3.4 Identification of DEPs by proteomic analysis
We conducted proteomic analysis based on tandem mass tag (TMT) quantification to identify DEPs between WT and OE-4 line treated with low N for 35 d (Figure 3). Principal component analysis (PCA) analyses confirmed the reliability of the replicated samples data (Figure 3a). By comparing the protein expression levels and applying a threshold value of FC > 1.2 and p-value < 0.05, we identified 253 downregulated proteins and 440 upregulated proteins were identified in the WT and OE-4 comparisons (Figure 3b,c). To gain a deeper understanding of DEPs in the sequencing data, the locations, characteristics, and functions of these proteins were annotated. Subcellular localization prediction indicated that 38.67% of DEPs were localized in the chloroplast, followed by the cytoplasm (26.12%), nucleus (18.76%), plasma membrane (8.08%), mitochondria (2.6%), other locations (5.77%) (Figure 3d).
The COG/KOG database was used to categorize the 502 DEPs into 22 groups based on their primary cellular functions (Figure 4a; Table S2). In the metabolism category, numerous DEPs were involved in carbohydrate, lipid, and amino acid transport and metabolism (G, I, and E), energy production and conversion (C), and secondary metabolites biosynthesis, transport, and catabolism. KEGG pathways analysis was also performed to predict potentially important pathways. We observed that the upregulated proteins were significantly enriched in pathways such as ‘alanine, aspartate and glutamate metabolism’ (map00250), ‘stilbenoid, diarylheptanoid and gingerol biosynthesis’ (map00945), ‘photosynthesis’ (map00196), ‘butanoate metabolism’ (man0060), and ‘flavonoid biosynthesis’ (map00941) (Figure 4b; Table S3). These findings suggest that DEPs between WT and OE-4 lines are involved in some processes associated with N deficiency such as photosynthesis, carbohydrate and amino acid metabolism, and energy production and conversion.