NMR Spectral Analysis of Ascorbate-Deficient Mutants of Arabidopsis Thaliana

Ascorbate is an important antioxidant, involved in the scavenging of reactive oxygen species (ROS) such as hydrogen peroxide hydrogen. It is also a cofactor for 2-oxoglutarate-dependent dioxygenases, which are involved in the biosynthesis of a number of metabolites. In the current study, Arabidopsis thaliana ascorbate-deficient mutants (vtc1, vtc2, vtc3, and vtc4) were used to investigate the effect of ascorbate deficiency on the metabolic changes. NMR was used as an analytical technique for the metabolite profiling of Arabidopsis thaliana vtc mutants. A total of 45 NMR spectra representing wild type and four mutants of two different growth stages (rosette and flowering) were collected and spectral processed for exploratory analysis. Analysis of variance (ANOVA) and multivariate data analysis (hierarchical clustering) were used to find the differences between different strains and metabolite clustering patterns. Combinations of different parameters were used to find the best method for the analysis of NMR-based metabolites data. The method was developed which allowed making multiple comparison such as two growth stages and five strains. It was found the vtc mutants grouped separately from WT which contained the low ascorbate showing ascorbate deficiency might have some effect on the metabolic level. Two interesting resonances 1.32 and 1.33ppm were separated which were negatively correlated with the ascorbate levels in the vtc mutants. These resonances (1.32 and 1.33) are putatively identified as threonine and lactate. Further investigations are needed to investigate the nature of relationship between ascorbate and threonine and lactate.