Metabolic profiling of Arabidopsis thaliana epidermal cells

Abstract

Metabolic phenotyping at cellular resolution may be considered one of the challenges in current plant physiology. A method is described which enables the cell type-specific metabolic analysis of epidermal cell types in Arabidopsis thaliana pavement, basal, and trichome cells. To achieve the required high spatial resolution, single cell sampling using microcapillaries was combined with routine gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) based metabolite profiling. The identification and relative quantification of 117 mostly primary metabolites has been demonstrated. The majority, namely 90 compounds, were accessible without analytical background correction. Analyses were performed using cell type-specific pools of 200 microsampled individual cells. Moreover, among these identified metabolites, 38 exhibited differential pool sizes in trichomes, basal or pavement cells. The application of an independent component analysis confirmed the cell type-specific metabolic phenotypes. Significant pool size changes between individual cells were detectable within several classes of metabolites, namely amino acids, fatty acids and alcohols, alkanes, lipids, N-compounds, organic acids and polyhydroxy acids, polyols, sugars, sugar conjugates and phenylpropanoids. It is demonstrated here that the combination of microsampling and GC-MS based metabolite profiling provides a method to investigate the cellular metabolism of fully differentiated plant cell types in vivo.

Fig. 1.

Electron micrograph of epidermal cell types. Shown are all the leaf epidermal cell types analysed in this study, namely pavement cells (PC), trichomes (T), and its supporting basal cells (BC).

Fig. 2.

(A, B) Arabidopsis thaliana trichomes sampled by leaf hair depilation (LHD). To confirm accuracy of the sampling and intactness of trichomes they were analysed under a binocular microscope. (A) Trichomes isolated by LHD (scale bar 250 μm) and (B) depicts an isolated trichome stained with DAPI. The trichome nucleus is visible as the bright spot in the centre of the image (scale bar 100 μm).

Fig. 3.

Heat map visualisation of relative differences in metabolite pools between different epidermal cells. Data were maximum normalized (cell types with the highest pool size were set to 100%). Each cell type is visualized in a single column (average of n=5–6) and each metabolite is represented by a single row. Red indicates high abundance, whereas low relative metabolites are deep blue (cf. scale above heat map). Samples and metabolites were submitted to the hierarchical clustering using Euclidian distance (metabolite clusters with similar cellular profiles are indicated by triangle shading). Trichomes shaved by LHD [TRI (sh)] and micro sampled trichomes (TRI), basal cells (BC), and pavement cells (PC).

Fig. 4.

(A, B) Independent component analysis. Independent component one (IC1) is plotted against independent component two (IC2). (A) The complete dataset is visualized; whereas (B) shows only the samples obtained by microsampling. The metabolite profiling dataset was a fingerprint of all observed mass fragments, which was normalized and transformed to represent log₁₀ of response ratios prior to independent component analysis (cf. methods section).

Fig. 5.

Comparative visualization of central metabolism as determined by metabolic profiling of trichomes, basal and pavement cells collected by micro sampling. The pathway connectivity and bar diagram visualisation of relative metabolite pool sizes (cf. Fig. 3) was performed using the software package VANTED (Junker et al., 2006). Empty squares indicate relevant central metabolites which were not detectable by GC-MS based profiling of single cell types. From left to right: trichomes, red; basal cells, blue; pavement cells, green.