Mixed-Linkage Glucan Is the Main Carbohydrate Source and Starch Is an Alternative Source during Brachypodium Grain Germination.

Seeds of the model grass Brachypodium distachyon are unusual because they contain very little starch and high levels of mixed-linkage glucan (MLG) accumulated in thick cell walls. It was suggested that MLG might supplement starch as a storage carbohydrate and may be mobilised during germination. In this work, we observed massive degradation of MLG during germination in both endosperm and nucellar epidermis. The enzymes responsible for the MLG degradation were identified in germinated grains and characterized using heterologous expression. By using mutants targeting MLG biosynthesis genes, we showed that the expression level of genes coding for MLG and starch-degrading enzymes was modified in the germinated grains of knocked-out cslf6 mutants depleted in MLG but with higher starch content. Our results suggest a substrate-dependent regulation of the storage sugars during germination. These overall results demonstrated the function of MLG as the main carbohydrate source during germination of Brachypodium grain. More astonishingly, cslf6 Brachypodium mutants are able to adapt their metabolism to the lack of MLG by modifying the energy source for germination and the expression of genes dedicated for its use.

Clinical Implications of Emotion Dysregulation in ADHD.

Emotion dysregulation is com- mon in patients with attention-deficit/hyperactive disorder (ADHD) and contributes substantially to ADHD related impairments and comorbidi- ties. The high prevalence of emotional dysregulation (ED) in individuals with ADHD highlights the need to understand the clinical implications of this association, neurobiological correlates, and potential treatment modalities. ED contributes significantly to ADHD adverse outcomes, such as occupational, driving, and legal consequences, and is associated with the development of psychiatric comorbidities. Neurobiological data suggest possible dysfunctions of various subcortical and/or cortical brain regions and an altered connectivity between those regions. Available data suggest that ED is responsive to pharmacological and psychotherapeutic interventions. This article overviews the prevalence of ED in ADHD samples, examines putative neurobiological processes, and discusses pharmacologic and psychotherapeutic strategies for treating ED symptoms of ADHD.

Mutation in Brachypodium caffeic acid O-methyltransferase 6 alters stem and grain lignins and improves straw saccharification without deteriorating grain quality.

Cereal crop by-products are a promising source of renewable raw material for the production of biofuel from lignocellulose. However, their enzymatic conversion to fermentable sugars is detrimentally affected by lignins. Here the characterization of the Brachypodium Bd5139 mutant provided with a single nucleotide mutation in the caffeic acid O-methyltransferase BdCOMT6 gene is reported. This BdCOMT6-deficient mutant displayed a moderately altered lignification in mature stems. The lignin-related BdCOMT6 gene was also found to be expressed in grains, and the alterations of Bd5139 grain lignins were found to mirror nicely those evidenced in stem lignins. The Bd5139 grains displayed similar size and composition to the control. Complementation experiments carried out by introducing the mutated gene into the AtCOMT1-deficient Arabidopsis mutant demonstrated that the mutated BdCOMT6 protein was still functional. Such a moderate down-regulation of lignin-related COMT enzyme reduced the straw recalcitrance to saccharification, without compromising the vegetative or reproductive development of the plant.

New Onset Seizure Disorder Following Electroconvulsive Therapy.

Electroconvulsive therapy (ECT) is an effective treatment for many psychiatric illnesses including refractory depression, catatonia, bipolar disorder, and schizophrenia, along with neuropsychiatric diseases such as dementia and Parkinson's disease. The emergence of a seizure disorder after the initiation of ECT is a rare but severe occurrence. It can become challenging for clinicians to determine whether ECT should be continued. We present a case of a 73-year-old female with schizoaffective disorder who received a total of 173 ECT treatments over the course of five years for breakthrough psychosis due to medication nonadherence. After five years of treatment, she experienced her first episode of tonic-clonic seizure activity. We review the current evidence and discuss factors that should be considered when treating such an individual.

The Deconstruction of Pectic Rhamnogalacturonan I Unmasks the Occurrence of a Novel Arabinogalactan Oligosaccharide Epitope.

