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.

BdERECTA controls vasculature patterning and phloem-xylem organization in Brachypodium distachyon.

BACKGROUND: The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses. RESULTS: We studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature stop codon in ERECTA, a LRR receptor-like serine/threonine-protein kinase. Mutations in this gene are pleiotropic indicating that it serves multiple roles during plant development. This mutant also displayed changes in cell wall composition, gene expression and hormone homeostasis. CONCLUSION: In summary, ERECTA has a pleiotropic role in Brachypodium. We propose a major role of ERECTA in vasculature anastomosis and vascular tissue organization in Brachypodium.

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.

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 [...].

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.

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.

Comparative study of wheat low-molecular-weight glutenin and α-gliadin trafficking in tobacco cells.

KEY MESSAGE : Wheat low-molecular-weight-glutenin and α-gliadin were accumulated in the endoplasmic reticulum and formed protein body-like structures in tobacco cells, with the participation of BiP chaperone. Possible interactions between these prolamins were investigated. Wheat prolamins are the major proteins that accumulate in endosperm cells and are largely responsible for the unique biochemical properties of wheat products. They are accumulated in the endoplasmic reticulum (ER) where they form protein bodies (PBs) and are then transported to the storage vacuole where they form a protein matrix in the ripe seeds. Whereas previous studies have been carried out to determine the atypical trafficking pathway of prolamins, the mechanisms leading to ER retention and PB formation are still not clear. In this study, we examined the trafficking of a low-molecular-weight glutenin subunit (LMW-glutenin) and α-gliadin fused to fluorescent proteins expressed in tobacco cells. Through transient transformation in epidermal tobacco leaves, we demonstrated that both LMW-glutenin and α-gliadin were retained in the ER and formed mobile protein body-like structures (PBLS) that generally do not co-localise with Golgi bodies. An increased expression level of BiP in tobacco cells transformed with α-gliadin or LMW-glutenin was observed, suggesting the participation of this chaperone protein in the accumulation of wheat prolamins in tobacco cells. When stably expressed in BY-2 cells, LMW-glutenin fusion was retained longer in the ER before being exported to and degraded in the vacuole, compared with α-gliadin fusion, suggesting the involvement of intermolecular disulphide bonds in ER retention, but not in PBLS formation. Co-localisation experiments showed that gliadins and LMW-glutenin were found in the same PBLS with no particular distribution, which could be due to their ability to interact with each other as indicated by yeast two-hybrid assays.

Resistance gene transfer during treatments for experimental avian colibacillosis.

An experiment was conducted in animal facilities to compare the impacts of four avian colibacillosis treatments-oxytetracycline (OTC), trimethoprim-sulfadimethoxine (SXT), amoxicillin (AMX), or enrofloxacin (ENR)-on the susceptibility of Escherichia coli in broiler intestinal tracts. Birds were first orally inoculated with rifampin-resistant E. coli strains bearing plasmid genes conferring resistance to fluoroquinolones (qnr), cephalosporins (bla(CTX-M) or bla(FOX)), trimethoprim-sulfonamides, aminoglycosides, or tetracyclines. Feces samples were collected before, during, and after antimicrobial treatments. The susceptibilities of E. coli strains were studied, and resistance gene transfer was analyzed. An increase in the tetracycline-resistant E. coli population was observed only in OTC-treated birds, whereas multiresistant E. coli was detected in the dominant E. coli populations of SXT-, AMX-, or ENR-treated birds. Most multiresistant E. coli strains were susceptible to rifampin and exhibited various pulsed-field gel electrophoresis profiles, suggesting the transfer of one of the multiresistance plasmids from the inoculated strains to other E. coli strains in the intestinal tract. In conclusion, this study clearly illustrates how, in E. coli, "old" antimicrobials may coselect antimicrobial resistance to recent and critical molecules.

Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions.

Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP 'core'. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions.

Clinical and microbial efficacy of antimicrobial treatments of experimental avian colibacillosis.

The clinical and microbial efficacy of antimicrobial treatments of avian colibacillosis was studied, using an experimental model on chickens previously inoculated with multiresistant commensal Escherichia coli strains. One E. coli with pMG252 plasmid containing bla(FOX5) and qnrA1 genes and another E. coli with pMG298 plasmid containing bla(CTX-M15) and qnrB1 genes were first orally inoculated to chickens Both isolates were also resistant to chloramphenicol, sulphamethoxazole, trimethoprim, streptomycin, gentamicin, kanamycin, and tetracycline. The birds were then experimentally infected with an avian pathogenic E. coli (APEC), via the air sac. Treatments (oxytetracycline (OTC), trimethoprim-sulfadimethoxin (SXT), amoxicillin (AMX) or enrofloxacin (ENR) were then offered at the therapeutic doses. Symptoms, lesions in dead or sacrificed birds, and isolation and characterization of APEC from internal organs were studied. Results showed that OTC, SXT or ENR treatments could control the pathology. AMX worsened the disease, possibly due to endotoxin shock. All APEC re-isolated from internal organs showed the same antimicrobial susceptibility as the APEC inoculated strain, except for one APEC isolate from an infected OTC-treated bird, which acquired tetracycline resistance only, and one APEC isolate recovered from the air sacs of a chicken in the infected SXT-treated group, which acquired the pMG252 plasmid and became multi-resistant. Thus three antimicrobials could control the disease but the experimental model enabled, to our knowledge, the first observation of plasmid transfer from a bacterium of the intestinal tract to a pathogenic isolate from the respiratory tract.

Persistence and spread of qnr, extended-spectrum beta-lactamase, and ampC resistance genes in the digestive tract of chickens.

The aim of this assay was to develop an experimental model of digestive colonization of chickens with bacteria harboring qnr, extended-spectrum beta-lactamase, or ampC genes. Specific pathogen-free chickens were orally inoculated with two Escherichia coli strains containing either the plasmid pMG252 bearing bla(FOX) and qnrA genes, or pMG298 bearing bla(CTX-M) and qnrB genes. Analysis of strains isolated from fecal samples showed that the two strains were able to persist for several weeks in the digestive flora of inoculated birds and could rapidly spread to noninoculated ones. However, the multi-resistant isolates were maintained as a small proportion of the overall enterobacterial population. The qnr, extended-spectrum beta-lactamase, and ampC resistance genes could be transferred, in vivo, in the absence of selective pressure, to other chicken E. coli or Klebsiella pneumoniae isolates.