Food system actor perspectives on future-proofing European food systems through plant breeding.

Crop improvement is a key innovation area in the pursuit of sustainable food systems. However, realising its potential requires integration of the needs and priorities of all agri-food chain stakeholders. In this study, we provide a multi-stakeholder perspective on the role of crop improvement in future-proofing the European food system. We engaged agri-business, farm- and consumer-level stakeholders, and plant scientists through an online survey and focus groups. Four of each group's top five priorities were shared and related to environmental sustainability goals (water, nitrogen and phosphorus efficiency, and heat stress). Consensus was identified on issues including considering existing alternatives to plant breeding (e.g. management strategies), minimising trade-offs, and addressing geographical variation in needs. We conducted a rapid evidence synthesis on the impacts of priority crop improvement options, highlighting the urgent need for further research examining downstream sustainability impacts to identify concrete targets for plant breeding innovation as a food systems solution.

A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2-diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products.

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1967) using PCR. After cloning and expression in E. coli, SDS-PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylgl ycerol, 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl]-1,2-diacylglycerol and 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[O-beta-D-glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as 14C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-beta-D-glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyl diacylglycerol or triglucosyl diacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in beta (1-->6) linkage to the product of the preceding reaction.

Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols.

A processive diacylglycerol glucosyltransferase has recently been identified from Bacillus subtilis [Jorasch, P., Wolter, F.P., Zähringer, U., and Heinz, E. (1998) Mol. Microbiol. 29, 419-430]. Now we report the cloning and characterization of two other genes coding for diacylglycerol glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana; only the S. aureus enzyme shows processivity similar to the B. subtilis enzyme. Both glycosyltransferases characterized in this work show unexpected acceptor specificities. We describe the isolation of the ugt106B1 gene (GenBank accession number Y14370) from the genomic DNA of S. aureus and the ugt81A1 cDNA (GenBank accession number AL031004) from A. thaliana by PCR. After cloning and expression of S. aureus Ugt106B1 in Escherichia coli, SDS/PAGE of total cell extracts showed strong expression of a protein having the predicted size of 44 kDa. Thin-layer chromatographic analysis of the lipids extracted from the transformed E. coli cells revealed several new glycolipids and phosphoglycolipids not present in the controls. These lipids were purified from lipid extracts of E. coli cells expressing the S. aureus gene and identified by NMR and mass spectrometry as 1, 2-diacyl-3-[O-beta-D-glucopyranosyl]-sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyrano-+ ++syl] -sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-( 1-->6)-O-beta-D-glucopyranosyl]-sn-glycerol, sn-3'-[O-beta-D-glucopyranosyl]-phosphatidylglycerol and sn-3'-[O-(6"'-O-acyl)-beta-D-glucopyranosyl-(1"'-->6")-O-beta-D-gluco pyranosyl]-sn-2'-acyl-phospha-tidylglycerol. A 1, 2-diacyl-3-[O-beta-D-galactopyranosyl]-sn-glycerol was isolated from extracts of E. coli cells expressing the ugt81A1 cDNA from A. thaliana. The enzymatic activities expected to catalyze the synthesis of these compounds were confirmed by in vitro assays with radioactive substrates. Experiments with several of the above described glycolipids as 14C-labeled sugar acceptors and unlabeled UDP-glucose as glucose donor, suggest that the ugt106B1 gene codes for a processive UDP-glucose:1, 2-diacylglycerol-3-beta-D-glucosyltransferase, whereas ugt81A1 codes for a nonprocessive diacylglycerol galactosyltransferase. As shown in additional assays with different lipophilic acceptors, both enzymes use diacylglycerol and ceramide, but Ugt106B1 also accepts glucosyl ceramide as well as cholesterol and cholesterol glucoside as sugar acceptors.