GO-PROMTO Illuminates Protein Membrane Topologies of Glycan Biosynthetic Enzymes in the Golgi Apparatus of Living Tissues

The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes

Evolution of Plant Nucleotide-Sugar Interconversion Enzymes

Nucleotide-diphospho-sugars (NDP-sugars) are the building blocks of diverse polysaccharides and glycoconjugates in all organisms. In plants, 11 families of NDP-sugar interconversion enzymes (NSEs) have been identified, each of which interconverts one NDP-sugar to another. While the functions of these enzyme families have been characterized in various plants, very little is known about their evolution and origin. Our phylogenetic analyses indicate that all the 11 plant NSE families are distantly related and most of them originated from different progenitor genes, which have already diverged in ancient prokaryotes. For instance, all NSE families are found in the lower land plant mosses and most of them are also found in aquatic algae, implicating that they have already evolved to be capable of synthesizing all the 11 different NDP-sugars. Particularly interesting is that the evolution of RHM (UDP-L-rhamnose synthase) manifests the fusion of genes of three enzymatic activities in early eukaryotes in a rather intriguing manner. The plant NRS/ER (nucleotide-rhamnose synthase/epimerase-reductase), on the other hand, evolved much later from the ancient plant RHMs through losing the N-terminal domain. Based on these findings, an evolutionary model is proposed to explain the origin and evolution of different NSE families. For instance, the UGlcAE (UDP-D-glucuronic acid 4-epimerase) family is suggested to have evolved from some chlamydial bacteria. Our data also show considerably higher sequence diversity among NSE-like genes in modern prokaryotes, consistent with the higher sugar diversity found in prokaryotes. All the NSE families are widely found in plants and algae containing carbohydrate-rich cell walls, while sporadically found in animals, fungi and other eukaryotes, which do not have or have cell walls with distinct compositions. Results of this study were shown to be highly useful for identifying unknown genes for further experimental characterization to determine their functions in the synthesis of diverse glycosylated molecules

A Genome Wide Association Study of arabinoxylan content in 2-row spring barley grain

In barley endosperm arabinoxylan (AX) is the second most abundant cell wall polysaccharide and in wheat it is the most abundant polysaccharide in the starchy endosperm walls of the grain. AX is one of the main contributors to grain dietary fibre content providing several health benefits including cholesterol and glucose lowering effects, and antioxidant activities. Due to its complex structural features, AX might also affect the downstream applications of barley grain in malting and brewing. Using a high pressure liquid chromatography (HPLC) method we quantified AX amounts in mature grain in 128 spring 2-row barley accessions. Amounts ranged from ~ 5.2 μg/g to ~ 9 μg/g. We used this data for a Genome Wide Association Study (GWAS) that revealed three significant quantitative trait loci (QTL) associated with grain AX levels which passed a false discovery threshold (FDR) and are located on two of the seven barley chromosomes. Regions underlying the QTLs were scanned for genes likely to be involved in AX biosynthesis or turnover, and strong candidates, including glycosyltransferases from the GT43 and GT61 families and glycoside hydrolases from the GH10 family, were identified. Phylogenetic trees of selected gene families were built based on protein translations and were used to examine the relationship of the barley candidate genes to those in other species. Our data reaffirms the roles of existing genes thought to contribute to AX content, and identifies novel QTL (and candidate genes associated with them) potentially influencing the AX content of barley grain. One potential outcome of this work is the deployment of highly associated single nucleotide polymorphisms markers in breeding programs to guide the modification of AX abundance in barley grain

Cutaneous lesions of the nose

Skin diseases on the nose are seen in a variety of medical disciplines. Dermatologists, otorhinolaryngologists, general practitioners and general plastic and dermatologic surgeons are regularly consulted regarding cutaneous lesions on the nose. This article is the second part of a review series dealing with cutaneous lesions on the head and face, which are frequently seen in daily practice by a dermatologic surgeon. In this review, we focus on those skin diseases on the nose where surgery or laser therapy is considered a possible treatment option or that can be surgically evaluated

The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides

Kanter U, Usadel B, Guerineau F, Li Y, Pauly M, Tenhaken R. The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides. Planta. 2005;221(2):243-254.The nucleotide sugar UDP-glucuronic acid (UDP-GlcA) is the principal precursor for galacturonic acid, xylose, apiose and arabinose residues of the plant cell-wall polymers. UDP-GlcA can be synthesized by two different functional pathways in Arabidopsis involving either UDP-glucose dehydrogenase or inositol oxygenase as the initial enzyme reaction to channel carbohydrates into a pool of UDP sugars used for cell-wall biosynthesis. The genes for the enzyme myo-inositol oxygenase (MIOX) were analyzed in Arabidopsis. They represent a small gene family containing four members. The transcription of all those members indicates a transient and organ-specific gene expression pattern in growing plant tissues as analyzed by RT-PCR and in promoter::GUS reporter gene lines. Two isoforms (MIOX1, MIOX2) are expressed in almost all tissues of the plant, whereas the expression of MIOX4 and MIOX5 is largely restricted to flowers, particularly maturing pollen. T-DNA insertion lines in MIOX genes were isolated; however, single knock-outs show growth phenotypes similar to the wild type. The monosaccharide composition of the cell wall in these mutants is not significantly changed compared to wild type plants. However, the incorporation of 3H-inositol into wall polymers of seedlings is greatly impaired in the mutant lines (Delta)MIOX1 and (Delta)MIOX2, which are the only isoforms that are expressed in seedlings

Does a Responsible University Need a Third Mission?

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The complement of soluble sugars in the Saccharum complex

The use of sugarcane as a biofactory and source of renewable biomass is being investigated increasingly due to its vigorous growth and ability to fix a large amount of carbon dioxide compared to other crops. The high biomass resulting from sugarcane production (up to 80 t/ha) makes it a candidate for genetic manipulation to increase the production of other sugars found in this research that are of commercial interest. Sucrose is the major sugar measured in sugarcane with hexoses glucose and fructose present in lower concentrations; sucrose can make up to 60% of the total dry weight of the culm. Species related to modern sugarcane cultivars were examined for the presence of sugars other than glucose, fructose and sucrose with the potential of this crop as a biofactory in mind. The species examined form part of the Saccharum complex, a closely-related interbreeding group. Extracts of the immature and mature internodes of six different species and a hybrid were analysed with gas chromatography mass spectrometry to identify mono-, di- and tri-saccharides, as well as sugar acids and sugar alcohols. Thirty two sugars were detected, 16 of which have previously not been identified in sugarcane. Apart from glucose, fructose and sucrose the abundance of sugars in all plants was low but the research demonstrated the presence of sugar pathways that could be manipulated. Since species from the Saccharum complex can be interbred, any genes leading to the production of sugars of interest could be introgressed into commercial Saccharum species or manipulated through genetic engineering