Carbon allocation of Spirodela polyrhiza under boron toxicity.

Pectic polysaccharides containing apiose, xylose, and uronic acids are excellent candidates for boron fixation. Duckweeds are the fastest-growing angiosperms that can absorb diverse metals and contaminants from water and have high pectin content in their cell walls. Therefore, these plants can be considered excellent boron (B) accumulators. This work aimed to investigate the relationship between B assimilation capacity with apiose content in the cell wall of Spirodela polyrhiza subjected to different boric acid concentrations. Plants were grown for 7 and 10 days in ½ Schenck-Hildebrandt media supplemented with 0 to 56 mg B.L-1, the non-structural and structural carbohydrates, and related genes were evaluated. The results showed that B altered the morphology and carbohydrate composition of this species during plant development. The optimum B concentration (1.8 mg B.L-1) led to the highest relative growth and biomass accumulation, reduced starch, and high pectin and apiose contents, together with increased expression of UDP-apiose/UDP-xylose synthase (AXS) and 1,4-α-galacturonosyltransferase (GAUT). The toxic state (28 and 56 mg B.L-1) increased the hexose contents in the cell wall with a concomitant reduction of pectins, apiose, and growth. The pectin content of S. polyrhiza was strongly associated with its growth capacity and regulation of B content within the cells, which have AXS as an important regulator. These findings suggest that duckweeds are suitable for B remediation, and their biomass can be used for bioenergy production.

Immobilization and Application of the Recombinant Xylanase GH10 of Malbranchea pulchella in the Production of Xylooligosaccharides from Hydrothermal Liquor of the Eucalyptus (Eucalyptus grandis) Wood Chips.

Xylooligosaccharides (XOS) are widely used in the food industry as prebiotic components. XOS with high purity are required for practical prebiotic function and other biological benefits, such as antioxidant and inflammatory properties. In this work, we immobilized the recombinant endo-1,4-ß-xylanase of Malbranchea pulchella (MpXyn10) in various chemical supports and evaluated its potential to produce xylooligosaccharides (XOS) from hydrothermal liquor of eucalyptus wood chips. Values >90% of immobilization yields were achieved from amino-activated supports for 120 min. The highest recovery values were found on Purolite (142%) and MANAE-MpXyn10 (137%) derivatives, which maintained more than 90% residual activity for 24 h at 70 °C, while the free-MpXyn10 maintained only 11%. In addition, active MpXyn10 derivatives were stable in the range of pH 4.0−6.0 and the presence of the furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan of various sources. Maximum values were observed for birchwood xylan at 8.6 mg mL−1 and wheat arabinoxylan at 8.9 mg mL−1, using Purolite-MpXyn10. Its derivative was also successfully applied in the hydrolysis of soluble xylan present in hydrothermal liquor, with 0.9 mg mL−1 of XOS after 3 h at 50 °C. This derivative maintained more than 80% XOS yield after six cycles of the assay. The results obtained provide a basis for the application of immobilized MpXyn10 to produce XOS with high purity and other high-value-added products in the lignocellulosic biorefinery field.

Biochemical composition of the pericarp cell wall of popcorn inbred lines with different popping expansion.

The popping expansion is a characteristic that is positively related with the quality of popcorn. A positive correlation between the volume of expansion and the thickness of the pericarp, and between the proportion of the opaque/shiny endosperm and the grain weight and volume, were postulated. However, there are no reports in the literature that address the importance of cell wall components in the popping expansion. Here, we investigate the biochemical composition of the pericarp cell walls of three inbred lines of popcorn with different popping expansion. Inbred lines GP12 (expansion volume >40 mL g-1), P11 (expansion volume 30 mL g-1) and P16 (expansion volume 14 mL g-1) were used for the analysis and quantification of monosaccharides by HPAEC-PAD, and ferulic and p-coumaric acids and lignin by HPLC. Our hypothesis is that the biochemical composition of the pericarp cell walls may be related to greater or lesser popping expansion. Our data suggest that the lignin content and composition contribute to popping expansion. The highest concentration of lignin (129.74 µg mg-1; 12.97%) was detected in the pericarp cell wall of the GP12 inbred line with extremely high popping expansion, and the lowest concentration (113.52 µg mg-1; 11.35%) was observed in the P16 inbred line with low popping expansion. These findings may contribute to indicating the quantitative trait locus for breeding programs and to developing other methods to improve the popping expansion of popcorn.

