Synthesis and Function of Complex Sphingolipid Glycosylation.

Glycosylinositol phosphorylceramides (GIPCs) constitute an important class of plasma-membrane lipids in plants. The complex glycan headgroups of GIPCs vary between plant species and tissues. Recent studies have shown that the structure of the glycan headgroup is important for plant development, abiotic stress tolerance, and interactions with pathogenic and symbiotic microorganisms

A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis.

Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants

Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies

ObjectivesSorghum is one of the most recalcitrant species for transformation. Considering the time and effort required for stable transformation in sorghum, establishing a transient system to screen the efficiency and full functionality of vector constructs is highly desirable.ResultsHere, we report an Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker. It also provides a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency. Given the simplicity and ease of transformation, high reproducibility, and ability to test large constructs, this method can be widely adopted to speed up functional genomic and genome editing studies

Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis.

All members of the DUF579 family characterized so far have been described to affect the integrity of the hemicellulosic cell wall component xylan: GXMs are glucuronoxylan methyltransferases catalyzing 4-O-methylation of glucuronic acid on xylan; IRX15 and IRX15L, although their enzymatic activity is unknown, are required for xylan biosynthesis and/or xylan deposition. Here we show that the DUF579 family members, AGM1 and AGM2, are required for 4-O-methylation of glucuronic acid of a different plant cell wall component, the highly glycosylated arabinogalactan proteins (AGPs)

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14.

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1-2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays, and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. The differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.The work presented in this paper was supported by grants from the BBSRC: BB/G016240/1 BBSRC Sustainable Energy Centre Cell Wall Sugars Programme (BSBEC) and grant BB/K005537/1. JCM’s work at the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE -AC02-05CH11231. NFB was supported by a PhD studentship from the Portuguese Foundation for Science and Technology. AN was supported by a summer studentship award from the Biochemical Society. The authors are grateful to the European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013) under the grant agreement no 251132.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/tpj.1289

Ten-Year Analysis of Bacterial Colonisation and Outcomes of Major Burn Patients with a Focus on Pseudomonas aeruginosa

A retrospective descriptive study included patients admitted with severe burns over the course of 10 years (2008–2018). Across all patients, there were 39 different species of bacteria, with 23 species being Gram-negative and 16 being Gram-positive bacteria, with also five different species of fungi cultured. Pseudomonas aeruginosa was the most commonly isolated organism, with 57.45% of patients having a positive culture. There was a significant difference in the number of P. aeruginosa isolated from patients that acquired their burns at work, in a garden, inside a vehicle, in a garage or in a public place. In patients that were positive for P. aeruginosa, the number of operations was higher (2.4) and the length of stay was significantly increased (80.1 days). Patients that suffered from substance abuse demonstrated significantly higher numbers of isolated P. aeruginosa (14.8%). Patients that suffered from both mental health illness and substance abuse demonstrated significantly higher numbers of P. aeruginosa isolated (18.5%). In the P. aeruginosa-negative group, there were significantly fewer patients that had been involved in a clothing fire. Furthermore, in the P. aeruginosa-negative patient cohort, the mortality rate was significantly higher (p = 0.002). Since the incidence of P. aeruginosa was also associated with a decreased mortality rate, it may be that patients admitted to hospital for shorter periods of time were less likely to be colonised with P. aeruginosa. This study demonstrates novel factors that may increase the incidence of P. aeruginosa isolated from burn patients

The Arabidopsis thaliana nucleotide sugar transporter GONST2 is a functional homolog of GONST1.

Glycosylinositolphosphorylceramides (GIPCs) are the predominant lipid in the outer leaflet of the plasma membrane. Characterized GIPC glycosylation mutants have severe or lethal plant phenotypes. However, the function of the glycosylation is unclear. Previously, we characterized Arabidopsis thaliana GONST1 and showed that it was a nucleotide sugar transporter which provides GDP-mannose for GIPC glycosylation. gonst1 has a severe growth phenotype, as well as a constitutive defense response. Here, we characterize a mutant in GONST1's closest homolog, GONST2. The gonst2-1 allele has a minor change to GIPC headgroup glycosylation. Like other reported GIPC glycosylation mutants, gonst1-1gonst2-1 has reduced cellulose, a cell wall polymer that is synthesized at the plasma membrane. The gonst2-1 allele has increased resistance to a biotrophic pathogen Golovinomyces orontii but not the necrotrophic pathogen Botrytis cinerea. Expression of GONST2 under the GONST1 promoter can rescue the gonst1 phenotype, indicating that GONST2 has a similar function to GONST1 in providing GDP-D-Man for GIPC mannosylation

Bi-functional glycosyltransferases catalyze both extension and termination of pectic galactan oligosaccharides

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side-chains of rhamnogalacturonan-I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP-Gal to growing β-1,4-galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP-α-d-Gal and the transfer of an arabinopyranose from UDP-β-l-Arap to galactan chains. The two substrates share a similar structure, but UDP-α-d-Gal is the preferred substrate, with a 10-fold higher affinity. Transfer of Arap to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage