Luisa Elias

The Genetic Map of Artemisia annua L. Identifies Loci Affecting Yield of the Antimalarial Drug Artemisinin

The Art of Artemisia As the malaria parasite, which is transmitted through mosquito vectors, develops resistance, previously useful control mechanisms are beginning to fail. Combination therapies based on the plant product artemisinin are a promising alternative. Graham et al. (p. 328; see the Perspective by Milhous and Weina) have now developed a genetic map of the plant Artemisia annua from which artemisinin is derived. The results lay the foundation for improving agricultural productivity of this natural product, which is becoming increasingly important in the fight against malaria. A linkage map for an important medicinal crop plant points to breeding targets for enhancing drug production. Artemisinin is a plant natural product produced by Artemisia annua and the active ingredient in the most effective treatment for malaria. Efforts to eradicate malaria are increasing demand for an affordable, high-quality, robust supply of artemisinin. We performed deep sequencing on the transcriptome of A. annua to identify genes and markers for fast-track breeding. Extensive genetic variation enabled us to build a detailed genetic map with nine linkage groups. Replicated field trials resulted in a quantitative trait loci (QTL) map that accounts for a significant amount of the variation in key traits controlling artemisinin yield. Enrichment for positive QTLs in parents of new high-yielding hybrids confirms that the knowledge and tools to convert A. annua into a robust crop are now available.

Overexpression of the UGT73C6 alters brassinosteroid glucoside formation in Arabidopsis thaliana

BackgroundBrassinosteroids (BRs) are signaling molecules that play essential roles in the spatial regulation of plant growth and development. In contrast to other plant hormones BRs act locally, close to the sites of their synthesis, and thus homeostatic mechanisms must operate at the cellular level to equilibrate BR concentrations. Whilst it is recognized that levels of bioactive BRs are likely adjusted by controlling the relative rates of biosynthesis and by catabolism, few factors, which participate in these regulatory events, have as yet been identified. Previously we have shown that the UDP-glycosyltransferase UGT73C5 of Arabidopsis thaliana catalyzes 23-O-glucosylation of BRs and that glucosylation renders BRs inactive. This study identifies the closest homologue of UGT73C5, UGT73C6, as an enzyme that is also able to glucosylate BRs in planta.ResultsIn a candidate gene approach, in which homologues of UGT73C5 were screened for their potential to induce BR deficiency when over-expressed in plants, UGT73C6 was identified as an enzyme that can glucosylate the BRs CS and BL at their 23-O-positions in planta. GUS reporter analysis indicates that UGT73C6 shows over-lapping, but also distinct expression patterns with UGT73C5 and YFP reporter data suggests that at the cellular level, both UGTs localize to the cytoplasm and to the nucleus. A liquid chromatography high-resolution mass spectrometry method for BR metabolite analysis was developed and applied to determine the kinetics of formation and the catabolic fate of BR-23-O-glucosides in wild type and UGT73C5 and UGT73C6 over-expression lines. This approach identified novel BR catabolites, which are considered to be BR-malonylglucosides, and provided first evidence indicating that glucosylation protects BRs from cellular removal. The physiological significance of BR glucosylation, and the possible role of UGT73C6 as a regulatory factor in this process are discussed in light of the results presented.ConclusionThe present study generates essential knowledge and molecular and biochemical tools, that will allow for the verification of a potential physiological role of UGT73C6 in BR glucosylation and will facilitate the investigation of the functional significance of BR glucoside formation in plants.

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.

Suppression of microRNA accumulation via RNA interference in Arabidopsis thaliana

MicroRNAs (miRNAs) are key regulatory molecules in plants. These small RNAs are processed in the nucleus from longer precursor transcripts that form distinct secondary structures. The miRNAs target specific messenger RNAs (mRNAs) and consequently down-regulate gene expression. The importance of these regulatory molecules is wide-ranging, however, few loss-of-function mutants have been identified in miRNA genes and understanding the biology of miRNA-target pairings has largely depended upon creating alterations in the sequences of the target genes. Here we demonstrate using Arabidopsis thaliana, that it is possible to use RNA interference (RNAi) to suppress accumulation of miRNAs. Significantly reduced accumulation of miR163 and miR171a was achieved using hairpin RNAi constructs that were designed to target both the primary miRNA transcripts and their promoters. The presence of DNA methylation in the targeted promoter regions suggests that inhibition of transcription of the miRNA precursors is responsible. Reduction of miRNA accumulation resulted in an increase in accumulation of the mRNA targets of these miRNAs. This demonstrates that knock-down of miRNA expression can be achieved, thereby providing a straightforward approach for disrupting miRNA-target pairings and studying miRNA functions.

An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion

Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.LPMOs catalyze the oxidative breakdown of polysaccharides, thereby facilitating biomass degradation. By analyzing the digestive proteome of firebrats, the authors here identify a yet uncharacterized LPMO family and provide phylogenetic, structural and biochemical insights into its origin and functions.

Characterization of the cellulolytic secretome of Trichoderma harzianum during growth on sugarcane bagasse and analysis of the activity boosting effects of swollenin

This study demonstrates the production of an active enzyme cocktail produced by growing Trichoderma harzianum on sugarcane bagasse. The component enzymes were identified by LCMS‐MS. Glycosyl hydrolases were the most abundant class of proteins, representing 67% of total secreted protein. Other carbohydrate active enzymes involved in cell wall deconstruction included lytic polysaccharide mono‐oxygenases (AA9), carbohydrate‐binding modules, carbohydrate esterases and swollenin, all present at levels of 1%. In total, proteases and lipases represented 5 and 1% of the total secretome, respectively, with the rest of the secretome being made up of proteins of unknown or putative function. This enzyme cocktail was efficient in catalysing the hydrolysis of sugarcane bagasse cellulolignin to fermentable sugars for potential use in ethanol production. Apart from mapping the secretome of T. harzianum, which is a very important tool to understand the catalytic performance of enzyme cocktails, the gene coding for T. harzianum swollenin was expressed in Aspergillus niger. This novel aspect in this work, allowed increasing the swollenin concentration by 95 fold. This is the first report about the heterologous expression of swollenin from T. harzianum, and the findings are of interest in enriching enzyme cocktail with this important accessory protein which takes part in the cellulose amorphogenesis. Despite lacking detectable glycoside activity, the addition of swollenin of T. harzianum increased by two‐fold the hydrolysis efficiency of a commercial cellulase cocktail.

A novel cellulase for biofuels production: structure of a marine GH7 cellobiohydrolase

There is strong pressure to diversify feedstocks used for biofuel generation, to avoid competition with food crops. In particular, there is increasing emphasis on the utilisation of woody (lignocellulosic) materials that are recalcitrant to degradation. Prospecting for enzymes capable of overcoming this recalcitrance has focused on the relatively few types of microorganisms and animals that have wood-degrading capability. Of particular interest are the GH7-family enzymes that convert cellulose polymers to cellobiose units, the central step in the production of glucose for downstream fermentation to bioethanol. Currently, fungal GH7 enzymes represent the main hydrolytic component in the majority of industrial cocktails, however, their high cost represents a significant barrier to the commercial viability of large-scale biofuel production. We are therefore exploring the rich resource of endogenous lignocellulose-degrading enzymes in wood boring crustaceans that achieve breakdown without the help of microbial mutualists. Unlike animals such as termites that employ a complex community of microbial flora to produce digestive enzymes, the marine crustacean Limnoria quadripunctata has a sterile gut and produces all the necessary enzymatic machinery to efficiently digest these difficult substrates. A successful collaboration between Portsmouth and York Universities, NREL in Golden, USA and Novozymes in Denmark has resulted in the detailed characterisation of the first animal cellobiohydrolase, LqCel7B. Following a transcriptomic analysis [1], we selected a GH7 family enzyme that was highly expressed in the hepatopancreas digestive gland. Biophysical analysis revealed a stable monomeric protein and extensive crystallisation trials produced well diffracting crystals. Four structures have been solved to date, one apo form and three with various bound ligands occupying the active site tunnel. A complex with the inhibitor thiocellobiose diffracted to 1.1 Å resolution. These structures provided the basis for structural comparisons and molecular dynamic simulations and have revealed a host of novel features with industrial potential [2].