The Effect of Sugarcane Straw Aging in the Field on Cell Wall Composition.

Cellulosic ethanol is an alternative for increasing the amount of bioethanol production in the world. In Brazil, sugarcane leads the bioethanol production, and to improve its yield, besides bagasse, sugarcane straw is a possible feedstock. However, the process that leads to cell wall disassembly under field conditions is unknown, and understanding how this happens can improve sugarcane biorefinery and soil quality. In the present work, we aimed at studying how sugarcane straw is degraded in the field after 3, 6, 9, and 12 months. Non-structural and structural carbohydrates, lignin content, ash, and cellulose crystallinity were analyzed. The cell wall composition was determined by cell wall fractionation and determination of monosaccharide composition. Non-structural carbohydrates degraded quickly during the first 3 months in the field. Pectins and lignin remained in the plant waste for up to 12 months, while the hemicelluloses and cellulose decreased 7.4 and 12.4%, respectively. Changes in monosaccharide compositions indicated solubilization of arabinoxylan (xylose and arabinose) and ß-glucans (ß-1,3 1,4 glucan; after 3 months) followed by degradation of cellulose (after 6 months). Despite cellulose reduction, the xylose:glucose ratio increased, suggesting that glucose is consumed faster than xylose. The degradation and solubilization of the cell wall polysaccharides concomitantly increased the level of compounds related to recalcitrance, which led to a reduction in saccharification and an increase in minerals and ash contents. Cellulose crystallinity changed little, with evidence of silica at the latter stages, indicating mineralization of the material. Our data suggest that for better soil mineralization, sugarcane straw must stay in the field for over 1 year. Alternatively, for bioenergy purposes, straw should be used in less than 3 months.

Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors.

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

Increased Malbranchea pulchella ß-glucosidase production and its application in agroindustrial residue hydrolysis: A research based on experimental designs.

ß-Glucosidases are a limiting factor in the conversion of cellulose to glucose for the subsequent ethanol production. Here, ß-glucosidase production by Malbranchea pulchella was optimized using Composite Central Designs and Response Surface Methodologies from a medium designed. The coefficient of determination (R2 ) was 0.9960, F-value was very high, and the lack of fit was found to be non-significant. This indicates a statistic valid and predictive result. M. pulchella enzymatic extract was successfully tested as an enzymatic cocktail in a mixture design using sugarcane bagasse, soybean hull and barley bagasse. We proved that the optimization of the ß-glucosidase production and the application in hydrolysis using unexpansive biomass and agricultural wastes can be accomplished by means of statistical methodologies. The strategy presented here can be useful for the improvement of enzyme production and the hydrolysis process, arising as an alternative for bioeconomy.

Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

A Highly Glucose Tolerant ß-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification.

ß-glucosidases catalyze the hydrolysis ß-1,4, ß-1,3 and ß-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl ß-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) ß-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source. Kinetic characterization revealed that the MpBgl3 was highly tolerant to glucose, which is in contrast to many Bgls that are completely inhibited by glucose. A 3D model of MpBgl3 was generated by molecular modeling and used for the evaluation of structural differences with a Bgl3 that is inhibited by glucose. Taken together, our results provide new clues to understand the glucose tolerance in GH3 ß-glucosidases.

Cell wall hydrolases act in concert during aerenchyma development in sugarcane roots.

BACKGROUND AND AIMS: Cell wall disassembly occurs naturally in plants by the action of several glycosyl-hydrolases during different developmental processes such as lysigenous and constitutive aerenchyma formation in sugarcane roots. Wall degradation has been reported in aerenchyma development in different species, but little is known about the action of glycosyl-hydrolases in this process. METHODS: In this work, gene expression, protein levels and enzymatic activity of cell wall hydrolases were assessed. Since aerenchyma formation is constitutive in sugarcane roots, they were assessed in segments corresponding to the first 5 cm from the root tip where aerenchyma develops. KEY RESULTS: Our results indicate that the wall degradation starts with a partial attack on pectins (by acetyl esterases, endopolygalacturonases, ß-galactosidases and α-arabinofuranosidases) followed by the action of ß-glucan-/callose-hydrolysing enzymes. At the same time, there are modifications in arabinoxylan (by α-arabinofuranosidases), xyloglucan (by XTH), xyloglucan-cellulose interactions (by expansins) and partial hydrolysis of cellulose. Saccharification revealed that access to the cell wall varies among segments, consistent with an increase in recalcitrance and composite formation during aerenchyma development. CONCLUSION: Our findings corroborate the hypothesis that hydrolases are synchronically synthesized, leading to cell wall modifications that are modulated by the fine structure of cell wall polymers during aerenchyma formation in the cortex of sugarcane roots.

Efficient hydrolysis of wine and grape juice anthocyanins by Malbranchea pulchella ß-glucosidase immobilized on MANAE-agarose and ConA-Sepharose supports.

ß-glucosidases (BGLs) hydrolyze short-chain cellulooligosaccharides. Some BGLs can hydrolyze anthocyanins and be applied in the clarification process of food industries, especially grape juice and wine. Enzyme immobilization is a valuable tool to increase enzyme stabilization. In this work, Malbranchea pulchella BGL was immobilized on Monoaminoethyl-N-ethyl-agarose ionic support, MANAE-agarose, and Concanavalin A-Sepharose affinity support, Con-A-Sepharose. The formed biocatalysts, denominated BLG-MANAE and BLG-ConA, were applied in the grape juice and red wine clarification. BGL-MANAE and BGL-ConA hyperactivated M. pulchella BGL 10- and 3-fold, respectively. Both biocatalysts showed at least 70% activity at pH range 2-11, until 24 h incubation. BGL-MANAE and BGL-ConA showed activity of 60% and 100%, respectively, at 50 °C, up to 24 h. Both biocatalysts were efficiently reused 20-fold. They were stable in the presence of up to 0.1 M glucose for 24 h incubation, and with 5%, 10% and 15% ethanol kept up to 70% activity. BGL-MANAE biocatalyst was 11% and 25% more efficient than BGL-ConA in clarification of concentrate and diluted wines, respectively. Likewise, BGL-MANAE biocatalysts were 14% and 33% more efficient than the BGL-ConA in clarification of diluted and concentrated juices, respectively. Therefore, the BGL-MANAE biocatalyst was especially effective in red wine and grape juice clarification.

The control of endopolygalacturonase expression by the sugarcane RAV transcription factor during aerenchyma formation.

The development of lysigenous aerenchyma starts with cell expansion and degradation of pectin from the middle lamella, leading to cell wall modification, and culminating with cell separation. Here we report that nutritional starvation of sugarcane induced gene expression along sections of the first 5 cm of the root and between treatments. We selected two candidate genes: a RAV transcription factor, from the ethylene response factors superfamily, and an endopolygalacturonase (EPG), a glycosyl hydrolase related to homogalacturonan hydrolysis from the middle lamella. epg1 and rav1 transcriptional patterns suggest they are essential genes at the initial steps of pectin degradation during aerenchyma development in sugarcane. Due to the high complexity of the sugarcane genome, rav1 and epg1 were sequenced from 17 bacterial artificial chromosome clones containing hom(e)ologous genomic regions, and the sequences were compared with those of Sorghum bicolor. We used one hom(e)olog sequence from each gene for transactivation assays in tobacco. rav1 was shown to bind to the epg1 promoter, repressing ß-glucuronidase activity. RAV repression upon epg1 transcription is the first reported link between ethylene regulation and pectin hydrolysis during aerenchyma formation. Our findings may help to elucidate cell wall degradation in sugarcane and therefore contribute to second-generation bioethanol production.

Nutrient and drought stress: implications for phenology and biomass quality in miscanthus.

