scholarly journals A Group of O-Acetyltransferases Catalyze Xyloglucan Backbone Acetylation and Can Alter Xyloglucan Xylosylation Pattern and Plant Growth When Expressed in Arabidopsis

2020 ◽  
Vol 61 (6) ◽  
pp. 1064-1079
Author(s):  
Ruiqin Zhong ◽  
Dongtao Cui ◽  
Dennis R Phillips ◽  
Elizabeth A Richardson ◽  
Zheng-Hua Ye
Keyword(s):  
Plant Growth ◽  
Recombinant Proteins ◽  
Cell Expansion ◽  
Cell Walls ◽  
Side Chain ◽  
Plant Cell Walls ◽  
Biochemical Mechanism ◽  
Wall Function ◽  
Severe Reduction

Abstract Xyloglucan is a major hemicellulose in plant cell walls and exists in two distinct types, XXXG and XXGG. While the XXXG-type xyloglucan from dicot species only contains O-acetyl groups on side-chain galactose (Gal) residues, the XXGG-type xyloglucan from Poaceae (grasses) and Solanaceae bears O-acetyl groups on backbone glucosyl (Glc) residues. Although O-acetyltransferases responsible for xyloglucan Gal acetylation have been characterized, the biochemical mechanism underlying xyloglucan backbone acetylation remains to be elucidated. In this study, we showed that recombinant proteins of a group of DUF231 members from rice and tomato were capable of transferring acetyl groups onto O-6 of Glc residues in cello-oligomer acceptors, indicating that they are xyloglucan backbone 6-O-acetyltransferases (XyBATs). We further demonstrated that XyBAT-acetylated cellohexaose oligomers could be readily xylosylated by AtXXT1 (Arabidopsis xyloglucan xylosyltransferase 1) to generate acetylated, xylosylated cello-oligomers, whereas AtXXT1-xylosylated cellohexaose oligomers were much less effectively acetylated by XyBATs. Heterologous expression of a rice XyBAT in Arabidopsis led to a severe reduction in cell expansion and plant growth and a drastic alteration in xyloglucan xylosylation pattern with the formation of acetylated XXGG-type units, including XGG, XGGG, XXGG, XXGG,XXGGG and XXGGG (G denotes acetylated Glc). In addition, recombinant proteins of two Arabidopsis XyBAT homologs also exhibited O-acetyltransferase activity toward cellohexaose, suggesting their possible role in mediating xyloglucan backbone acetylation in vivo. Our findings provide new insights into the biochemical mechanism underlying xyloglucan backbone acetylation and indicate the importance of maintaining the regular xyloglucan xylosylation pattern in cell wall function.

A Single Xyloglucan Xylosyltransferase is Sufficient for Generation of the XXXG Xylosylation Pattern of Xyloglucan

2021 ◽  
Author(s):  
Ruiqin Zhong ◽  
Dennis R Phillips ◽  
Zheng-Hua Ye
Keyword(s):  
Recombinant Proteins ◽  
Cell Walls ◽  
Degree Of Polymerization ◽  
Primary Cell ◽  
Hek293 Cells ◽  
Biochemical Mechanism ◽  
The Third ◽  
Insight Into

Abstarct Xyloglucan is the most abundant hemicellulose in the primary cell walls of dicots. Dicot xyloglucan is the XXXG-type consisting of repeating units of three consecutive xylosylated Glc residues followed by one unsubstituted Glc. Its xylosylation is catalyzed by xyloglucan 6-xylosyltransferases (XXTs) and there exist five XXTs (AtXXT1-5) in Arabidopsis. While AtXXT1and AtXXT2 have been shown to add the first two Xyl residues in the XXXG repeat, which XXTs are responsible for the addition of the third Xyl residue remains elusive although AtXXT5 was a proposed candidate. In this report, we generated recombinant proteins of all five Arabidopsis XXTs and one rice XXT (OsXXT1) in the mammalian HEK293 cells and investigated their ability to sequentially xylosylate Glc residues to generate the XXXG xylosylation pattern. We found that like AtXXT1/2, AtXXT4 and OsXXT1 could efficiently xylosylate the cellohexaose (G6) acceptor to produce mono- and di-xylosylated G6, whereas AtXXT5 was only barely capable of adding one Xyl onto G6. When AtXXT1-catalyzed products were used as acceptors, AtXXT1/2/4 and OsXXT1 but not AtXXT5 were able to xylosylate additional Glc residues to generate tri- and tetra-xylosylated G6. Further characterization of the tri- and tetra-xylosylated G6 revealed that they had the sequence of GXXXGG and GXXXXG with three and four consecutive xylosylated Glc residues, respectively. In addition, we have found that although tri-xylosylation occurred on G6, cello-oligomers with a degree of polymerization of 3 to 5 could only be mono- and di-xylosylated. Together, these results indicate that each of AtXXT1/2/4 and OsXXT1 is capable of sequentially adding Xyl onto three contiguous Glc residues to generate the XXXG xylosylation pattern and these findings provide new insight into the biochemical mechanism underlying xyloglucan biosynthesis.

Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro and in the cell walls of ripening pear fruit

2001 ◽  
Vol 357 (3) ◽  
pp. 729-737 ◽  
Author(s):  
Stephen C. FRY ◽  
Jo C. DUMVILLE ◽  
Janice G. MILLER
Keyword(s):  
Hydroxyl Radicals ◽  
Cell Walls ◽  
Plant Cell Walls ◽  
Pear Fruit ◽  
Connective Tissues ◽  
Softening Process ◽  
Radical Attack

Hydroxyl radicals (•OH) may cause non-enzymic scission of polysaccharides in vivo, e.g. in plant cell walls and mammalian connective tissues. To provide a method for detecting the action of endogenous •OH in vivo, we investigated the products formed when polysaccharides were treated with •OH (generated in situ by ascorbate-H2O2-Cu2+ mixtures) followed by NaB3H4. Treatment with •OH increased the number of NaB3H4-reacting groups present in citrus pectin, homogalacturonan and tamarind xyloglucan. This increase is attributed partly to the formation of glycosulose and glycosulosuronic acid residues, which are then reduced back to the original (but radioactive) sugar residues and their epimers by NaB3H4. The glycosulose and glycosulosuronic acid residues were stable for > 16h at 20°C in ethanol or buffer (pH4.7), but were destroyed in alkali. Driselase-digestion of the radiolabelled polysaccharides yielded characteristic patterns of 3H-products, which included galactose and galacturonate from pectin, and isoprimeverose, galactose, glucose and arabinose from xyloglucan. Pectin yielded at least eight 3H-labelled anionic products, separable by electrophoresis at pH3.5. The patterns of radioactive products form useful ‘fingerprints’ by which •OH-attacked polysaccharides may be recognized. Applied to the cell walls of ripening pear (Pyrus communis) fruit, the method gave evidence for progressive •OH radical attack on polysaccharides during the softening process.

Structure of the oligogalacturonate-specific KdgM porin

2014 ◽  
Vol 70 (6) ◽  
pp. 1770-1778 ◽  
Author(s):  
C. A. J. Hutter ◽  
R. Lehner ◽  
Ch. Wirth ◽  
G. Condemine ◽  
C. Peneff ◽  
...  
Keyword(s):  
Cell Walls ◽  
Circular Cross Section ◽  
Erwinia Chrysanthemi ◽  
Plant Cell Walls ◽  
Negative Bacterium ◽  
Dickeya Dadantii ◽  
Outer Membrane Porin ◽  
Minimal Radius ◽  
Arginine Residues

The phytopathogenic Gram-negative bacteriumDickeya dadantii(Erwinia chrysanthemi) feeds on plant cell walls by secreting pectinases and utilizing the oligogalacturanate products. An outer membrane porin, KdgM, is indispensable for the uptake of these acidic oligosaccharides. Here, the crystal structure of KdgM determined to 1.9 Å resolution is presented. KdgM is folded into a regular 12-stranded antiparallel β-barrel with a circular cross-section defining a transmembrane pore with a minimal radius of 3.1 Å. Most of the loops that would face the cell exteriorin vivoare disordered, but nevertheless mediate contact between densely packed membrane-like layers in the crystal. The channel is lined by two tracks of arginine residues facing each other across the pore, a feature that is conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides.

scholarly journals In Vivo Competitions between Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminoccus albus in a Gnotobiotic Sheep Model Revealed by Multi-Omic Analyses