Rhamnogalacturonan I (RGI) is a pectic polysaccharide composed of a backbone of alternating rhamnose and galacturonic acid residues with side chains containing galactose and/or arabinose residues. The structure of these side chains and the degree of substitution of rhamnose residues are extremely variable and depend on species, organs, cell types and developmental stages. Deciphering RGI function requires extending the current set of monoclonal antibodies (mAbs) directed to this polymer. Here, we describe the generation of a new mAb that recognizes a heterogeneous subdomain of RGI. The mAb, INRA-AGI-1, was produced by immunization of mice with RGI oligosaccharides isolated from potato tubers. These oligomers consisted of highly branched RGI backbones substituted with short side chains. INRA-AGI-1 bound specifically to RGI isolated from galactan-rich cell walls and displayed no binding to other pectic domains. In order to identify its RGI-related epitope, potato RGI oligosaccharides were fractionated by anion-exchange chromatography. Antibody recognition was assessed for each chromatographic fraction. INRA-AGI-1 recognizes a linear chain of (1→4)-linked galactose and (1→5)-linked arabinose residues. By combining the use of INRA-AGI-1 with LM5, LM6 and INRA-RU1 mAbs and enzymatic pre-treatments, evidence is presented of spatial differences in RGI motif distribution within individual cell walls of potato tubers and carrot roots. These observations raise questions about the biosynthesis and assembly of pectin structural domains and their integration and remodeling in cell walls.

Endomembrane proteomics reveals putative enzymes involved in cell wall metabolism in wheat grain outer layers.

Cereal grain outer layers fulfil essential functions for the developing seed such as supplying energy and providing protection. In the food industry, the grain outer layers called 'the bran' is valuable since it is rich in dietary fibre and other beneficial nutriments. The outer layers comprise several tissues with a high content in cell wall material. The cell wall composition of the grain peripheral tissues was investigated with specific probes at a stage of active cell wall synthesis. Considerable wall diversity between cell types was revealed. To identify the cellular machinery involved in cell wall synthesis, a subcellular proteomic approach was used targeting the Golgi apparatus where most cell wall polysaccharides are synthesized. The tissues were dissected into outer pericarp and intermediate layers where 822 and 1304 proteins were identified respectively. Many carbohydrate-active enzymes were revealed: some in the two peripheral grain fractions, others only in one tissue. Several protein families specific to one fraction and with characterized homologs in other species might be related to the specific detection of a polysaccharide in a particular cell layer. This report provides new information on grain cell walls and its biosynthesis in the valuable outer tissues, which are poorly studied so far. A better understanding of the mechanisms controlling cell wall composition could help to improve several quality traits of cereal products (e.g. dietary fibre content, biomass conversion to biofuel).

Exploring architecture of xyloglucan cellulose nanocrystal complexes through enzyme susceptibility at different adsorption regimes.

Xyloglucan (XG) is believed to act as a cementing material that contributes to the cross-linking and mechanical properties of the cellulose framework in plant cell walls. XG can adsorb to the cellulose nanocrystal (CNC) surface in vitro in order to simulate this in vivo relationship. The target of our work was to investigate the sorption behavior of tamarind seed XG on CNC extracted from cotton linters at different XG/CNC concentration ratios, that is, different adsorption regimes regarding the XG-CNC complex organization and the enzymatic susceptibility of XG. First, we determined the adsorption isotherm. Second, XG-CNC complexes were enzymatically hydrolyzed using a xyloglucan-specific endoglucanase in order to quantify the different XG fractions involved in binding to CNC and to determine adsorption regimes, that is, presence of loops, tails, and trains. Finally, the architecture of the XG-CNC complex was investigated by transmission electron microscopy imaging of negatively stained XG-CNC suspensions and XG immunolabeled suspensions at different XG/CNC concentration ratios, both before and after xyloglucanase hydrolysis process. This study revealed that an increasing XG/CNC concentration ratio led to a change in the XG binding organization to CNC. At low XG/CNC concentration ratios, almost all XG chains were bound as trains to the CNC surface. In contrast, at increasing XG/CNC concentration ratios, the proportion of loops and tails increases. The organization change induces CNC aggregation to form a cellulose/XG network at low XG/CNC regimes, whereas CNC remains in the form of individual particles at higher XG/CNC regimes. Results are discussed both regarding the biological role of XG in plant cell walls and in the perspective of designing new biobased materials.

In situ imaging of LPMO action on plant tissues.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Several studies have reported LPMO action in synergy with other carbohydrate-active enzymes (CAZymes) for the degradation of lignocellulosic biomass but direct LPMO action at the plant tissue level remains challenging to investigate. Here, we have developed a MALDI-MS imaging workflow to detect oxidised oligosaccharides released by a cellulose-active LPMO at cellular level on maize tissues. Using this workflow, we imaged LPMO action and gained insight into the spatial variation and relative abundance of oxidised and non-oxidised oligosaccharides. We reveal a targeted action of the LPMO related to the composition and organisation of plant cell walls.

New Growth-Related Features of Wheat Grain Pericarp Revealed by Synchrotron-Based X-ray Micro-Tomography and 3D Reconstruction.

Wheat (Triticum aestivum L.) is one of the most important crops as it provides 20% of calories and proteins to the human population. To overcome the increasing demand in wheat grain production, there is a need for a higher grain yield, and this can be achieved in particular through an increase in the grain weight. Moreover, grain shape is an important trait regarding the milling performance. Both the final grain weight and shape would benefit from a comprehensive knowledge of the morphological and anatomical determinism of wheat grain growth. Synchrotron-based phase-contrast X-ray microtomography (X-ray µCT) was used to study the 3D anatomy of the growing wheat grain during the first developmental stages. Coupled with 3D reconstruction, this method revealed changes in the grain shape and new cellular features. The study focused on a particular tissue, the pericarp, which has been hypothesized to be involved in the control of grain development. We showed considerable spatio-temporal diversity in cell shape and orientations, and in tissue porosity associated with stomata detection. These results highlight the growth-related features rarely studied in cereal grains, which may contribute significantly to the final grain weight and shape.

Interactions between proteins and cellulose in a liquid crystalline media: Design of a droplet based experimental platform.

Model systems are needed to provide controlled environment for the understanding of complex phenomena. Interaction between polysaccharides and proteins in dense medium are involved in numerous complex systems such as biomass conversion or plant use for food processing or biobased materials. In this work, cellulose nanocrystals (CNCs) were used to study proteins in a dense and organized cellulosic environment. This environment was designed within microdroplets using a microfluidic setup, and applied to two proteins, bovine serum albumin (BSA) and a GH7 endoglucanase, relevant to food and plant science, respectively. The CNC at 56.5 g/L organized in liquid crystalline structure and the distribution of the proteins was probed using synchrotron deep-UV radiation. The proteins were homogeneously distributed throughout the volume, but BSA significantly disturbed the droplet global organization, preferring partition in hydrophilic external micelles. In contrast, GH7 partitioned with the CNCs showing stronger non-polar interaction but without disruption of the system organization. Such results pave the road for the development of more complex polysaccharides - proteins in-vitro models.

Real-time imaging of enzymatic degradation of pretreated maize internodes reveals different cell types have different profiles.

This work presents a dynamic view of the enzymatic degradation of maize cell walls, and sheds new light on the recalcitrance of hot water pretreated maize stem internodes. Infra-red microspectrometry, mass spectrometry, fluorescence recovery after photobleaching and fluorescence imaging were combined to investigate enzymatic hydrolysis at the cell scale. Depending on their polymer composition and organisation, cell types exhibits different extent and rate of enzymatic degradation. Enzymes act sequentially from the cell walls rich in accessible cellulose to the most recalcitrant cells. This phenomenon can be linked to the heterogeneous distribution of enzymes in the liquid medium and the adsorption/desorption mechanisms that differ with the type of cell.

Spatial correlation of water distribution and fine structure of arabinoxylans in the developing wheat grain.

This study was to investigate the distribution of water and arabinoxylan structures in growing wheat grain using two complementary imaging techniques, magnetic resonance microimaging (µMRI) and mass spectrometry imaging (MSI). µMRI showed an inhomogeneous water distribution, particularly at early stages. This heterogeneity revealed histological differences that corresponded, within the limits of resolution of µMRI, to tissues with specific physiological functions, including the vascular bundles, the cavity and the endosperm periphery. All of these tissues had a higher water content than the central endosperm. MSI revealed distinct xylan structures in these regions with high levels of Araf substitution around the cavity and acetylated xylans concentrated at the endosperm periphery. For the first time, acetylation and Araf substitution of arabinoxylans were found by image processing to spatially correlate with water distribution in planta. Acetylation and Araf substitution of xylans, which alter chain-chain interactions and increase wall porosity, decreased as the grain matured.

The endosperm cavity of wheat grains contains a highly hydrated gel of arabinoxylan.

