Proteomic Investigation of Molecular Mechanisms in Response to PEG-Induced Drought Stress in Soybean Roots

Abstract

Roots are generally the critical drought sensors, but little is known about their molecular response to drought stress. We used the drought-tolerant soybean variety 'Jiyu 47' to investigate the differentially expressed proteins (DEPs) in soybean roots during the seedling stage based on the tandem mass tag (TMT) proteomics analysis. Various expression patterns were observed in a total of six physiological parameters. A total of 468 DEPs (144 up-regulated and 324 down-regulated) among a total of 8687 proteins were identified in response to drought stress in 24 h. The expression of DEPs was further validated based on quantitative real-time PCR of a total of five genes (i.e., GmGSH, GmGST1, GmGST2 k GmCAT, and Gm6PGD) involved in the glutathione biosynthesis. Results of enrichment analyses revealed a coordinated expression pattern of proteins involved in various cellular metabolisms responding to drought stress in soybean roots. Our results showed that drought stress caused significant alterations in the expression of proteins involved in several metabolic pathways in soybean roots, including carbohydrate metabolism, metabolism of the osmotic regulation substances, and antioxidant defense system (i.e., the glutathione metabolism). Increased production of reduced glutathione (GSH) enhanced the prevention of the damage caused by reactive oxygen species and the tolerance of the abiotic stress. The glutathione metabolism played a key role in modifying the antioxidant defense system in response to drought stress in soybean roots. Our proteomic study suggested that the soybean plants responded to drought stress by coordinating their protein expression during the vegetative stage, providing novel insights into the molecular mechanisms regulating the response to abiotic stress in plants.

Keywords: antioxidant; drought stress; enrichment analysis; gene ontology; glutathione; physiological response; proteomics; soybean root; tandem mass tag.

Figure 1

Physiological variations in soybean roots showing the contents of (A) peroxidase (POD), (B) catalase (CAT), (C) proline (PRO), (D) malondialdehyde (MDA), (E) reduced glutathione (GSH), and (F) oxidized glutathione (GSSH). Data are represented as mean ± standard deviation (SD) of three biological replicates. Different letters a, b, c, and d indicate significant differences set at p value of 0.05.

Figure 2

Proteomic profiling of the distributions of the mass spectra (A) and the differentially expressed proteins (DEPs) (B) induced by drought stress in soybean roots.

Figure 3

GO annotation of differentially expressed proteins (DEPs) (A) and COG functional classifications of total DEPs (B), of up-regulated DEPs (C), and of down-regulated DEPs (D) induced by drought stress in soybean roots.

Figure 4

Subcellular localization of the total differentially expressed proteins (DEPs) (A), of up-regulated proteins (B), and of down-regulated proteins (C) induced by drought stress in soybean roots.

Figure 5

KEGG enrichment analysis of all differentially expressed proteins (DEPs) induced by drought stress in soybean roots (A), of up-regulated DEPs (B), of down-regulated DEPs (C), of up-regulated proteins of molecular functions (D), and of down-regulated proteins of molecular functions (E).

Figure 6

KEGG pathway enrichment of differentially expressed proteins (DEPs) induced by drought stress in soybean roots in clusters of groups Q1 to Q4 (A) and in combination of groups Q1 to Q4 (B).

Figure 7

Glutathione metabolism in soybean roots under drought stress. Enzymes showing up-regulation are indicated in red. OPLAH, 5-oxoprolinase (ATP-hydrolysis); GCLC, glutamate-cysteine ligase catalytic unit; GSS, glutathione synthase; GPX, glutathione peroxidase; GR, glutathione reductase; 6PGD, 6-phosphogluconate dehydrogenase; GST, glutathione S-transferase; GGT, gamma-glutamyl transpeptidase; ANPEP, aminopeptidase N; NAT8, N-acetyltransferase 8.

Figure 8

Temporal expression of GmGSH (A), GmGST1 (B), GmGST2 (C), GmCAT (D), and Gm6PGD (E) in soybean roots under drought stress. Data are represented as mean ± standard deviation (SD) of three biological replicates. Different letters a, b, c, and d indicate the significant difference set at p value of 0.05.

Figure 9

Three metabolic pathways (i.e., carbohydrate pathway, glycolytic pathway, and tricarboxylic acid pathway) in soybean roots under drought stress. Enzymes showing up-regulation and down-regulation are indicated in red and blue, respectively. CES, cellulose synthase; G-1-PG, glucose-1-phosphate guanylyltransferase; SUS, surcose synthase; UGP2, UTP-glucose-1-phosphate uridylyltransferase; PGM, phosphoglucomutase; GPI, glucose-6-phosphate isomerase; ALDO, fructose-bisphosphate aldolase; PGK, phosphoglycerate kinase; PGAM, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; PK, pyruvate kinase; aceE, pyruvate dehydrogenase E1; DLAT, dihydrolipoamide acetyltransferase; PC, pyruvate carboxylase; MDH1, malate dehydrogenase; fumA, fumarate hydratase; SDHA, succinate dehydrogenase (ubiquinone) flavoprotein; sucD, succinyl-CoA synthetase; sucL, succinyl-CoA ligase; a-keDC, a-ketoglutarate dehydrogenase complex; IDH1, isocitrate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; ACO, aconitate hydratase; CS, citrate synthase.