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Carl J. Yeoman ◽  
Christopher J. Fields ◽  
Pascale Lepercq ◽  
Philippe Ruiz ◽  
Evelyne Forano ◽  
...  
Keyword(s):  
Plant Cell ◽  
Cell Walls ◽  
Fibrobacter Succinogenes ◽  
Plant Cell Walls ◽  
Cellulolytic Bacteria ◽  
Ruminococcus Albus ◽  
Ruminococcus Flavefaciens ◽  
Content Type

ABSTRACT Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens are the three predominant cellulolytic bacterial species found in the rumen. In vitro studies have shown that these species compete for adherence to, and growth upon, cellulosic biomass. Yet their molecular interactions in vivo have not heretofore been examined. Gnotobiotically raised lambs harboring a 17-h-old immature microbiota devoid of culturable cellulolytic bacteria and methanogens were inoculated first with F. succinogenes S85 and Methanobrevibacter sp. strain 87.7, and 5 months later, the lambs were inoculated with R. albus 8 and R. flavefaciens FD-1. Longitudinal samples were collected and profiled for population dynamics, gene expression, fibrolytic enzyme activity, in sacco fibrolysis, and metabolite profiling. Quantitative PCR, metagenome and metatranscriptome data show that F. succinogenes establishes at high levels initially but is gradually outcompeted following the introduction of the ruminococci. This shift resulted in an increase in carboxymethyl cellulase (CMCase) and xylanase activities but not in greater fibrolysis, suggesting that F. succinogenes and ruminococci deploy different but equally effective means to degrade plant cell walls. Expression profiles showed that F. succinogenes relied upon outer membrane vesicles and a diverse repertoire of CAZymes, while R. albus and R. flavefaciens preferred type IV pili and either CBM37-harboring or cellulosomal carbohydrate-active enzymes (CAZymes), respectively. The changes in cellulolytics also affected the rumen metabolome, including an increase in acetate and butyrate at the expense of propionate. In conclusion, this study provides the first demonstration of in vivo competition between the three predominant cellulolytic bacteria and provides insight on the influence of these ecological interactions on rumen fibrolytic function and metabolomic response. IMPORTANCE Ruminant animals, including cattle and sheep, depend on their rumen microbiota to digest plant biomass and convert it into absorbable energy. Considering that the extent of meat and milk production depends on the efficiency of the microbiota to deconstruct plant cell walls, the functionality of predominant rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, has been extensively studied in vitro to obtain a better knowledge of how they operate to hydrolyze polysaccharides and ultimately find ways to enhance animal production. This study provides the first evidence of in vivo competitions between F. succinogenes and the two Ruminococcus species. It shows that a simple disequilibrium within the cellulolytic community has repercussions on the rumen metabolome and fermentation end products. This finding will have to be considered in the future when determining strategies aiming at directing rumen fermentations for animal production.

Exploration of cell wall architecture with the rapid-freeze deep-etch technique

1993 ◽  
Vol 51 ◽  
pp. 128-129
Author(s):  
Béatrice Satiat-Jeunemaitre ◽  
Chris Hawes
Keyword(s):  
Plant Cell ◽  
Cell Walls ◽  
Cell Biology ◽  
Molecular Model ◽  
Three Dimensional ◽  
Secretory Activity ◽  
Wall Structure ◽  
Specimen Preparation ◽  
Molecular Architecture ◽  
Plant Cell Walls

The comprehension of the molecular architecture of plant cell walls is one of the best examples in cell biology which illustrates how developments in microscopy have extended the frontiers of a topic. Indeed from the first electron microscope observation of cell walls it has become apparent that our understanding of wall structure has advanced hand in hand with improvements in the technology of specimen preparation for electron microscopy. Cell walls are sub-cellular compartments outside the peripheral plasma membrane, the construction of which depends on a complex cellular biosynthetic and secretory activity (1). They are composed of interwoven polymers, synthesised independently, which together perform a number of varied functions. Biochemical studies have provided us with much data on the varied molecular composition of plant cell walls. However, the detailed intermolecular relationships and the three dimensional arrangement of the polymers in situ remains a mystery. The difficulty in establishing a general molecular model for plant cell walls is also complicated by the vast diversity in wall composition among plant species.

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