Quantitative iTRAQ Proteomics Revealed Possible Roles for Antioxidant Proteins in Sorghum Aluminum Tolerance

Abstract

Aluminum (Al) toxicity inhibits root growth and limits crop yields on acid soils worldwide. However, quantitative information is scarce on protein expression profiles under Al stress in crops. In this study, we report on the identification of potential Al responsive proteins from root tips of Al sensitive BR007 and Al tolerant SC566 sorghum lines using a strategy employing iTRAQ and 2D-liquid chromatography (LC) coupled to MS/MS (2D-LC-MS/MS). A total of 771 and 329 unique proteins with abundance changes of >1.5 or <0.67-fold were identified in BR007 and SC566, respectively. Protein interaction and pathway analyses indicated that proteins involved in the antioxidant system were more abundant in the tolerant line than in the sensitive one after Al treatment, while opposite trends were observed for proteins involved in lignin biosynthesis. Higher levels of ROS accumulation in root tips of the sensitive line due to decreased activity of antioxidant enzymes could lead to higher lignin production and hyper-accumulation of toxic Al in cell walls. These results indicated that activities of peroxidases and the balance between production and consumption of ROS could be important for Al tolerance and lignin biosynthesis in sorghum.

Keywords: aluminum tolerance; antioxidant system; lignin biosynthesis; proteomics; sorghum.

Figure 1

Relative Net Root Growth (%RNRG) of SC566 and BR007. Daily %RNRG was calculated as net root growth of +Al treatment / net root growth of −Al treatment at the indicated day of Al exposure. n = 20 seedlings.

Figure 2

Experimental design and schematic diagram of the workflow. HpRP, High-pH reversed-phase chromatography; std, Internal standard which was pooled by equal amounts of all samples in the experiment; MGF by PD, use Proteome Discover software to convert the spectra to MGF format peak list files.

Figure 3

Comparison of Log2 iTRAQ ratios for all proteins identified in three biological and analytical replicates of each experiment. This figure shows an example of comparison of ratios from BR007 5D Al treated sample #1, #2, and #3 vs. average control. (A) #1 vs. #2; (B) #2 vs. #3; (C) #3 vs. #1.

Figure 4

Number of up- and down-regulated DEPs of SC566 and BR007 in functional categorization. Functional categorization of 1.5-fold differentially expressed proteins was analyzed for both SC566 (A,B) and BR007 (C,D) treated with Al at indicated time points using the Blast2GO 2.8 software with default NCBI nr/nt Blast databases and settings.

Figure 5

Enzymatic activities. Root tip samples (0.5 g FW) were homogenized in the HEPES-KOH buffer + 0.1 mM EDTA (pH 7.8). Supernatant of each sample was collected for enzymatic activity assays: (A) Superoxide dismutase (SOD), (B) Peroxidase (POD), and (C) Catalase (CAT) in root tips of BR007 and SC566 with Al or without Al (CK) treatment. n = 3.

Figure 6

Root tip lignin production. Root tip samples (0.5 g FW) were homogenized in 95% EtOH. Lignin contents of the washed and air-dried pellet samples were determined for (A) SC566 and (B) BR007 under Al or control (CK) treatment. n = 3. ^*p < 0.05; ^**p < 0.01.

Figure 7

STRING-based protein-protein interaction analysis with high confidence interaction score of 0.700. (A) STRING analysis for up-regulated differentially expressed proteins (DEPs) in SC566 at 5D Al treatment. The circled nodes are Ribosomal family proteins indicating that protein biosynthesis is highly active for the Al tolerance line under Al stress. (B) STRING analysis for up-regulated DEPs in SC566 at 3D Al treatment. (C) STRING analysis for down-regulated DEPs in BR007 at 3D and 5D Al treatment. The circled nodes in (B,C) are peroxidase family proteins.