•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 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.
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.
Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to ...accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.
Sorghum (Sorghum bicolor (L.) Moench) is the fifth most important grain produced in the world. Interest for cultivating sorghum is increasing all over the world in the context of climate change, due ...to its low input and water requirements. Like other cultivated cereals, sorghum has significant nutritional value thanks to its protein, carbohydrate and dietary fiber content, these latter mainly consisting of cell wall polysaccharides. This work describes for the first time a transcriptomic analysis dedicated to identify the genes involved in the biosynthesis and remodelling of cell walls both in the endosperm and outer layers of sorghum grain during its development. Further analysis of these transcriptomic data will improve our understanding of cell wall assembly, which is a key component of grain quality.
This research delineates the steps of our analysis, starting with the cultivation conditions and the grain harvest at different stages of development, followed by the laser microdissection applied to separate the endosperm from the outer layers. It also describes the procedures implemented to generate RNA libraries and to obtain a normalized and filtered table of transcript counts, and finally determine the number of putative cell wall-related genes already listed in literature.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Apple fruit mealiness is one of the most important textural problems that results from an undesirable ripening process during storage. This phenotype is characterized by textural deterioration ...described as soft, grainy and dry fruit. Despite several studies, little is known about mealiness development and the associated molecular events. In this study, we integrated phenotypic, microscopic, transcriptomic and biochemical analyses to gain insights into the molecular basis of mealiness development.
Instrumental texture characterization allowed the refinement of the definition of apple mealiness. In parallel, a new and simple quantitative test to assess this phenotype was developed.
These data support the role of PME in cell wall remodelling during apple fruit development and ripening and suggest a local action of these enzymes. Mealiness may partially result from qualitative and spatial variations of pectin microarchitecture rather than quantitative pectin differences, and these changes may occur early in fruit development. The specific MdPME2 gene highlighted in this study could be a good early marker of texture unfavourable trait in apple.
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.
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.
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