Time-Course Comparative Metabolite Profiling under Osmotic Stress in Tolerant and Sensitive Tibetan Hulless Barley

Abstract

Tibetan hulless barley is widely grown in the extreme environmental conditions of the Qinghai-Tibet Plateau which is characterized by cold, high salinity, and drought. Osmotic stress always occurs simultaneously with drought and its tolerance is a vital part of drought tolerance. The diversity of metabolites leading to osmotic stress tolerance was characterized using widely-targeted metabolomics in tolerant (XL) and sensitive (D) accessions submitted to polyethylene glycol. XL regulated a more diverse set of metabolites than D, which may promote the establishment of a robust system to cope with the stress in XL. Compounds belonging to the group of flavonoids, amino acids, and glycerophospholipids constitute the core metabolome responsive to the stress, despite the tolerance levels. Moreover, 8 h appeared to be a critical time point for stress endurance involving a high accumulation of key metabolites from the class of nucleotide and its derivative which provide the ultimate energy source for the synthesis of functional carbohydrates, lipids, peptides, and secondary metabolites in XL. This intrinsic metabolic adjustment helped XL to efficiently alleviate the stress at the later stages. A total of 22 diverse compounds were constantly and exclusively regulated in XL, representing novel stress tolerance biomarkers which may help improving stress tolerance, especially drought, in hulless barley.

Figure 1

Quantification of leaf malondialdehyde (MDA) content. MDA was measured at 0, 0.5, 1, 2, 4, 8, 12, 24, and 48 h in the accession XL (blue bar) and D (red bar). t test comparison was performed between the two accessions. ^∗Significant at p ≤ 0.05. ^∗∗Significant at p ≤ 0.01. ^∗∗∗Significant at p ≤ 0.001. ns, nonsignificant at p > 0.05.

Figure 2

Heatmap hierarchical clustering of detected metabolite pools. Hierarchical trees were drawn based on detected metabolites in leaves of XL and D at 0, 1, 4, 8, 24, and 48 h in control (CK) and stress treatment (S). Columns correspond to accessions at different time points, while rows represent different metabolites.

Figure 3

Temporal changes in metabolic reprogramming in XL and D under osmotic stress. (a) Differentially changed metabolites at 1, 4, 8, 24, and 48 h in XL (blue line) and D (red line) under osmotic stress. (b) Down- and upregulated metabolites in D at 1, 4, 8, 24, and 48 h under osmotic stress. (c) Down- and upregulated metabolites in XL at 1, 4, 8, 24, and 48 h under osmotic stress.

Figure 4

Overview of top regulated metabolites between XL and D under short-term osmotic stress. (a) Top 20 down- and up-accumulated metabolites between XL and D at 1 h. (b) Top 20 down- and up- accumulated metabolites between XL and D at 4 h.

Figure 5

Overview of top regulated metabolites between XL and D under mid-term osmotic stress. (a) Top 20 down- and up-accumulated metabolites between XL and D at 8 h. (b) Top 20 down- and up-accumulated metabolites between XL and D at 24 h.

Figure 6

Overview of top regulated metabolites between XL and D under long-term osmotic stress. (a) Top 20 down- and up-accumulated metabolites between XL and D at 48 h. (b) Venn diagram representing the distribution of shared and common down-accumulated metabolites at different time points between XL and D. (c) Venn diagram representing the distribution of shared and common up-accumulated metabolites at different time points between XL and D.