A sister group contrast using untargeted global metabolomic analysis delineates the biochemical regulation underlying desiccation tolerance in Sporobolus stapfianus

Abstract

Understanding how plants tolerate dehydration is a prerequisite for developing novel strategies for improving drought tolerance. The desiccation-tolerant (DT) Sporobolus stapfianus and the desiccation-sensitive (DS) Sporobolus pyramidalis formed a sister group contrast to reveal adaptive metabolic responses to dehydration using untargeted global metabolomic analysis. Young leaves from both grasses at full hydration or at 60% relative water content (RWC) and from S. stapfianus at lower RWCs were analyzed using liquid and gas chromatography linked to mass spectrometry or tandem mass spectrometry. Comparison of the two species in the fully hydrated state revealed intrinsic differences between the two metabolomes. S. stapfianus had higher concentrations of osmolytes, lower concentrations of metabolites associated with energy metabolism, and higher concentrations of nitrogen metabolites, suggesting that it is primed metabolically for dehydration stress. Further reduction of the leaf RWC to 60% instigated a metabolic shift in S. stapfianus toward the production of protective compounds, whereas S. pyramidalis responded differently. The metabolomes of S. stapfianus leaves below 40% RWC were strongly directed toward antioxidant production, nitrogen remobilization, ammonia detoxification, and soluble sugar production. Collectively, the metabolic profiles obtained uncovered a cascade of biochemical regulation strategies critical to the survival of S. stapfianus under desiccation.

Figure 1.

Drying Curve and the Relationship of RWC to Water Content on a Gram Dry Weight Basis for Both S. stapfianus (s) and S. pyramidalis (•). Each point in the drying curve (A) is an average of a minimum of four samples from individual plants; the vertical lines through each point represents the standard deviation from the mean. Each point in the RWC to water content plot (B) is an individual sample taken from individual plants from multiple drying experiments (solid line, S. pyramidalis; dashed line, S. stapfianus).

Figure 2.

Statistical Comparison Design. Statistical comparisons of samples from each species, indicated by arrows, were conducted between hydrated (Hyd), 60, 50, 40, 20, and ~5% RWC (Dry) within S. stapfianus, Hyd and 60% RWC within S. pyramidalis, and Hyd and 60% RWC between species. [See online article for color version of this figure.]

Figure 3.

Global Metabolite Comparison at 100% Hydrated and at 60% RWC between S. stapfianus and S. pyramidalis. PLS-DA model is constructed from 105 variables (i.e., metabolites) generating a three-PLS-DA component model with R2 = 65.2, and Q2 = 61.0. S. pyramidalis: squares, 100% hydrated; crosses, 60% RWC. S. stapfianus: asterisks, 100% hydrated; upside down triangles, 60% RWC. [See online article for color version of this figure.]

Figure 4.

Differences of the Metabolites in Glycolysis/TCA Cycle and Amino Acids between S. stapfianus and S. pyramidalis. (A) Amino acid biosynthetic pathway and glycolysis/TCA cycle. The metabolites in red indicate higher levels in S. stapfianus. The metabolites in blue indicate lower levels in S. stapfianus. The metabolites in black indicate that there were no significant differences between S. stapfianus and S. pyramidalis. The metabolites in gray indicate that they are below detection level (not detected). (B) Heat map showing the ratio of the metabolite levels between S. stapfianus and S. pyramidalis and their statistical significance of the differences. Cells shaded with red indicate higher levels in S. stapfianus with P < 0.05. Cells shaded with green indicate lower levels in S. stapfianus with P < 0.05. Cells not shaded indicate that the difference between S. stapfianus and S. pyramidalis are not statistically significant (P > 0.05). The number in each cell indicates the fold change between S. stapfianus and S. pyramidalis.

Figure 5.

The Relative Amounts of Asn, Gln, and Allantoin between S. pyramidalis and S. stapfianus under Fully Hydrated Condition by Box Plots. The box represents the middle 50% of the distribution and upper and lower whiskers represent the entire spread of the data. The hyphen refers to the median. The outlier determined by the statistical program R, if any, is represented by a circle. The y axis is the median scaled value (relative level). The P values for all comparisons are referenced in the Supplemental Data online. P, S. pyramidalis; S, S. stapfianus.

Figure 6.

The Relative Amounts of Several Osmolytes between S. pyramidalis and S. stapfianus under Fully Hydrated Conditions by Box Plots. The P values for all comparisons are referenced in the Supplemental Data online. P, S. pyramidalis; S, S. stapfianus.

Figure 7.

Heat Map of Metabolic Responses of S. stapfianus and S. pyramidalis to the Dehydration Stress Reducing RWC from Fully Hydrated to 60%. Cells shaded with red indicate higher levels in 60% RWC conditions with P < 0.05. Cells shaded with green indicate lower levels in 60% RWC conditions with P < 0.05. Cells not shaded indicate that the difference between 60% and fully hydrated are not statistically significant (P > 0.05). The number in each cell indicates the fold change between 60% and fully hydrated.

Figure 8.

Elevation of Glutathione Biosynthesis in S. stapfianus in Response to Desiccation. The enzymes γ-glutamylcysteine synthetase (GCS) and glutathione synthetase (GS) are shared between glutathione biosynthesis and ophthalmate biosynthesis. The metabolites in red italics indicate higher levels during the dehydration process. The relative levels of the significantly altered metabolites were displayed using box plots. The P values for all comparisons are referenced in the Supplemental Data online. The x axis for each plot is RWC as a percentage. H, fully hydrated sample. [See online article for color version of this figure.]

Figure 9.

The Relative Amounts of γ-Glutamyl Amino Acids in Response to Severe Desiccation in T. Ruralis and Selaginella Lepidophylla By Box Plots. The P-values for all comparisons are referenced in the Supplemental Data online. The x axis for each plot is RWC as a percentage. HYD, fully hydrated sample; DRY, dried sample.

Figure 10.

The Relative Amounts of Tocopherols in Response to Severe Desiccation in S. stapfianus by Box Plots. The P values for all comparisons are referenced in the Supplemental Data online. The x axis for each plot is RWC as a percentage. H, fully hydrated sample.

Figure 11.

The Relative Amounts of Lysolipids in Response to Severe Desiccation in S. stapfianus by Box Plots. The P values for all comparisons are referenced in the Supplemental Data online. The x axis for each plot is RWC as a percentage. H, fully hydrated sample; GPC, glycerophosphocholine; GPI, glycerophosphoinositol.

Figure 12.

Heat Map of the Amino Acid Changes in S. stapfianus in Response to the Dehydration. There were five dehydration stages to reduce RWC to 60, 50, 40, and 20% and dry. Cells shaded with red indicate higher levels in comparison to 100% RWC conditions, with P < 0.05. Cells shaded with green indicate lower levels in comparison to 100% RWC conditions, with P < 0.05. Cells not shaded indicate that the differences were not statistically significant (P > 0.05). The number in each cell indicates the fold change.

Figure 13.

The Relative Amounts of Nitrogen Storage Metabolites in Response to Severe Desiccation in S. stapfianus by Box Plots. The P values for all comparisons are referenced in the Supplemental Data online. The x axis for each plot is RWC as a percentage. H, fully hydrated sample.

Figure 14.

Elevation of Raffinose Pathways in S. stapfianus in Response to Severe Desiccation. The metabolites in red indicate higher amounts during the dehydration process of reducing RWC. The relative abundance of the significantly altered metabolites was displayed using box plots. The P values for all comparisons are referenced in the Supplemental Data online. The x axis for each plot is RWC as a percentage. H, fully hydrated sample. [See online article for color version of this figure.]