Functional and Integrative Analysis of the Proteomic Profile of Radish Root under Pb Exposure

Abstract

Lead (Pb) is one of the most abundant heavy metal (HM) pollutants, which can penetrate the plant through the root and then enter the food chain causing potential health risks for human beings. Radish is an important root vegetable crop worldwide. To investigate the mechanism underlying plant response to Pb stress in radish, the protein profile changes of radish roots respectively upon Pb(NO₃)₂ at 500 mg L^-1(Pb500) and 1000 mg L^-1(Pb1000), were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification). A total of 3898 protein species were successfully detected and 2141 were quantified. Among them, a subset of 721 protein species were differentially accumulated upon at least one Pb treatment, and 135 ones showed significantly abundance changes under both two Pb-stressed conditions. Many critical protein species related to protein translation, processing, and degradation, reactive oxygen species (ROS) scavenging, photosynthesis, and respiration and carbon metabolism were successfully identified. Gene Ontology (GO) and pathway enrichment analysis of the 135 differential abundance protein species (DAPS) revealed that the overrepresented GO terms included "cell wall," "apoplast," "response to metal ion," "vacuole," and "peroxidase activity," and the critical enriched pathways were involved in "citric acid (TCA) cycle and respiratory electron transport," "pyruvate metabolism," "phenylalanine metabolism," "phenylpropanoid biosynthesis," and "carbon metabolism." Furthermore, the integrative analysis of transcriptomic, miRNA, degradome, metabolomics and proteomic data provided a strengthened understanding of radish response to Pb stress at multiple levels. Under Pb stress, many key enzymes (i.e., ATP citrate lyase, Isocitrate dehydrogenase, fumarate hydratase and malate dehydrogenase) involved in the glycolysis and TCA cycle were severely affected, which ultimately cause alteration of some metabolites including glucose, citrate and malate. Meanwhile, a series of other defense responses including ascorbate (ASA)-glutathione (GSH) cycle for ROS scavenging and Pb-defense protein species (glutaredoxin, aldose 1-epimerase malate dehydrogenase and thioredoxin), were triggered to cope with Pb-induced injuries. These results would be helpful for further dissecting molecular mechanism underlying plant response to HM stresses, and facilitate effective management of HM contamination in vegetable crops by genetic manipulation.

Keywords: heavy metal; iTRAQ; lead stress; proteomics; radish.

Figure 1

The Venn diagram of differential abundance protein species (DAPS) involved in radish root under Pb500 and Pb1000 exposure.

Figure 2

GO classification of the DAPS of radish root in response to Pb stress.

Figure 3

The representative pathways of the DAPS involved in radish root response to Pb exposure.

Figure 4

The putative genetic regulatory network of Pb stress response in radish root. 12-oxo-phytodienoic acid reductase-1 (OPR-1), 12-oxo-phytodienoic acid reductase-3 (OPR-3), Ascorbate (ASA)–Glutathione (GSH), Calreticulin-3-like isoform x1 (CRT3), Glutaredoxin (GRX), thioredoxin (TRX), Leucine-rich repeat family protein (LRR), Monodehydroascorbate reductase (MDHAR), Pectinesterases (PE), Peroxidase (POD), Peroxiredoxin (PRX), Polygalacturonases (PG), Protein disulfide isomerase (PDI), Translation initiation factor (TIF).