultivation of miscanthus and 4) miscanthus has many characteristics that make it a crop with a good potential for cultivation on marginal soils, where abiotic stresses such as drought may prevail. Drought stress was shown to result in a large reduction in cell wall and cellulose content and a substantial increase in hemicellulosic polysaccharides and cellulose conversion rates. We hypothesized that the reduction in cellulose content was due to an increase in the production of osmolytes, which are well-known for their role in plant protection against drought. The results indicated that drought stress had a positive effect on the cell wall degradability of miscanthus biomass. Overall the compendium of knowledge generated within the framework of this thesis provided insights into the variation in biomass quality properties in miscanthus, increased our understanding of the molecular, genetic and environmental factors influencing its conversion efficiency into biofuel and provided tools to exploit these factors to expand the use of miscanthus as a lignocellulose feedstock.

Two transgenic potato lines, csr2-1 and csr4-8, containing two different antisense genes, csr2 and csr4, respectively, were crossed to investigate the possibility of achieving reduction in cellulose content in the tuber cell walls of the progeny. The progeny containing both transgenes (double csr2/csr4 transformant) exhibited reduction of 63% in cell wall cellulose content, while the single transformants csr2 and csr4 had a 44% and 22% reduction in cellulose content, respectively. Keywords : Cellulose content, cellulose synthase, double transformant, genetic crossing, Solanum tuberosum Cameroon Journal of Experimental Microbiology Vol. 4 (2) 2008: pp. 80-83

Ancymidol inhibits the incorporation of cellulose into cell walls of maize cell cultures in a gibberellin-independent manner, impairing cell growth; the reduction in the cellulose content is compensated with xylans. Ancymidol is a plant growth retardant which impairs gibberellin biosynthesis. It has been reported to inhibit cellulose synthesis by tobacco cells, based on its cell-malforming effects. To ascertain the putative role of ancymidol as a cellulose biosynthesis inhibitor, we conducted a biochemical study of its effect on cell growth and cell wall metabolism in maize cultured cells. Ancymidol concentrations ≤500µM progressively reduced cell growth and induced globular cell shape without affecting cell viability. However, cell growth and viability were strongly reduced by ancymidol concentrations≥1.5mM. The I50 value for the effect of ancymidol on FW gain was 658µM. A reversal of the inhibitory effects on cell growth was observed when 500µM ancymidol-treated cultures were supplemented with 100µM GA3. Ancymidol impaired the accumulation of cellulose in cell walls, as monitored by FTIR spectroscopy. Cells treated with 500µM ancymidol showed a ~60% reduction in cellulose content, with no further change as the ancymidol concentration increased. Cellulose content was partially restored by 100µM GA3. Radiolabeling experiments confirmed that ancymidol reduced the incorporation of [14C]glucose into α-cellulose and this reduction was not reverted by the simultaneous application of GA3. RT-PCR analysis indicated that the cellulose biosynthesis inhibition caused by ancymidol is not related to a downregulation of ZmCesA gene expression. Additionally, ancymidol treatment increased the incorporation of [3H]arabinose into a hemicellulose-enriched fraction, and up-regulated ZmIRX9 and ZmIRX10L gene expression, indicating an enhancement in the biosynthesis of arabinoxylans as a compensatory response to cellulose reduction.

Two transgenic potato lines, csr2-1 and csr4-8, containing two different antisense genes: csr2 and csr4, respectively, were crossed to investigate the possibility of achieving reduction in cellulose content in the tuber cell walls of the progeny. The progeny containing both transgenes (double csr2/csr4 transformant) exhibited reduction of 63% in cell wall cellulose content, while the single transformants csr2 and csr4 had a 44 and 22% reduction in cellulose content, respectively. Key words: Cellulose content, cellulose synthase, double transformant, genetic crossing, Solanum tuberosum.

