Conference link: https://atspb2018.uni-graz.at/de/

Abstract:

Quantifying the Subcellular Plant Metabolome

Lisa Fürtauer ⁽¹⁾, Wolfram Weckwerth ^(1,2) and Thomas Nägele^(1,2,3)

Compartmentation is a key feature of eukaryotic cells though biological research is frequently limited by methods allowing for a subcellular resolution of the metabolome. It is discussed and accepted since decades that cellular biochemical research and metabolic regulation can only be resolved if subcellular compartments are taken into account. Technical challenges have frequently limited data reproducibility and sample throughput significantly affecting statistical robustness of subcellular metabolite data. Here, we present a method which is based on the non-aqueous fractionation technique enabling the assignment of metabolites to their subcellular compartment, e.g. vacuole, cytosol or plastid [1]. The method is applicable to resolve subcellular metabolite dynamics in a precise and statistically robust manner. Adapted to a benchtop standard equipment the method is based on the separation of cellular fractions via density gradients consisting of organic solvents. Determination of relative distributions of compartment specific marker enzymes together with metabolite profiles over the density gradient facilitates estimations of compartment specific metabolite concentrations by correlation.

The method was developed, tested and validated in a cold acclimation experiment of three natural accessions of Arabidopsis thaliana. Accessions were chosen due to their differential cold acclimation output to unravel different strategies during the metabolic acclimation process. Cold sensitivity was found to particularly correlate with shuffling of amino acids between subcellular compartments. In contrast, cold tolerance was associated with subcellular accumulation of primary metabolites and, hence, we suggest that the intensity of subcellular rearrangements of primary metabolites is negatively correlated with freezing tolerance.

Finally, we conclude that subcellular metabolome analysis is essential to unambiguously unravel different regulatory strategies being involved in plant-environment interactions.

[1] Fürtauer L., Weckwerth W., Nägele T., 2016. A Benchtop Fractionation Procedure for Subcellular Analysis of the Plant Metabolome, Frontiers in Plant Science, 7:1912.