Global Profiling of Phosphorylation Reveals the Barley Roots Response to Phosphorus Starvation and Resupply

Abstract

Phosphorus (P) deficiency is a major threat to the crop production, and for understanding the response mechanism of plant roots, P stress may facilitate the development of crops with increased tolerance. Phosphorylation plays a critical role in the regulation of proteins for plant responses to biotic and abiotic stress; however, its functions in P starvation/resupply are largely unknown for barley (Hordeum vulgare) growth. Here, we performed a global review of phosphorylation in barley roots treated by P starvation/resupply. We identified 7,710 phosphorylation sites on 3,373 proteins, of which 76 types of conserved motifs were extracted from 10,428 phosphorylated peptides. Most phosphorylated proteins were located in the nucleus (36%) and chloroplast (32%). Compared with the control, 186 and 131 phosphorylated proteins under P starvation condition and 156 and 111 phosphorylated proteins under P resupply condition showed significant differences at 6 and 48 h, respectively. These proteins mainly participated in carbohydrate metabolism, phytohormones, signal transduction, cell wall stress, and oxidases stress. Moreover, the pathways of the ribosome, RNA binding, protein transport, and metal binding were significantly enriched under P starvation, and only two pathways of ribosome and RNA binding were greatly enriched under Pi resupply according to the protein–protein interaction analysis. The results suggested that the phosphorylation proteins might play important roles in the metabolic processes of barley roots in response to Pi deficiency/resupply. The data not only provide unique access to phosphorylation reprogramming of plant roots under deficiency/resupply but also demonstrate the close cooperation between these phosphorylation proteins and key metabolic functions.

Global proteome analyses of phosphorylation and succinylation of barley root proteins in response to phosphate starvation and recovery

“…The recent developments of high-resolution mass spectrometry, antibody based affinity enrichment proteomic technology, and powerful bioinformatic tools have substantially contributed to the global analysis of protein phosphorylation and succinylation in barley. In our study in the last year, a large number of phosphoryl-and succinyl-proteins with diverse biological functions were identified to be responsive to Pi stress and recovery in barley roots (Ma et al, 2021;Wang et al, 2021).…”

“…The recent developments of high-resolution mass spectrometry, antibody based affinity enrichment proteomic technology, and powerful bioinformatic tools have substantially contributed to the global analysis of protein phosphorylation and succinylation in barley. In our study in the last year, a large number of phosphoryl- and succinyl-proteins with diverse biological functions were identified to be responsive to Pi stress and recovery in barley roots ( Ma et al, 2021 ; Wang et al, 2021 ). Although these studies contributed to our understanding of the mechanisms involved in responses to Pi starvation and recovery, they were limited to phosphoryl- and succinyl-proteins that were characterized separately, thus failing to capture any global crosstalk between phosphorylation and succinylation in the regulation of Pi homeostasis.…”

“…Although these studies contributed to our understanding of the mechanisms involved in responses to Pi starvation and recovery, they were limited to phosphoryl- and succinyl-proteins that were characterized separately, thus failing to capture any global crosstalk between phosphorylation and succinylation in the regulation of Pi homeostasis. In addition, such studies on phosphoryl- and succinyl-protein pathway enrichment were analyzed by a ranked differential protein list filtered by a particular threshold with a p value < 0.05 and fold-change > 1.5 ( Ma et al, 2021 ; Wang et al, 2021 ), which is more dependent on differential proteins and has a certain subjectivity ( Reimand et al, 2019 ). Here, in order to overcome the above defects, we used a threshold-free, GSEA approach to explore the underlying mechanisms by which protein phosphorylation and succinylation were involved in responses to Pi starvation and recovery in barley roots.…”

“…Further details on protein extraction, digestion, phosphopeptide and succinyl-peptide enrichment, liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis, and database searching are outlined in the our previously articles ( Ma et al, 2021 ; Wang et al, 2021 ). In summary, for phosphopeptide enrichment, tryptic peptides were dissolved in a 6% (v/v) TFA/50% ACN (v/v) buffer and the supernatant was incubated with an immobilized metal affinity column that bound phosphopeptides.…”

“…However, compared with other crops, research on PTMs during the phosphate starvation response in barley is still very limited. We have pioneered research on the identification of phosphorylation and lysine succinylation in barley in response to Pi starvation and the recovery process, respectively ( Ma et al, 2021 ; Wang et al, 2021 ). Although these studies revealed that phosphorylated and succinylated proteins were involved in a wide variety of biological processes, an understanding of PTM-mediated crosstalk between protein phosphorylation and succinylation is still largely unknown in barley.…”