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.