Quantitative phosphoproteomics analysis of nitric oxide-responsive phosphoproteins in cotton leaf

Abstract

Knowledge of phosphorylation events and their regulation is crucial to understanding the functional biology of plant proteins, but very little is currently known about nitric oxide-responsive phosphorylation in plants. Here, we report the first large-scale, quantitative phosphoproteome analysis of cotton (Gossypium hirsutum) treated with sodium nitroprusside (nitric oxide donor) by utilizing the isobaric tag for relative and absolute quantitation (iTRAQ) method. A total of 1315 unique phosphopeptides, spanning 1528 non-redundant phosphorylation sites, were detected from 1020 cotton phosphoproteins. Among them, 183 phosphopeptides corresponding to 167 phosphoproteins were found to be differentially phosphorylated in response to sodium nitroprusside. Several of the phosphorylation sites that we identified, including RQxS, DSxE, TxxxxSP and SPxT, have not, to our knowledge, been reported to be protein kinase sites in other species. The phosphoproteins identified are involved in a wide range of cellular processes, including signal transduction, RNA metabolism, intracellular transport and so on. This study reveals unique features of the cotton phosphoproteome and provides new insight into the biochemical pathways that are regulated by nitric oxide.

Figure 1. Functional classifications (A) and protein subcellular locations (B) of all 1020 phosphoproteins.

Figure 2. Analysis of the 183 phosphopeptides whose levels changed after exposure to SNP.

N/a, not applicable, indicates that the phosphopeptides were not detected in this group and were only expressed in other treatment groups.

Figure 3. Functional classifications (A) and protein subcellular locations (B) of the 167 phosphoproteins.

Figure 4. Fold changes of the phosphopeptides involved in spliceosome significantly altered following SNP.

Protein identification numbers refer to the CGP database. To make the figure easier to read, the protein ID is represented only by the numbers in the key (i.e., remove “Cotton_D_gene_”).

Figure 5. Fold changes of the phosphopeptides involved in mRNA surveillance significantly altered following SNP.

Protein identification numbers refer to the CGP database. To make the figure easier to read, the protein ID is represented only by the numbers in the key (i.e., remove “Cotton_D_gene_”).

Figure 6. Fold changes of the phosphopeptides involved in RNA transport significantly altered following SNP.

Protein identification numbers refer to the CGP database. To make the figure easier to read, the protein ID is represented only by the numbers in the key (i.e., remove “Cotton_D_gene_”).

Figure 7. Fold changes of the phosphopeptides involved in plant-pathogen interaction significantly altered following SNP.

Protein identification numbers refer to the CGP database. To make the figure easier to read, the protein ID is represented only by the numbers in the key (i.e., remove “Cotton_D_gene_”).