Metabolomic response of Calotropis procera growing in the desert to changes in water availability

Abstract

Water availability is a major limitation for agricultural productivity. Plants growing in severe arid climates such as deserts provide tools for studying plant growth and performance under extreme drought conditions. The perennial species Calotropis procera used in this study is a shrub growing in many arid areas which has an exceptional ability to adapt and be productive in severe arid conditions. We describe the results of studying the metabolomic response of wild C procera plants growing in the desert to a one time water supply. Leaves of C. procera plants were taken at three time points before and 1 hour, 6 hours and 12 hours after watering and subjected to a metabolomics and lipidomics analysis. Analysis of the data reveals that within one hour after watering C. procera has already responded on the metabolic level to the sudden water availability as evidenced by major changes such as increased levels of most amino acids, a decrease in sucrose, raffinose and maltitol, a decrease in storage lipids (triacylglycerols) and an increase in membrane lipids including photosynthetic membranes. These changes still prevail at the 6 hour time point after watering however 12 hours after watering the metabolomics data are essentially indistinguishable from the prewatering state thus demonstrating not only a rapid response to water availability but also a rapid response to loss of water. Taken together these data suggest that the ability of C. procera to survive under the very harsh drought conditions prevailing in the desert might be associated with its rapid adjustments to water availability and losses.

Figure 1. Experimental Setup and plants chosen.

(a) Representative photo of the plants chosen for this experiment growing in its natural habitat in Saudi Arabia near to Jeddah. For this study representative species of similar size and performance were chosen. (b) Experimental set-up. At day 1 (control) leaves of three independent plants were harvested 1 h post-dawn, at midday and 1 h pre-dusk. One day later (Day 2), plants were watered at dawn and leaves were harvested 1 h post dawn, at midday and 1 h pre-dusk. Harvested leaves were frozen immediately in liquid –N and processes as described in Experimental procedures.

Figure 2. Principal Component Analysis (PCA) and ANOVA for metabolomic analysis of leaf samples from control and watered plants.

(a) PCA (upper part) and ANOVA (lower part) for primary metabolites. (b) PCA (upper part) and ANOVA (lower part) for complex lipids. (c) PCA (upper part) and ANOVA (lower part) for secondary metabolites. Shown are always three independent samples per time point (dawn (1 hour post dawn/after watering), midday (6 hours after dawn/after watering) and pre-dusk (12 hours after dawn/after watering). Watered samples are shown in blue, non-watered in red. The lower part shows the results of a Bonferroni corrected ANOVA displaying the influence of treatment (watering) for all samples and of harvesting time for primary and secondary metabolites.

Figure 3. Boxplots and pathway visualization of representative primary metabolites.

(a) and (b): Boxplot-visualizations for a subset of amino acids (A) and sugars and sugar alcohols(B) as determined for the three independent samples for the different time points and treatments as indicated on the x-axis. (b) Pathway mapping of a number of primary metabolites visualized as their averaged log2-foldchange ratio of rehydration versus control (green  =  decrease; red  =  increase).

Figure 4. Clustered heatmap visualization of different lipid classes.

Shown is the average abundance of several complex lipids visualized in a false-color heatmap at the three time points before and after watering ordered according to their presence in photosynthetic membranes, in cellular membranes or representing storage lipids.

Figure 5. Boxplots of representative species of photosynthetic, structural and storage lipids.

Boxplot-visualizations for a subset of complex lipids as determined for the three independent samples for the different time points and treatments as indicated on the x-axis of three replicates that were harvested at 1 h post-dawn, midday and 1 h pre-dusk for control and rehydrated plants.

Figure 6. Relative water content of leaves of C. procera plants taken at dawn, midday and one hour pre-dusk one day before watering and up to three days after watering.