Cereal grains provide a substantial part of the calories for humans and animals. The main quality determinants of grains are polysaccharides (mainly starch but also dietary fibers such as arabinoxylans, mixed-linkage glucans) and proteins synthesized and accumulated during grain development in a specialized storage tissue: the endosperm. In this study, the composition of a structure localized at the interface of the vascular tissues of the maternal plant and the seed endosperm was investigated. This structure is contained in the endosperm cavity where water and nutrients are transferred to support grain filling. While studying the wheat grain development, the cavity content was found to autofluoresce under UV light excitation. Combining multispectral analysis, Fourier-Transform infrared spectroscopy, immunolabeling and laser-dissection coupled with wet chemistry, we identified in the cavity arabinoxylans and hydroxycinnamic acids. The cavity content forms a "gel" in the developing grain, which persists in dry mature grain and during subsequent imbibition. Microscopic magnetic resonance imaging revealed that the gel is highly hydrated. Our results suggest that arabinoxylans are synthesized by the nucellar epidermis, released in the cavity where they form a highly hydrated gel which might contribute to regulate grain hydration.

The TaCslA12 gene expressed in the wheat grain endosperm synthesizes wheat-like mannan when expressed in yeast and Arabidopsis.

Mannan is a class of cell wall polysaccharides widespread in the plant kingdom. Mannan structure and properties vary according to species and organ. The cell walls of cereal grains have been extensively studied due to their role in cereal processing and to their beneficial effect on human health as dietary fiber. Recently, we showed that mannan in wheat (Triticum aestivum) grain endosperm has a linear structure of ß-1,4-linked mannose residues. The aim of this work was to study the biosynthesis and function of wheat grain mannan. We showed that mannan is deposited in the endosperm early during grain development, and we identified candidate mannan biosynthetic genes expressed in the endosperm. The functional study in wheat was unsuccessful therefore our best candidate genes were expressed in heterologous systems. The endosperm-specificTaCslA12 gene expressed in Pichia pastoris and in an Arabidopsis thaliana mutant depleted in glucomannan led to the production of wheat-like linear mannan lacking glucose residues and with moderate acetylation. Therefore, this gene encodes a mannan synthase and is likely responsible for the synthesis of wheat endosperm mannan.

Spatial and temporal distribution of cell wall polysaccharides during grain development of Brachypodium distachyon.

Brachypodium distachyon (Brachypodium) is now well considered as being a suitable plant model for studying temperate cereal crops. Its cell walls are phylogenetically intermediate between rice and poaceae, with a greater proximity to these latter. By microscopic and biochemical approaches, this work gives an overview of the temporal and spatial distribution of cell wall polysaccharides in the grain of Brachypodium from the end of the cellularization step to the maturation of grain. Variation in arabinoxylan chemical structure and distribution were demonstrated according to development and different grain tissues. In particular, the kinetic of arabinoxylan feruloylation was shown occuring later in the aleurone layers compared to storage endosperm. Mixed linked ß-glucan was detected in whole the tissues of Brachypodium grain even at late stage of development. Cellulose was found in both the storage endosperm and the outer layers. Homogalacturonan and rhamnogalacturonan I epitopes were differentially distributed within the grain tissues. LM5 galactan epitope was restricted to the aleurone layers contrary to LM6 arabinan epitope which was detected in the whole endosperm. A massive deposition of highly methylated homogalacturonans in vesicular bodies was observed underneath the cell wall of the testa t2 layer at early stage of development. At maturity, low-methylated homogalacturonans totally fulfilled the lumen of the t2 cell layer, suggesting pectin remodeling during grain development. Xyloglucans were only detected in the cuticle above the testa early in the development of the grain while feruloylated arabinoxylans were preferentially deposited into the cell wall of t1 layer. Indeed, the circumscribed distribution of some of the cell wall polysaccharides raises questions about their role in grain development and physiology.

Use of X-ray micro computed tomography imaging to analyze the morphology of wheat grain through its development.

BACKGROUND: Wheat is one of the most important staple source in the world for human consumption, animal feed and industrial raw materials. To deal with the global and increasing population demand, enhancing crop yield by increasing the final weight of individual grain is considered as a feasible solution. Morphometric analysis of wheat grain plays an important role in tracking and understanding developmental processes by assessing potential impacts on grains properties, size and shape that are major determinants of final grain weight. X-ray micro computed tomography (µCT) is a very powerful non-invasive imaging tool that is able to acquire 3D images of an individual grain, enabling to assess the morphology of wheat grain and of its different compartments. Our objective is to quantify changes of morphology during growth stages of wheat grain from 3D µCT images. METHODS: 3D µCT images of wheat grains were acquired at various development stages ranging from 60 to 310 degree days after anthesis. We developed robust methods for the identification of outer and inner tissues within the grains, and the extraction of morphometric features using 3D µCT images. We also developed a specific workflow for the quantification of the shape of the grain crease. RESULTS: The different compartments of the grain could be semi-automatically segmented. Variations of volumes of the compartments adequately describe the different stages of grain developments. The evolution of voids within wheat grain reflects lysis of outer tissues and growth of inner tissues. The crease shape could be quantified for each grain and averaged for each stage of development, helping us understand the genesis of the grain shape. CONCLUSION: This work shows that µCT acquisitions and image processing methodologies are powerful tools to extract morphometric parameters of developing wheat grain. The results of quantitative analysis revealed remarkable features of wheat grain growth. Further work will focus on building a computational model of wheat grain growth based on real 3D imaging data.

