Exploring the molecular mechanism of rice tolerance to heavy metal stress by proteome + PRM validation

PM protein in rice roots after 7 days of treatment in experimental group (T) and control group (CK), three biological replicates in each group

 Technical route

Step 1: The effect of zinc on the accumulation of zinc and cadmium in rice seedlings treated with low cadmium;

Step 2: Quantitative proteomic detection under zinc stress;

Step 3: Bioinformatics analysis of differentially expressed proteins;

Step 4: Research on zinc-cadmium transport-related proteins;

Step 5: PRM validation of candidate proteins

 Research result

1.

Low concentrations of Zn promoted the uptake and accumulation of Cd in rice seedlings, resulting in increased Cd content and inhibited the growth of rice seedlings (synergistic effect); moderate concentrations of Zn inhibited the uptake and accumulation of Cd and promoted the growth of seedlings (antagonistic effect).

2.

PM proteins were quantitatively detected by iTRAQ proteomics technology, and rice roots treated with CK and T4 for 7 days were analyzed by bioinformatics

3.

Through hierarchical clustering analysis, it can be seen that every three replicates of the control group and the treatment group are clustered together, with high repeatability

Figure 3 GO annotation and enrichment analysis of differentially expressed proteins

KEGG pathway analysis found that DEPs related to plant metabolism were mainly involved in glutathione metabolism, phenylpropionic acid biosynthesis, and carbon fixation in photosynthetic organisms

Figure 4 Annotation and enrichment analysis of differentially expressed proteins KEGG pathway

4.

^{Seven DEPs involved in Zn 2+} and Cd ²⁺ transport were recorded in GO analysis and KEGG pathway analysis
.
Among them, 3 DEPs were up-regulated (A2XQ09, B8B248 and B8AAT90); 4 DEPs were down-regulated (B8AB68, A0A1B1ELU7, A2XPS1 and B8BN76)
.
PPI interaction analysis revealed that 5 of these 7 DEPs interacted with each other
.

Figure 5 PPI interaction network of differentially expressed proteins

5.
PRM validation of candidate proteins

A PRM detection method for 4 candidate proteins (B8AB68, B8AAT9, A0A1B1ELU7 and B8BN76) was established for targeted validation
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The results showed that the protein A0A1B1ELU7 had the same expression changes in different treatments
.

Figure 6 PRM quantification results of candidate proteins

 Xiaobian summary

In conclusion, relative quantitative proteomics and PRM targeting validation were used to reveal the mechanism of Zn/Cd interaction and transmembrane transport in stress response at the protein level, and to find potential targets related to heavy metal uptake and transport in rice roots.
protein
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This study provides basic data reference for the screening of heavy metal stress response protein biomarkers in rice roots and the detoxification mechanism of heavy metal stress resistance
.