Developing Metabolomics-Type Techniques for the High-Throughput Analysis of Carbohydrate Polymers to Improve Our Understanding of Fructan Biosynthesis in Grasses

Abstract

Non structural polymeric water soluble carbohydrates such as fructooligosaccharides (fructans) accumulate in many important crops and forage plants. Fructans in Lolium perenne (perennial ryegrass) are complex mixtures of four fructan classes belonging to the (neo)inulin and (neo)levan series with degree of polymerisation (DP) > 100. Sufficient understanding of fructan biosynthesis in L. perenne is hampered by the lack of rapid, high-throughput, and quantitative methods for the analysis of the full fructan complement as well as by the unavailability of fructan standards. A major goal of this project was therefore the development of novel methods overcoming current limitations. Several chromatographic systems were tested and chromatography on porous graphitised carbon columns was shown to be superior for the separation of the different isomeric fructan series and a wide range of DP. To detect the separated fructans, methods using electrospray ionisation mass spectrometry in profile mode were developed for the analysis of the full fructan complement in L. perenne extracts. While the mass range of ion trap MS instruments is limited, it was shown that monitoring selected multiply charged ions can be used for the detection and quantification of high mass fructan isomers (DP>20). Linear ion trap MS instruments (LTQ) as used in this project have a low mass resolution and are therefore limited to the analysis of fructans with a DP up to 49. Using high resolution orbitrap MS (Exactive) greatly improved the signal-to-noise ratio and allowed the detection of fructans up to DP=100. These methods can be used with standard high pressure liquid chromatography pumps and allow separation and quantification of isomeric fructan oligomers ranging from degree of polymerisation (DP) 3 to 117 and were used to show that recently developed L. perenne high sugar grass cultivars accumulate fructans with much higher DP compared to cultivars with normal sugar levels. In vitro incubations of recombinant L. perenne fructosyltransferases (Lp6G-FFT/ 1-FFT, Lp6-SFT) expressed in Pichia pastoris with labelled 13C sucrose allowed the production of labelled fructans belonging to the four major classes of fructans found in ryegrass. The activities of the enzymes were confirmed and reaction products up to DP6 were synthesised that were also detected in ryegrass extracts. However, examination of both the total ion chromatogram (TIC) and the extracted ion chromatograms (XIC) also showed that additional oligosaccharide peaks are present in planta, possibly indicating additional enzyme activities. LC-MSn analysis of 13C labelled fructan oligomers produced by recombinant L. perenne fructosyltransferases showed that specific MSn fragmentation patterns are associated with β 1-2 (inulins and neoinulins) and β 2-6 (levans and neolevans) fructans. This method allows to account for the observed fragmentation patterns in terms of preferential cleavage of the glycosidic bond between O- and fructose C2 in both inulins and levans, and to differentiate reducing-end from non-reducing end cross ring cleavages in levans. We propose that higher order MS fragmentation patterns can be used to distinguish between the four major classes of fructans present in ryegrass, i.e. inulins, neoinulins, levans, and neolevans without the need for authentic standards. This project has developed LC-ESI-MSn methods and demonstrated these to be the most promising techniques for accurate profiling and quantitation of the full complement of ryegrass fructans providing considerable technical advantages over previously established techniques. Coupled with UHPLC instrumentation allows high-throughput analysis of large sample sets required by large scale biological experiments, e.g. population studies with genetical metabolomics. The unambiguous identification of the different fructan isomers based on their specific MS fragmentation patterns now paves the way for the routine analysis of fructan distribution in a wide range of plants.