Phenolic distribution in apple epidermal and outer cortex tissue by multispectral deep-UV autofluorescence cryo-imaging.

Phenolic compounds in fruit are involved in responses to biotic and abiotic stresses and are responsible for organoleptic properties. To establish the distribution of these secondary metabolites at the tissue and sub-cellular scales, mapping of fluorescence in apple epidermis and outer cortex tissue in cryogenic condition was performed after deep-UV excitation at 275 nm. Douce Moën and Guillevic cider apple varieties were sampled and frozen after harvest, after 30 days at 4 °C and after 20 days at room temperature. Image analysis of fluorescence emission images acquired between 300 and 650 nm allowed the assignment of fluorescence signals to phenolic compound families based on reference molecules. Emission attributed to monomeric and/or condensed flavanol was localized in whole tissue with major fluorescence in the cuticle region. Hydroxycinnamic acids were found predominantly in the outer cortex and appeared in the cell wall. Fluorescent pigments were mostly found in the epidermis. The distribution of flavanols in the sub-cuticle and phenolic acids in the outer cortex distinguished apple varieties. Storage conditions had no impact on phenolic distribution. The proposed fluorescent imaging and analysis approach enables studies on phenolic distribution in relation to fruit development, biotic/abiotic stress resistance and quality.

Mobility of pectin methylesterase in pectin/cellulose gels is enhanced by the presence of cellulose and by its catalytic capacity.

The pectin methylesterase action is usually studied in a homogeneous aqueous medium in the presence of a large excess of soluble substrate and water. However in the cell wall, the water content is much lower, the substrate is cross-linked with itself or with other polymers, and the enzyme has to diffuse through the solid matrix before catalysing the linkage breakdown. As plant primary cell walls can be considered as cellulose-reinforced hydrogels, this study investigated the diffusion of a fungal pectin methylesterase in pectin/cellulose gels used as cell wall-mimicking matrix to understand the impact of this matrix and its (micro) structure on the enzyme's diffusion within it. The enzyme mobility was followed by synchrotron microscopy thanks to its auto-fluorescence after deep-UV excitation. Time-lapse imaging and quantification of intensity signal by image analysis revealed that the diffusion of the enzyme was impacted by at least two criteria: (i) only the active enzyme was able to diffuse, showing that the mobility was related to the catalytic ability, and (ii) the diffusion was improved by the presence of cellulose in the gel.

Cannabis for peripheral neuropathy: The good, the bad, and the unknown.

Cannabis may be an effective alternative or adjunctive treatment for peripheral neuropathy, an often debilitating condition for which standard treatments often provide little relief. Most studies show moderately improved pain from inhaled cannabis use, but adverse effects such as impaired cognition and respiratory problems are common, especially at high doses. Data on the long-term safety of cannabis treatments are limited. Until risk-benefit profiles are better characterized, doctors in states where cannabis therapy is legal should recommend it for peripheral neuropathy only after careful consideration.

Ferulate and lignin cross-links increase in cell walls of wheat grain outer layers during late development.

Important biological, nutritional and technological roles are attributed to cell wall polymers from cereal grains. The composition of cell walls in dry wheat grain has been well studied, however less is known about cell wall deposition and modification in the grain outer layers during grain development. In this study, the composition of cell walls in the outer layers of the wheat grain (Triticum aestivum Recital cultivar) was investigated during grain development, with a focus on cell wall phenolics. We discovered that lignification of outer layers begins earlier than previously reported and long before the grain reaches its final size. Cell wall feruloylation increased in development. However, in the late stages, the amount of ferulate releasable by mild alkaline hydrolysis was reduced as well as the yield of lignin-derived thioacidolysis monomers. These reductions indicate that new ferulate-mediated cross-linkages of cell wall polymers appeared as well as new resistant interunit bonds in lignins. The formation of these additional linkages more specifically occurred in the outer pericarp. Our results raised the possibility that stiffening of cell walls occur at late development stages in the outer pericarp and might contribute to the restriction of the grain radial growth.