In higher plants, cellulose is synthesized by membrane-spanning large protein complexes named cellulose synthase complexes (CSCs). In this study, the Arabidopsis PASTICCINO2 (PAS2) was identified as an interacting partner of cellulose synthases. PAS2 was previously characterized as the plant 3-hydroxy-acyl-CoA dehydratase, an ER membrane-localized dehydratase that is essential for very-long-chain-fatty acid (VLCFA) elongation. The pas2-1 mutants show defective cell elongation and reduction in cellulose content in both etiolated hypocotyls and light-grown roots. Although disruption of VLCFA synthesis by a genetic alteration had a reduction in VLCFA in both etiolated hypocotyls and light-grown roots, it had a differential effect on cellulose content in the two systems, suggesting the threshold level of VLCFA for efficient cellulose synthesis may be different in the two biological systems. pas2-1 had a reduction in both CSC delivery rate and CSC velocity at the PM in etiolated hypocotyls. Interestingly, Golgi but not post-Golgi endomembrane structures exhibited a severe defect in motility. Experiments using pharmacological perturbation of VLCFA content in etiolated hypocotyls strongly indicate a novel function of PAS2 in the regulation of CSC and Golgi motility. Through a combination of genetic, biochemical and cell biology studies, our study demonstrated that PAS2 as a multifunction protein has an important role in the regulation of cellulose biosynthesis in Arabidopsis hypocotyl.

Early cell-wall modifications of maize cell cultures during habituation to dichlobenil

Leaves play a crucial role as ornamental organs in Spathiphyllum, exhibiting distinct differences across various Spathiphyllum varieties. Leaf development is intricately linked to processes of cell proliferation and expansion, with cell morphology often regulated by plant cell walls, primarily composed of cellulose. Alterations in cellulose content can impact cell morphology, subsequently influencing the overall shape of plant organs. Although cellulases have been shown to affect cellulose levels in plant cells, genetic evidence linking them to the regulation of leaf shape remains limited. This study took the leaves of Spathiphyllum 'Mojo' and its somatic variants as the research objects. We screened four cellulase gene family members from the transcriptome and then measured the leaf cellulose content, cellulase activity, and expression levels of cellulase-related genes. Correlation analysis pinpointed the gene SpGH9A3 as closely associated with leaf shape variations in the mutant. Green fluorescent fusion protein assays revealed that the SpGH9A3 protein was localized to the cell membrane. Notably, the expression of the SpGH9A3 gene in mutant leaves peaked during the early spread stage, resulting in smaller overall leaf size and reduced cellulose content upon overexpression in Arabidopsis.

Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection

Increasing evidence has revealed that membrane trafficking is highly associated with cell wall metabolism. Factors involved in vesicle delivery, e.g. cytoskeleton and motor proteins, have showed regulatory effects on cell wall structure and components. However, little is known about the involvement of other trafficking components in distribution of cell wall-related compartments. Dynamins are important proteins functioning in membrane tubulation and vesiculation. Recently, we have reported characterization of the rice dynamin-related protein 2B (OsDRP2B). Mutation in OsDRP2B causes a significant reduction in cellulose content. Its association with the trans-Golgi network (TGN) and clathrin-coated vesicles and the reduced CESA4 abundance at the bc3 plasma membrane suggest that BC3/OsDRP2B is involved in the transport of essential elements for cellulose synthesis. Here, we provide additional evidence for BC3 subcellular localization via observing OsDRP2B-GFP in living root hairs of transgenic plants. Uronic acid and fractional composition analyses further confirm that the amount of arabinoxylan and other noncellulosic polysaccharides is increased in bc3. However, three putative xylan synthesis genes are down-regulated in mutant plant revealed by real-time PCR analysis. These results imply that compartments delivered by OsDRP2B are specifically responsible for cellulose biosynthesis.

The uppermost internode is one of the fastest elongating organs in rice, and is expected to require an adequate supply of cell-wall materials and enzymes to the cell surface to enhance mechanical strength. Although it has been reported that the phenotype of shortened uppermost internode 1 (sui1) is caused by mutations in PHOSPHATIDYLSERINE SYNTHASE (OsPSS), the underlying mechanism remains unclear. Here we show that the OsPSS-1, as a gene expressed predominantly in elongating cells, regulates post-Golgi vesicle secretion to intercellular spaces. Mutation of OsPSS-1 leads to compromised delivery of CESA4 and secGFP towards the cell surface, resulting in weakened intercellular adhesion and disorganized cell arrangement in parenchyma. The phenotype of sui1-4 is caused largely by the reduction in cellulose contents in the whole plant and detrimental delivery of pectins in the uppermost internode. We found that OsPSS-1 and its potential product PS (phosphatidylserine) localized to organelles associated with exocytosis. These results together suggest that OsPSS-1 plays a potential role in mediating cell expansion by regulating secretion of cell wall components.

