Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum

Abstract

One of the remarkable adaptive features of the halophyte Mesembryanthemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF) and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples identified 352 significantly differing metabolites (268 after correction for FDR). Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na(+) and Cl(-) ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggests large alterations in M. crystallinum epidermal bladder cells.

Keywords: Crassulacean acid metabolism; Mesembryanthemum crystallinum; epidermal bladder cells; halophyte; metabolomics; salinity; salt-tolerance; trichomes.

Figure 1

Top 10 down- and up-regulated metabolites in EBC extracts from salt-treated plants compared to control plants. Metabolites which were significantly down-regulated (A) and upregulated (B), as determined by a two-sample t-Test. The probability or significance level for the test statics (i.e., p-values) were adjusted for the multiple hypotheses tested to allow for a maximum 5% probability (q = 0.05) of false positives. Box and whiskers plots showing natural logarithm transformed values are used to visualize the group means and standard deviations.

Figure 2

Heatmap displaying similarities between biological samples organized by HCA. Multivariate sample similarities, displayed as a heatmap in which columns represent samples and rows metabolites. The heatmap visualization is used to encode individual measurements for each sample (autoscaled) as colors (red, relative increase; blue, relative decrease), as indicated by the color bar at top right of figure, all of which are organized using HCA. Metabolites are ordered on the heatmap in the order they appear in the list in Supplementary Table 2.

Figure 3

Metabolic changes associated with salinity. Principle component analysis (PCA) plot (x – first component, y – second component) of control (pink) and salt-treated (blue) EBC extract of GC/MS based metabolites. Symbol numbers represent the different biological replicates (1–7, control replicates; 8–14, salt-treated replicates). Colored ellipses (based on the Hotelling's T2 95% confidence interval) are used to display experimental group scores.

Figure 4

Classification of the significantly salt-regulated EBC metabolites based on their known function and chemical attributes. (A) A large proportion of the identified metabolites could have roles as compatible solutes in the EBC, including those classified as amino acids, sugars, organic acids, and polyols. (B) The pie graph shows the relative composition of the identified metabolites. (C) Representation of proline biosynthesis pathway showing the location in the pathway of identified metabolites and indicating the enzymes in the pathway which have been shown to be up-regulated in RNAseq analysis of EBC (Oh et al., 2015).