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.

Correction: Cherkaoui, M., Lollier, V., Geairon, A., Bouder, A., Larré, C., Rogniaux, H., Jamet, E., Guillon, F. and Francin-Allami, M. Cell Wall Proteome of Wheat Grain Endosperm and Outer Layers at Two Key Stages of Early Development. Int. J. Mol. Sci. 2020, 21, 239.

The authors wish to make the following corrections to this paper [...].

Cell Wall Proteome of Wheat Grain Endosperm and Outer Layers at Two Key Stages of Early Development.

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.

Cell Wall Proteome Investigation of Bread Wheat (Triticum Aestivum) Developing Grain in Endosperm and Outer Layers.

The remodeling of cell wall polysaccharides is controlled by cell wall proteins (CWPs) during the development of wheat grain. This work describes for the first time the cell wall proteomes of the endosperm and outer layers of the wheat developing grain, which have distinct physiological functions and technological uses. Altogether 636 nonredundant predicted CWPs are identified with 337 proteins in the endosperm and 594 proteins in the outer layers, among which 295 proteins are present in both tissues, suggesting both common and tissue specific remodeling activities. These proteins are distributed into eight functional classes. Approximatively a quarter of them were predicted to act on cell wall polysaccharides, with many glycosylhydrolases and also expansin, lyases, and carbohydrate esterases. Therefore, these results provide crucial data to go further in the understanding of relationship between tissue-specific morphogenesis and cell wall remodeling in cereals. Data are available via ProteomeXchange with identifier PXD010367.

High-energy photon activation tandem mass spectrometry provides unprecedented insights into the structure of highly sulfated oligosaccharides extracted from macroalgal cell walls.

Extreme ultraviolet photon activation tandem mass spectrometry (MS) at 69 nm (18 eV) was used to characterize mixtures of oligo-porphyrans, a class of highly sulfated oligosaccharides. Porphyrans, hybrid polymers whose structures are far from known, continue to provide a challenge for analytical method development. Activation by 18 eV photons led to a rich fragmentation of the oligo-porphyrans, with many cross-ring and glycosidic cleavages. In contrast to multistage MSn strategies such as activated electron photodetachment dissociation, a single step of irradiation by energetic UV of multiply charged anions led to a complete fragmentation of the oligo-porphyrans. In both ionization modes, the sulfate groups were retained on the backbone, which allowed the pattern of these modifications along the porphyran backbone to be described in unprecedented detail. Many structures released by the enzymatic degradation of the porphyran were completely resolved, including isomers. This work extends the existing knowledge of the structure of porphyrans. In addition, it provides a new demonstration of the potential of activation by high-energy photons for the structural analysis of oligosaccharides, even in unseparated mixtures, with a particular focus on sulfated compounds.

TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells.

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity in vitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.

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.

Functional exploration of Pseudoalteromonas atlantica as a source of hemicellulose-active enzymes: Evidence for a GH8 xylanase with unusual mode of action.

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 ß-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.

Spatial mapping of lichen specialized metabolites using LDI-MSI: chemical ecology issues for Ophioparma ventosa.

Imaging mass spectrometry techniques have become a powerful strategy to assess the spatial distribution of metabolites in biological systems. Based on auto-ionisability of lichen metabolites using LDI-MS, we herein image the distribution of major secondary metabolites (specialized metabolites) from the lichen Ophioparma ventosa by LDI-MSI (Mass Spectrometry Imaging). Such technologies offer tremendous opportunities to discuss the role of natural products through spatial mapping, their distribution patterns being consistent with previous chemical ecology reports. A special attention was dedicated to miriquidic acid, an unexpected molecule we first reported in Ophioparma ventosa. The analytical strategy presented herein offers new perspectives to access the sharp distribution of lichen metabolites from regular razor blade-sectioned slices.

Amaranth (Amaranthus hypochondriacus) vicilin subunit structure.

The 7S-globulin fraction is a minor component of the amaranth storage proteins. The present work provides new information about this protein. The amaranth 7S-globulin or vicilin presented a sedimentation coefficient of 8.6 ± 0.6 S and was composed of main subunits of 66, 52, 38, and 16 kDa. On the basis of mass spectrometry (MS) analysis of tryptic fragments, the 52, 38, and 16 kDa subunits presented sequence homology with sesame vicilin, whereas the 66 kDa subunit showed sequence similarity with a putative vicilin. Several characteristics of the 66 kDa subunit were similar to members of the convicilin family. Results support the hypothesis that the 7S-globulin molecules are composed of subunits coming from at least two gene families with primary products of 66 and 52 kDa, respectively. According to the present information, amaranth vicilin may be classified into the vicilin group that includes pea, broad bean, and sesame vicilins, among others.

Disruption of Dnmt1/PCNA/UHRF1 interactions promotes tumorigenesis from human and mice glial cells.

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.

Globulin-p and 11S-globulin from amaranthus Hypochondriacus: are two isoforms of the 11S-globulin.

Amaranth is an ancient crop with a high content of good quality proteins. Globulins are some of the most abundant storage proteins of amaranth grain. They contain two fractions distinguishable according to their different solubility: the salt-soluble 7S and 11S-globulins and the globulin-p soluble in mild-alkaline, low-ionic-strength solutions. As part of the amaranth proteins characterization, in this work we investigated the structural characteristics responsible for the different physicochemical properties of these globulins. We studied certain conformational parameters of the purified aggregates (AMGp) and individual molecules (IMGp) of globulin-p and of the partially purified globulin (ppGb) and compared the AMGp polypeptide sequences with the sequence of the 11S-globulin propolypeptide from Amaranthus (gi|122726601). The results indicated that the AMGp aggregates are responsible for the different solubility of globulin-p. Subtle conformational differences as determined by fluorescence spectroscopy and urea sensitivity were found between the molecules studied: The AMGp showed some surface differences from the IMGp and the ppGb; the AMGp also had a lower affinity for the hydrophobic fluorescent probe 1,8-aniline-naphthalene-sulfonate and a higher ionic charge than the ppGb and the IMGp, characteristics that might cause their lower solubility. In addition, we have demonstrated differences between the AMGp polypeptide sequences and that reported for amaranth 11S-globulin. These differences suggest that the globulin-p and 11S-globulin are two 11S-globulin isoforms comprised of polypeptides coming from different legumin-gene subfamilies.