The cell wall is an important compartment in grain cells that fulfills both structural and functional roles. It has a dynamic structure that is constantly modified during development and in response ...to biotic and abiotic stresses. Non-structural cell wall proteins (CWPs) are key players in the remodeling of the cell wall during events that punctuate the plant life. Here, a subcellular and quantitative proteomic approach was carried out to identify CWPs possibly involved in changes in cell wall metabolism at two key stages of wheat grain development: the end of the cellularization step and the beginning of storage accumulation. Endosperm and outer layers of wheat grain were analyzed separately as they have different origins (maternal and seed) and functions in grains. Altogether, 734 proteins with predicted signal peptides were identified (CWPs). Functional annotation of CWPs pointed out a large number of proteins potentially involved in cell wall polysaccharide remodeling. In the grain outer layers, numerous proteins involved in cutin formation or lignin polymerization were found, while an unexpected abundance of proteins annotated as plant invertase/pectin methyl esterase inhibitors were identified in the endosperm. In addition, numerous CWPs were accumulating in the endosperm at the grain filling stage, thus revealing strong metabolic activities in the cell wall during endosperm cell differentiation, while protein accumulation was more intense at the earlier stage of development in outer layers. Altogether, our work gives important information on cell wall metabolism during early grain development in both parts of the grain, namely the endosperm and outer layers. The wheat cell wall proteome is the largest cell wall proteome of a monocot species found so far.
Seeds of the model grass
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
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,
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.
•Mannan appears early in the developing endosperm of wheat grain.•However, the first cell walls of the developing endosperm are devoid of mannan.•Mannan related genes were identified in wheat ...endosperm.•TaCslA12 produced pure wheat-like mannan when expressed in yeast and Arabidopsis.•Glucomannan were replaced by pure mannan in Arabidopsis stem without visible effect.
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 β-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.
Global DNA hypomethylation is a hallmark of cancer cells, but its molecular mechanisms have not been elucidated. Here, we show that the disruption of Dnmt1/PCNA/UHRF1 interactions promotes a global ...DNA hypomethylation in human gliomas. We then demonstrate that the Dnmt1 phosphorylations by Akt and/or PKC abrogate the interactions of Dnmt1 with PCNA and UHRF1 in cellular and acellular studies including mass spectrometric analyses and the use of primary cultured patient-derived glioma. By using methylated DNA immunoprecipitation, methylation and CGH arrays, we show that global DNA hypomethylation is associated with genes hypomethylation, hypomethylation of DNA repeat element and chromosomal instability. Our results reveal that the disruption of Dnmt1/PCNA/UHRF1 interactions acts as an oncogenic event and that one of its signatures (i.e. the low level of mMTase activity) is a molecular biomarker associated with a poor prognosis in GBM patients. We identify the genetic and epigenetic alterations which collectively promote the acquisition of tumor/glioma traits by human astrocytes and glial progenitor cells as that promoting high proliferation and apoptosis evasion.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•Distribution of polysaccharides during Brachypodium grain development.•Fine structure of arabinoxylans varies according to tissue and grain development.•Amazing distribution of polysaccharides ...highlighted into the testa of Brachypodium grain.
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 β-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.
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.
Soaking and boiling can improve the digestibility of seeds by reducing anti-nutritional factors and the hard-to-cook phenomenon. The use of pre-treated seeds in protein fractionation is thus of ...interest for the development of protein-rich ingredients. In this work, soaked and soaked-boiled seeds were applied prior to aqueous fractionation of pigeon pea proteins. Pre-treatments impact the particle size distribution in the flours, with boiling leading to aggregate formation (>1000 μm) due to starch gelatinization. The soaked-boiled flour showed a lower lipid content, higher content in soluble polysaccharides, and loss of proteins with low molecular weight compared to the control. The use of soaked-boiled seeds for protein fractionation reduced trypsin inhibitor activity by 95%, demonstrating a positive effect in improving nutritional quality. Protein fractionation also resulted in a 3-fold accumulation of lipids, and the mannose content, notably, doubled in protein concentrates compared to their flour counterparts. A lower intensity of tryptophan fluorescence emission was observed in protein concentrates, which may be related to chemical degradation or interactions with other molecules. The protein concentrate from soaked-boiled seeds exhibited higher emulsion stabilization properties compared to the others protein concentrates. The decrease in foaming properties caused by the soaking can be attributed to the leaching of minor compounds that also act as foaming agents, such as saponins. This work brings new insights into the effects of soaking and boiling-based pre-treatments on the composition of pulse protein ingredients, and in turn, on their techno-functionalities as exemplified with their emulsifying, foaming and gelling properties.
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•Physical treatments affected size and morphology of pigeon pea flour particles.•Mannose in protein concentrates suggests the presence of glycosylated proteins.•Phospholipids were removed during fractionation of soaked-boiled seeds.•Seed pre-treatments led to a loss of low molecular weight proteins (<37 kDa).•Protein concentrates from boiled seeds were more efficient at stabilizing emulsions.
•Pseudoalteromonas atlantica produced a high diversity of polysaccharide-degrading enzymes.•The marine bacterium Pseudoalteromonas atlantica secreted enzymes active towards hemicelluloses from ...terrestrial plants.•β-xylosidase, α-arabinofuranosidase and acetylesterase activities were evidenced in the secretome of Pseudoalteromonas atlantica.•The GH8 protein present in Pseudoalteromonas atlantica genome is a xylanase with unusual product profile.
To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a β-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end.
•The wheat grain central endosperm cavity is filled with a gel-like structure.•The gel-like structure autofluoresces under UV excitation.•It contains fluorescent, water-absorbing and gel-forming ...feruloylated arabinoxylans.•High and stable cavity water content suggests a role in regulating grain hydration.
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.
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