Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development

Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.     

An inter-laboratory comparison demonstrates that [1H]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection

In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [¹H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [¹H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories.     

Assessment of 1H NMR spectroscopy and multivariate analysis as a technique for metabolite fingerprinting of Arabidopsis thaliana

An approach to metabolite fingerprinting of crude plant extracts that utilizes 1H nuclear magnetic resonance (NMR) spectroscopy and multivariate statistics has been tested. Using ecotypes of Arabidopsis thaliana as experimental material, a method has been developed for the rapid analysis of unfractionated polar plant extracts, enabling the creation of reproducible metabolite fingerprints. These fingerprints could be readily stored and compared by a variety of chemometric methods. Comparison by principal component analysis using SIMCA-P allowed the generation of residual NMR spectra of the compounds that contributed significantly to the differences between samples. From these plots, conclusions were drawn with respect to the identity and relative levels of metabolites differing between samples.     

Thiazolidinediones block fatty acid release by inducing glyceroneogenesis in fat cells

Thiazolidinediones are used to treat type 2 diabetes mellitus because they decrease plasma glucose, insulin, triglyceride, and fatty acid levels. Thiazolidinediones are agonists for peroxisome proliferator-activated receptor gamma, a nuclear receptor that is highly expressed in fat tissue. We identify glyceroneogenesis as a target of thiazolidinediones in cultured adipocytes and fat tissues of Wistar rats. The activation of glyceroneogenesis by thiazolidinediones occurs mainly in visceral fat, the same fat depot that is specifically implicated in the progression of obesity to type 2 diabetes. The increase in glyceroneogenesis is a result of the induction of its key enzyme, phosphoenolpyruvate carboxykinase, whose gene expression is peroxisome proliferator-activated receptor gamma-dependent in adipocytes. The main role of this metabolic pathway is to allow the re-esterification of fatty acids via a futile cycle in adipocytes, thus lowering fatty acid release into the plasma. The importance of such a fatty acid re-esterification process in the control of lipid homeostasis is highlighted by the existence of a second thiazolidinedione-induced pathway involving glycerol kinase. We show that glyceroneogenesis accounts for at least 75% of the whole thiazolidinedione effect. Because elevated plasma fatty acids promote insulin resistance, these results suggest that the glyceroneogenesis-dependent fatty acid-lowering effect of thiazolidinediones could be an essential aspect of the antidiabetic action of these drugs.