BACKGROUND AND AIMS: The cultivation of dedicated biomass crops, including miscanthus, on marginal land provides a promising approach to the reduction of dependency on fossil fuels. However, little is known about the impact of environmental stresses often experienced on lower-grade agricultural land on cell-wall quality traits in miscanthus biomass crops. In this study, three different miscanthus genotypes were exposed to drought stress and nutrient stress, both separately and in combination, with the aim of evaluating their impact on plant growth and cell-wall properties. METHODS: Automated imaging facilities at the National Plant Phenomics Centre (NPPC-Aberystwyth) were used for dynamic phenotyping to identify plant responses to separate and combinatorial stresses. Harvested leaf and stem samples of the three miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus and Miscanthus × giganteus) were separately subjected to saccharification assays, to measure sugar release, and cell-wall composition analyses. KEY RESULTS: Phenotyping showed that the M. sacchariflorus genotype Sac-5 and particularly the M. sinensis genotype Sin-11 coped better than the M. × giganteus genotype Gig-311 with drought stress when grown in nutrient-poor compost. Sugar release by enzymatic hydrolysis, used as a biomass quality measure, was significantly affected by the different environmental conditions in a stress-, genotype- and organ-dependent manner. A combination of abundant water and low nutrients resulted in the highest sugar release from leaves, while for stems this was generally associated with the combination of drought and nutrient-rich conditions. Cell-wall composition analyses suggest that changes in fine structure of cell-wall polysaccharides, including heteroxylans and pectins, possibly in association with lignin, contribute to the observed differences in cell-wall biomass sugar release. CONCLUSIONS: The results highlight the importance of the assessment of miscanthus biomass quality measures in addition to biomass yield determinations and the requirement for selecting suitable miscanthus genotypes for different environmental conditions.

Correlation of Apiose Levels and Growth Rates in Duckweeds.

The carbon assimilated by photosynthesis in plants can be partitioned into starch, soluble sugars, and cell wall polymers. Higher levels of starch accumulation in leaves are usually correlated with a lower growth capacity. Duckweeds are fast-growing aquatic monocot plants that can accumulate high levels of starch. They are an unusual group because their cell wall has very low levels of lignin while accumulating apiogalacturonan, a pectic polysaccharide that could be involved with boron assimilation. In this work, five duckweed species from different genera (Spirodela polyrhiza, Landoltia punctata, Lemna gibba, Wolffiella caudata, and Wolffia borealis) were cultivated under two light intensities (20 and 500 µmoles of photons m-2 s-1) to evaluate the effects of growth rate on carbohydrate metabolism. A comparative analysis was performed by measuring their relative growth rates (RGR), and their content for starch, as well as soluble and cell wall carbohydrates. We found that the faster-growing species (the Lemnoideae) accumulate lower starch and higher soluble sugars than the slower-growing species within the Wolffioideae. Interestingly, analysis of the cell wall monosaccharides revealed that the slower-growing species displayed lower content of apiose in their walls. Our results indicate that higher accumulation of apiose observed in cell walls of the Lemnoideae species, which likely correlates with a higher proportion of apiogalacturonan, may lead to higher efficiency in the assimilation of boron. This is consistent with the increased RGR observed under conditions with higher apiose in the cell wall, such as higher light intensity. Consistent with their lower growth capacity, the Wolffioideae species we studied shows higher starch accumulation in comparison with the Lemnoideae species. We suggest that apiose levels could be good biomarkers for growth capacity of duckweeds and suggest that boron uptake could be an important factor for growth control in this aquatic plant family.

Diurnal variation in gas exchange and nonstructural carbohydrates throughout sugarcane development.

Photosynthesis and growth are dependent on environmental conditions and plant developmental stages. However, it is still not clear how the environment and development influence the diurnal dynamics of nonstructural carbohydrates production and how they affect growth. This is particularly the case of C4 plants such as sugarcane (Saccharum spp.). Aiming to understand the dynamics of leaf gas exchange and nonstructural carbohydrates accumulation in different organs during diurnal cycles across the developmental stages, we evaluated these parameters in sugarcane plants in a 12-month field experiment. Our results show that during the first 3 months of development, light and vapour pressure deficit (VPD) were the primary drivers of photosynthesis, stomatal conductance and growth. After 6 months, in addition to light and VPD, drought, carbohydrate accumulation and the mechanisms possibly associated with water status maintenance were also likely to play a role in gas exchange and growth regulation. Carbohydrates vary throughout the day in all organs until Month 9, consistent with their use for growth during the night. At 12 months, sucrose is accumulated in all organs and starch had accumulated in leaves without any diurnal variation. Understanding of how photosynthesis and the dynamics of carbohydrates are controlled might lead to strategies that could increase sugarcane's biomass production.

The evolution of the Glycomic Codes of extracellular matrices.