Sum frequency generation (SFG) vibration spectroscopy can selectively detect crystalline cellulose without spectral interference from cell wall matrix components. Here, we show that the cellulose SFG spectrum is sensitive to cellulose microfibril alignment and packing within the cell wall. SFG intensity at 2,944 cm(-1) correlated well with crystalline cellulose contents of various regions of the Arabidopsis (Arabidopsis thaliana) inflorescence, while changes in the 3,320/2,944 cm(-1) intensity ratio suggest subtle changes in cellulose ordering as tissues mature. SFG analysis of two cellulose synthase mutants (irx1/cesa8 and irx3/cesa7) indicates a reduction in cellulose content without evidence of altered cellulose structure. In primary cell walls of Arabidopsis, cellulose exhibited a characteristic SFG peak at 2,920 and 3,320 cm(-1), whereas in secondary cell walls, it had peaks at 2,944 and 3,320 cm(-1). Starch (amylose) gave an SFG peak at 2,904 cm(-1) (CH methine) whose intensity increased with light exposure prior to harvest. Selective removal of matrix polysaccharides from primary cell walls by acid hydrolysis resulted in an SFG spectrum resembling that of secondary wall cellulose. Our results show that SFG spectroscopy is sensitive to the ordering of cellulose microfibrils in plant cell walls at the meso scale (nm to μm) that is important for cell wall architecture but cannot be probed by other spectroscopic or diffraction techniques.

Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ∼25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged.

Nanocrystalline cellulose has emerged as a substantial nanomaterial in recent years due to its peculiar characteristics such as bio renewability, sustainability, and low toxicity while having high mechanical strengths, optical transparency, and much more. Meanwhile, pineapple leaves (PL) as by-products after fruit harvest exhibit a huge potential in cellulose and nanocellulose extraction due to their high cellulose content (approx. more than 36%). The latest studies have successfully recovered cellulose from pineapple leaves, in which the bleaching stage greatly affects the properties of the produced cellulose. In some cases, this stage can result in a reduction in cellulose content because of the excessive use of chemicals. However, the effects of influential factors such as solid-liquid ratio, reaction time, and reagent concentration in the bleaching stage have not been widely investigated although they are necessary to scale up the cellulose recovery process. In this study, cellulose was extracted from PL using alkali treatment with sodium hydroxide and bleaching with hydrogen peroxide before synthesizing nanocellulose. The characterization of PL, cellulose, and nanocellulose was performed by Thermogravimetry Analysis (TGA), Fourier Transform spectroscopy (FTIR), X-ray diffraction (XRD), Transmission Electron Microscope (TEM) and Dynamic Light Scattering (DLS). The results indicated that bleaching with H2O2 at 6% after 60 min at a solid-liquid ratio of 1:20 yielded an impressively high cellulose content of 94.25%. Obtained nanocellulose possessed high crystallinity index of approx. 80% with a diameter in the range of 15-30 nm. Along with further research related to the application of organic nanoparticles, this study has a great impact on the proposing processes with better stability, which is meaningful in terms of green chemistry towards sustainable development by satisfying most principles of this theory.

The aim of this study was to investigate changes in cell wall chemical composition and polymer size in the root tip of intact cotton seedlings (Gossypium hirsutum L. cv. Acala SJ-2) grown in saline environments, in order to relate the interaction between high salinity and root growth to possible changes in cell wall metabolism. Cotton seedlings were grown in modified Hoagland nutrient solution with various combinations of NaCl and CaCl 2 . Cell walls were fractionated into four fractions (pectin, hemicellulose 1 and 2, cellulose), and analysed for their total sugar content, neutral sugar composition and size of polysaccharides. At 1 mol m −3 Ca, 150 mol m −3 NaCl resulted in a significant increase in the cell wall uronic acid content, but a reduction in cellulose content on a per unit dry weight basis

Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.