Arabidopsis thaliana non aqueous fractionation LC-MS/MS analysis of genotypes Ler (Landsberg erecta) and gin2-1 (glucose insensitive (hexokinase 1)) time series analysis under normal, chilling temperature and highlight+heat.

ABSTRACT: Plant cells are characterized by a high degree of compartmentalization and a diverse proteome and metabolome. Only a very limited number of studies has addressed combined subcellular proteomics and metabolomics which strongly limits biochemical and physiological interpretation of large-scale omics data. Our study presents a methodological combination of non-aqueous fractionation, shotgun proteomics, enzyme kinetics and metabolomics to reveal subcellular diurnal dynamics of plant metabolism. Subcellular marker protein sets were identified and enzymatically validated to resolve metabolism in a 4-compartment model comprising chloroplasts, cytosol, vacuole and mitochondria. These marker sets are now available for future studies which aim to monitor subcellular metabolome and proteome dynamics. Comparing subcellular dynamics in wild type plants and (hexokinase) HXK1-deficient gin2-1 mutants revealed a strong impact of HXK1 activity on metabolome dynamics in multiple compartments. Glucose accumulation in the cytosol of gin2-1 was accompanied by diminished vacuolar glucose levels. Subcellular dynamics of pyruvate, succinate and fumarate concentrations were significantly affected in gin2-1 which coincided with differential mitochondrial proteome dynamics. Lowered mitochondrial glycine and serine concentrations in gin2-1 together with reduced abundance of photorespiratory proteins indicated an effect of the gin2-1 mutation on photorespiratory capacity. Our findings highlight the necessity to resolve plant metabolism to a subcellular level in order to provide a causal relationship between metabolites, proteins and metabolic pathway regulation. --- In an attempt to draw a full picture of abiotic stress responses a complete set of time resolved, compartmentalized metabolomics and proteomics data under different stress conditions was generated. To investigate interactions of stress responses with primary metabolism, the gin2-1 mutant, was compared to its wildtype Landsberg erecta (Ler) under high irradiance and at low temperature. Chlorophyll fluorescence parameters proved the well-known sensitivity of the gin2-1 mutant to high irradiance. Dynamics of subcellular metabolite redistribution upon stress were delayed in gin2-1, and this correlated with massive reduction in proteins belonging to the ATP producing electron transport chain. Evidence for compounds specifically protecting compartments, e.g. maltose in plastids and myo-inositol in mitochondria, was obtained in Ler, but gin2-1 failed to respond or responded only slowly to high irradiance, while no delay was obtained in the cold. Whole-cell concentrations of the amino acids glycine, and serine, provided strong evidence for an important role of the photorespiratory pathway during stress exposure and may contribute to the slow growth of the gin2-1 mutant under high irradiance.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Photosynthetic Cell, Leaf

SUBMITTER: Lisa Fürtauer  

LAB HEAD: Thomas Nägele

PROVIDER: PXD013646 | Pride | 2019-08-05

REPOSITORIES: Pride