We furthermore harness wide genetic diversity in order to understand the genetics of metabolite accumulation and are currently developing highly sensitive analytical tools to determine metabolic fluxes. Various species of tomato, maize and Arabidopsis thaliana are the primary model systems used.
Enzyme-enzyme assemblies metabolons (substrate channeling) and their roles in plant metabolism
Despite the fact that a huge number of metabolons have been claimed to exist in plants most of these claims are erroneous since only a handful of these have been proven to channel metabolites. To our knowledge, in plants only the pathways of glycolysis, the TCA cycle, the upper pathway of phenylpropanoid biosynthesis and the cyanogenic glucoside biosynthetic pathway meet these criteria. Using the protein-protein several interaction methods and the isotope dilution assay, both the glycolysis (Zhang et al., 2020) and TCA cycle (Zhang et al., 2017) metabolons were proved as essential metabolites flux regulation mechanism. In addition, plants produce various secondary metabolites which play an important role in plant defense and human health as medicines, flavorings, pigments, we are interested in investigating the secondary metabolism pathway metabolon which is also recognized as a mechanism to control the production of specialized metabolites.
Manipulation of the dominant fluxes of carbon metabolism
In potato (Solanum tuberosum), we have taken multiple strategies to adjust carbon flux into starch. Interestingly, in all cases, glycolysis increases while starch synthesis decreases in these transgenic plants. We are currently also engaged in analysing the sucrose-starch transition in the tomato (Solanum lycopersicum). Where our interests include both sucrose transport and its use in sink organs.Driven by the unexpected metabolic shift towards respiration in plants exhibiting enhanced sucrolysis, we initiated a project concerned with understanding better the contribution of the TCA cycle enzymes to metabolic regulation (also in tomato). Intriguingly, these studies revealed very tight links between mitochondrial and photosynthetic metabolism which we are investigating further.
Analytical and experimental tool development
These intriguing results compelled us to develop a wide range of analytical tools to better study the intricacies of cellular biosynthetic machinery. We have perfected non-aqueous subcellular fractionation techniques in order to separate chloroplasts and vacuoles from cytosol. We are operating a metabolite profiling system, using GC-MS, which allows us to distinguish among large numbers of metabolites within each of these samples (subcellular fractions or tissue samples). In excess of 300 compounds can be profiled in this way > 100 of these compounds having known chemical structures. A further experimental development that we are exploring is the use of chemically-inducible promoters to drive transgene expression in a controlled manner in order to study perturbations of metabolism on a temporal basis. In recent years we have additionally established an RT-PCR platform for tomato transcription factors and sensitive methods for following the metabolism of stable isotope labeled substrate and an LC-MS based platform for the analysis of plant phenylpropanoids and targeted hormone analysis.
Metabolic profiling in Solanaceous species
Metabolic profiling using gas chromatography mass spectrometry (GC-MS) technologies represents both a rapid and robust methodology for multiparallel metabolite analysis and a largely untapped potential in the field of functional genomics. We are currently in the process of using this technique to profile the primary metabolism of genetically and environmentally diverse Solanaceous plant systems (both potato and tomato). Use of this technique in tandem with bioinformatic tools for data mining allows comprehensive analysis of metabolic phenotypes and the identification of metabolic phenocopies (i.e. two differently manipulated systems that closely resemble one another on the basis of their metabolic complements). Moreover, the fact that this method yields information on many metabolites within a single extract facilitates the application of an extensive correlation analysis between the various metabolites and thus allows many conclusions to be drawn concerning metabolic interactions within these systems.
We have undertaken a large project in collaboration with Prof. Dani Zamir (Hebrew University of Jerusalem, Rehovot) in which we have profiled the metabolic complement of a series of over 80 Solanum lycopersicum introgression lines each harbouring defined and distinct substitutions from Solanum pennelli that cover the entire genome. Profiling of these lines will include MS analysis alongside analysis of polymeric compounds such as starch, protein, and cell wall components. Recently, we have expanded these at the compound, tissue and species level to give a much higher resolution of the genetic control of metabolism and how pathways and even plant organs compete for substrate under a range of environmental conditions.
Also in tomato fruit we have started to unravel functional network associated with transcription factors and have projects running on the metabolic engineering and metabolomics assisted breeding of phenolics (which are well known it have health benefits for humans as well as to aid in stress responses within the plants themselves). In Arabidopsis we are following a similar approach with regard to phenolics – paying particular attention to the isolation, identification and functional characterization of novel phenylpropanoids.
Other group activities
The Group of Alisdair Fernie is also, via funding from the EU Horizon2020 TEAMING PROJECT PlantaSyst (SGA-CSA No 664621 and No 739582 under FPA No. 664620) currently involved in aiding the establishment of the Centre of Plant Systems Biology and Biotechnology in Plovdiv, Bulgaria.