In recent years, the impressive development of molecular genetics tools, the sequencing of the Arabidopsis thaliana genome, the availability of DNA or transposon tagged mutants, and the multiple possibilities offered by stable transformation with DNA in sense and antisense orientation have enabled the application of a strategy of gain or loss of function to study the sterol biosynthesis pathway. Here we describe the results obtained with these techniques. The results essentially confirm data obtained previously with sterol biosynthesis inhibitors (SBIs) and enable the precise dissection of biosynthetic pathways. We discuss the advantages and disadvantages of molecular genetics techniques as applied to sterol metabolism. The greater selectivity of these techniques constitutes an invaluable advantage and has led to the discovery of a role for sterols in plant development.

Biosynthesis and accumulation of sterols

Defensins from Insects and Plants Interact with Fungal Glucosylceramides

Glycosyltransferases (GTs) (EC 2.4.x.y) catalyze the transfer of sugar moieties to a wide range of acceptor molecules, such as sugars, lipids, proteins, nucleic acids, antibiotics and other small molecules, including plant secondary metabolites. These enzymes can be classified into at least 92 families, of which family 1 glycosyltransferases (GT1), often referred to as UDP glycosyltransferases (UGTs), is the largest in the plant kingdom. To understand how UGTs expanded in both number and function during evolution of land plants, we screened genome sequences from six plants (Physcomitrella patens, Selaginella moellendorffii, Populus trichocarpa, Oryza sativa, Arabidopsis thaliana and Arabidopsis lyrata) for the presence of a conserved UGT protein domain. Phylogenetic analyses of the UGT genes revealed a significant expansion of UGTs, with lineage specificity and a higher duplication rate in vascular plants after the divergence of Physcomitrella. The UGTs from the six species fell into 24 orthologous groups that contained genes derived from the common ancestor of these six species. Some orthologous groups contained multiple UGT families with known functions, suggesting that UGTs discriminate compounds as substrates in a lineage-specific manner. Orthologous groups containing only a single UGT family tend to play a crucial role in plants, suggesting that such UGT families may have not expanded because of evolutionary constraints.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2011.04493.x

An evolutionary view of functional diversity in family 1 glycosyltransferases.

Specific lipid requirements of membrane proteins—a putative bottleneck in heterologous expression

Plant sphingolipids: structural diversity, biosynthesis, first genes and functions

http://www.jbc.org/content/274/19/13048.full.pdf

Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum.

The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis. Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis, S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are beta-glucosyl ceramides consisting of a saturated alpha-hydroxy or non-hydroxy fatty acid and a Delta4,8-diunsaturated, C9-methyl-branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol-beta-D-glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S. cerevisiae. We suggest P. pastoris and two plant pathogenic fungi to be selected for this approach.

Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress.

Annette Fahl

Papers

Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum.

Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress.