Metabolomic Profiling of Soybeans (Glycine max L.) Reveals the Importance of Sugar and Nitrogen Metabolism under Drought and Heat Stress

Abstract

Soybean is an important crop that is continually threatened by abiotic stresses, especially drought and heat stress. At molecular levels, reduced yields due to drought and heat stress can be seen as a result of alterations in metabolic homeostasis of vegetative tissues. At present an incomplete understanding of abiotic stress-associated metabolism and identification of associated metabolites remains a major gap in soybean stress research. A study with a goal to profile leaf metabolites under control conditions (28/24 °C), drought [28/24 °C, 10% volumetric water content (VWC)], and heat stress (43/35 °C) was conducted in a controlled environment. Analyses of non-targeted metabolomic data showed that in response to drought and heat stress, key metabolites (carbohydrates, amino acids, lipids, cofactors, nucleotides, peptides and secondary metabolites) were differentially accumulated in soybean leaves. The metabolites for various cellular processes, such as glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, and starch biosynthesis, that regulate carbohydrate metabolism, amino acid metabolism, peptide metabolism, and purine and pyrimidine biosynthesis, were found to be affected by drought as well as heat stress. Computationally based regulatory networks predicted additional compounds that address the possibility of other metabolites and metabolic pathways that could also be important for soybean under drought and heat stress conditions. Metabolomic profiling demonstrated that in soybeans, keeping up with sugar and nitrogen metabolism is of prime significance, along with phytochemical metabolism under drought and heat stress conditions.

Keywords: crop yield; drought stress; heat stress; metabolomic profiling; phytochemicals; soybean.

Figure 1

Metabolomics data of soybean leaves under drought and heat stress. (A) Classification of differentially expressed metabolites into chemical groups, in response to both drought and heat stress, based on their metabolic designations. Percentage values symbolize abundance of metabolites in each functional class. (B) Principal component analysis (PCA) biplot of all the variable metabolites originating from three different treatments i.e., control, drought and heat is shown here, (cos2 = the quality of the individuals on the factor map displayed as color intensity). (C) Scatter plot distribution of all groups of metabolites revealed that there is a linear positive correlation between the expression levels of control metabolites, and drought stress and heat stress-responsive metabolites. The best scatter of the metabolites was observed at the base of both the plot, which is visualized with an intensity level gradient ranging from 0.1 to 0.5. (Plot created by using R 3.3.1 software with the ggplot package).

Figure 2

Effects of drought and heat stresses on the metabolites of glycolysis and the tricarboxylic acid (TCA) cycle. (A) Glycolysis pathway showing the metabolites that had abundances significantly affected by stresses in the study; (B) Box plots showing the relative abundances of glucose, dihydroxyacetone and pyruvate under the control, drought, and heat stress conditions. (C) TCA cycle showing metabolites that were found to have significant effect on their abundance of stress (D) Box plots showing relative abundances of metabolites of TCA cycle under various stressed conditions [the Y axis defines the relative abundances of specific metabolite and X axis defines the treatment group; the red lightning sign in panels (A,C) indicates heat stress; green box plot: control, orange box plot: drought and pink box plot: heat]. Inside the box plots, “+” indicates mean value, and “—“ indicates median value.

Figure 3

Effect of drought and heat stresses on metabolites related to the pentose phosphate pathway. (A) The pink colored metabolites indicate downregulation in response to heat stress, orange colored metabolites indicate downregulation in response to drought stress. (B) Box plots showing relative abundances of PPP metabolites and sugar compounds under control, drought, and heat stress conditions (pink highlights indicate heat stress-affected; box plot description is the same as Figure 2).

Figure 4

Effect of drought and heat stresses on metabolites of aromatic amino acid metabolism. (A) Pathway for aromatic amino acid metabolism showing the metabolites that were found to have significant effect of stresses on their abundance in the study (B) Box plots showing the relative abundances of aromatic amino acids in control, drought, and heat stress conditions (C) Effect of drought and heat stress on pyruvate and oxaloacetate-derived amino acid biosynthesis (D) Box plots showing relative levels of pyruvate and oxaloacetate-derived amino acids in control, drought and heat stress conditions. (Box plot description is the same as in Figure 2; red and yellow lightning signs in (A) and (C) indicate the effects of heat and drought stress, respectively).

Figure 5

Differential abundance of peptides under drought and heat stresses. Heat map showing the differences in the abundance of the various peptides in soybean leaves under control (C.), drought (D.), and heat stress (H.) conditions.

Figure 6

Effect of heat stress on regulation of pyrimidine metabolism. (A) Pathway for pyrimidine metabolism showing the metabolites that were found to have a significant effect of stresses on their abundance in the study. (B) Box plot showing major purine and pyrimidine bases and their differential abundances under control, drought and heat stress (Box plot description is the same as Figure 2; red lightning sign in (A) indicates heat stress).

Figure 7

Effect of drought and heat stress on secondary metabolites. (A) Heat map showing differential expressions of various secondary metabolites during drought and heat stress compared to the control treatment. (B) Bar diagram showing relative expression of various phytochemicals in response to heat stress (blue) compared to the control (green). Various phytochemicals are shown on the X-axis, and the Y-axis has the scaled intensity of the respective phytochemical. Each value represents the mean ± S.E., and the asterisks designate the significance of changes from their corresponding control (p < 0.05).

Figure 8

A proposed model shows the metabolomic responses of soybean leaves during heat stress. The figure shows the effects of heat stress on key metabolites in key cell organelles. Single and double downward-pointing arrows (based on scaled quantity of metabolites) indicate metabolites reduced due to heat stress. There was no metabolite found in this investigation that showed increased abundance during heat stress. The red lightning arrows on the corners of the cell represent heat stress.