Comparative quantitative phosphoproteomic and parallel reaction monitoring analysis of soybean roots under aluminum stress identify candidate phosphoproteins involved in aluminum resistance capacity

Aluminum (Al) toxicity adversely impacts soybean (Glycine max) growth in acidic soil. Reversible protein phosphorylation plays an important role in adapting to adverse environmental conditions by regulating multiple physiological processes including signal transduction, energy coupling and metabolism adjustment in higher plant. This study aimed to reveal the Al-responsive phosphoproteins to understand their putative function and involvement in the regulation of Al resistance in soybean root. We used immobilized metal affinity chromatography to enrich the key phosphoproteins from soybean root apices at 0, 4, or 24 h Al exposure. These phosphoproteins were detected using liquid chromatography-tandem mass spectrometry measurement, verified by parallel reaction monitoring (PRM), and functionally characterized via overexpression in soybean hairy roots. A total of 638 and 686 phosphoproteins were identified as differentially enriched between the 4-h and 0-h, and the 24-h and 0-h Al treatment comparison groups, respectively. Typically, the phosphoproteins involved in biological processes including cell wall modification, and RNA and protein metabolic regulation displayed patterns of decreasing enrichment (clusters 3, 5 and 6), however, the phosphoproteins involved in the transport and metabolic processes of various substrates, and signal transduction pathways showed increased enrichment after 24 h of Al treatment. The enrichment of phosphoproteins in organelle organization bottomed after 4 h of Al treatment (cluster 1). Next, we selected 26 phosphoproteins from the phosphoproteomic profiles, assessed their enrichment status using PRM, and detected enrichment patterns similar to those observed via phosphoproteomic analysis. Among them, 15 phosphoproteins were found to reduce the accumulation of Al and callose in Al-stressed soybean root apices when their corresponding genes were individually overexpressed in soybean hairy roots. In summary, the findings of this study facilitated a comprehensive understanding of the protein phosphorylation events involved in Al resistance responses and revealed some critical phosphoproteins that enhance Al resistance in soybean roots. [Display omitted] •More phosphoproteins exhibiting decreased enrichment in soybean root apices under Al stress.•A total of 6213 phosphorylation sites in 3267 phosphoproteins from soybean root apices were identified.•Thirty differentially phosphorylated sites were detected in 26 proteins by parallel re