The extracellular matrices (ECMs) of living organisms are compartments responsible for maintenance of cell shape, cell adhesion, and cell communication. They are also involved in cell signaling and defense against the attack of pathogens. The plant cell walls have been recently defined as encoded structures that combine polysaccharides with other encoded structures (proteins and phenolic compounds). The term Glycomic Code has been used to define the set of mechanisms that generate cell wall architecture (the combination of polymers of different types) and biological function. Here, the composition of the extracellular matrices of archaea, bacteria, animals, fungi, algae, and plants was compared to understand how the Glycomic Code of these different organisms operate to produce polysaccharides and therefore how the Glycomic Code may have evolved in nature. It was found that the heterotrophs display EMC polysaccharides containing aminosugars (nitrogen-based polysaccharides) whereas the photosynthetic organisms have cellulose-based walls, with polymers that hardly present aminosugars in its composition. Another subgroup is of the organisms containing EMCs with sulfated polysaccharides (animals and red algae). The main hemicellulose found in plants (xyloglucan) is used as a case study along with other seed cell wall storage polysaccharides of plants to exemplify the evolution of the Glycomic Code in plants. Overall, the trends observed in this work shows for the first time how the Glycomic Code in ECMs of living organisms may have evolved and diversified in nature.

Corrigendum: Pectins, Endopolygalacturonases, and Bioenergy.

[This corrects the article on p. 1401 in vol. 7, PMID: 27703463.].

Carbohydrate-mediated responses during zygotic and early somatic embryogenesis in the endangered conifer, Araucaria angustifolia.

Three zygotic developmental stages and two somatic Araucaria angustifolia cell lines with contrasting embryogenic potential were analyzed to identify the carbohydrate-mediated responses associated with embryo formation. Using a comparison between zygotic and somatic embryogenesis systems, the non-structural carbohydrate content, cell wall sugar composition and expression of genes involved in sugar sensing were analyzed, and a network analysis was used to identify coordinated features during embryogenesis. We observed that carbohydrate-mediated responses occur mainly during the early stages of zygotic embryo formation, and that during seed development there are coordinated changes that affect the development of the different structures (embryo and megagametophyte). Furthermore, sucrose and starch accumulation were associated with the responsiveness of the cell lines. This study sheds light on how carbohydrate metabolism is influenced during zygotic and somatic embryogenesis in the endangered conifer species, A. angustifolia.

Pectins, Endopolygalacturonases, and Bioenergy.

The precise disassembly of the extracellular matrix of some plant species used as feedstocks for bioenergy production continues to be a major barrier to reach reasonable cost effective bioethanol production. One solution has been the use of pretreatments, which can be effective, but increase even more the cost of processing and also lead to loss of cell wall materials that could otherwise be used in industry. Although pectins are known to account for a relatively low proportion of walls of grasses, their role in recalcitrance to hydrolysis has been shown to be important. In this mini-review, we examine the importance of pectins for cell wall hydrolysis highlighting the work associated with bioenergy. Here we focus on the importance of endopolygalacturonases (EPGs) discovered to date. The EPGs cataloged by CAZy were screened, revealing that most sequences, as well as the scarce structural work performed with EPGs, are from fungi (mostly Aspergillus niger). The comparisons among the EPG from different microorganisms, suggests that EPGs from bacteria and grasses display higher similarity than each of them with fungi. This compilation strongly suggests that structural and functional studies of EPGs, mainly from plants and bacteria, should be a priority of research regarding the use of pectinases for bioenergy production purposes.

Do plant cell walls have a code?

A code is a set of rules that establish correspondence between two worlds, signs (consisting of encrypted information) and meaning (of the decrypted message). A third element, the adaptor, connects both worlds, assigning meaning to a code. We propose that a Glycomic Code exists in plant cell walls where signs are represented by monosaccharides and phenylpropanoids and meaning is cell wall architecture with its highly complex association of polymers. Cell wall biosynthetic mechanisms, structure, architecture and properties are addressed according to Code Biology perspective, focusing on how they oppose to cell wall deconstruction. Cell wall hydrolysis is mainly focused as a mechanism of decryption of the Glycomic Code. Evidence for encoded information in cell wall polymers fine structure is highlighted and the implications of the existence of the Glycomic Code are discussed. Aspects related to fine structure are responsible for polysaccharide packing and polymer-polymer interactions, affecting the final cell wall architecture. The question whether polymers assembly within a wall display similar properties as other biological macromolecules (i.e. proteins, DNA, histones) is addressed, i.e. do they display a code?