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. 2021 Mar 31:12:649147.
doi: 10.3389/fpls.2021.649147. eCollection 2021.

Dynamic Responses of Barley Root Succinyl-Proteome to Short-Term Phosphate Starvation and Recovery

Juncheng Wang  1   2 , Zengke Ma  1   2 , Chengdao Li  3 , Panrong Ren  1   2 , Lirong Yao  1   2 , Baochun Li  4 , Yaxiong Meng  1   2 , Xiaole Ma  1   2 , Erjing Si  1   2 , Ke Yang  1   2 , Xunwu Shang  2 , Huajun Wang  1   2
Affiliations

Dynamic Responses of Barley Root Succinyl-Proteome to Short-Term Phosphate Starvation and Recovery

Juncheng Wang et al. Front Plant Sci. .

Abstract

Barley (Hordeum vulgare L.)-a major cereal crop-has low Pi demand, which is a distinct advantage for studying the tolerance mechanisms of phosphorus deficiency. We surveyed dynamic protein succinylation events in barley roots in response to and recovery from Pi starvation by firstly evaluating the impact of Pi starvation in a Pi-tolerant (GN121) and Pi-sensitive (GN42) barley genotype exposed to long-term low Pi (40 d) followed by a high-Pi recovery for 10 d. An integrated proteomics approach involving label-free, immune-affinity enrichment, and high-resolution LC-MS/MS spectrometric analysis was then used to quantify succinylome and proteome in GN121 roots under short-term Pi starvation (6, 48 h) and Pi recovery (6, 48 h). We identified 2,840 succinylation sites (Ksuc) across 884 proteins; of which, 11 representative Ksuc motifs had the preferred amino acid residue (lysine). Furthermore, there were 81 differentially abundant succinylated proteins (DFASPs) from 119 succinylated sites, 83 DFASPs from 110 succinylated sites, 93 DFASPs from 139 succinylated sites, and 91 DFASPs from 123 succinylated sites during Pi starvation for 6 and 48 h and during Pi recovery for 6 and 48 h, respectively. Pi starvation enriched ribosome pathways, glycolysis, and RNA degradation. Pi recovery enriched the TCA cycle, glycolysis, and oxidative phosphorylation. Importantly, many of the DFASPs identified during Pi starvation were significantly overexpressed during Pi recovery. These results suggest that barley roots can regulate specific Ksuc site changes in response to Pi stress as well as specific metabolic processes. Resolving the metabolic pathways of succinylated protein regulation characteristics will improve phosphate acquisition and utilization efficiency in crops.

Keywords: Hordeum vulgare L.; Pi stress; germplasm; metabolism; succinylated protein.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Root characteristics of two barley lines with different low-Pi tolerance in response to Pi starvation and recovery. (A) GN121 (Pi-tolerant) and GN42 (Pi-sensitive) seedlings grown in normal P (+, 0.39 mM Pi) and low P (–, 0.039 mM Pi) for 10, 20, 30, or 40 d, followed by recovery P (+, 0.39 mM P) for 10 d; (B) total root length, (C) root surface area, and (D) root volume of GN121 seedlings; (E) total root length, (F) root surface area, and (G) root volume of GN42 seedlings; (H,I) are plant and root fresh weights of GN121 and GN42 seedlings, respectively. Data are means ± SD (n = 5); * Indicates significant differences (one-way ANOVA, Duncan, P ≤ 0.05) between normal and Pi treatments.
Figure 2
Figure 2
Dynamics of protein succinylation in roots of GN121 seedlings under Pi starvation and recovery using the anti-suc-lysine antibody. For immunoblot results, proteins were collected from (A) low-Pi (0.039 mM Pi) at 0, 3, 6, 12, 24, 48, 72 h, 5 d, and 7 d, and (B) Pi recovery (0.39 mM Pi) at 3, 6, 12, 24, 48, and 72 h. The same amount of protein (25 μg per lane) was loaded in each panel.
Figure 3
Figure 3
Workflow used to analyze lysine succinylation in seedling roots in response to Pi starvation and recovery.
Figure 4
Figure 4
Properties of the succinylated peptides in barley roots. (A) Summary of the succinylome identified and quantified; (B) motif analysis of identified succinylated sequence by Motif-X software. The motifs with high significance (P < 0.000001) are shown; (C) position-specific amino acid composition around the succinylation sites. The –log10 (Fisher's exact test P-value) for every amino acid in each position (from −10 to +10) is shown; (D) overview of the differentially succinylated sites and proteins (fold change > 1.5, P < 0.05).
Figure 5
Figure 5
KEGG pathway enrichment analysis of DFASPs in roots under Pi starvation at 6 h (A) and 48 h (B) and Pi recovery at 6 h (C) and 48 h (D).
Figure 6
Figure 6
Summary of the differentially succinylated proteins in response to Pi starvation. (A,B) Venn diagram of difffrentially abundant succinylated proteins (sites) in response to Pi starvation; (C) functional clustering analyses (HCL) of the differentially succinylated sites based on the relative succinylation intensity, relative to the control. Cluster identification and number of profiles included in each cluster are indicated on the left. A detailed view of individual profiles is in Supplementary Figure 6; (D) KEGG pathway enrichment analysis of DFASPs in each cluster.
Figure 7
Figure 7
Summary of differentially succinylated proteins in response to Pi recovery. (A,B) Venn diagram of difffrentially abundant succinylated proteins (sites) in response to Pi recovery; (C) functional clustering analyses (HCL) of the differentially succinylated sites based on the relative succinylation intensity, relative to the control. Cluster identification and number of profiles included in each cluster are indicated on the left. Detailed view of individual profiles is in Supplementary Table 6; (D) KEGG pathway enrichment analysis of DFASPs in each cluster.
Figure 8
Figure 8
Protein–protein interaction (PPI) network of succinylated proteins in response to Pi starvation. All DFASPs were searched against the STRING database (version 10.5) for PPIs and visualized using Cytoscape (version 3.6.1; http://www.cytoscape.org/). We collated all interactions with a high confidence score (>0.7). A graph-theoretical clustering algorithm, molecular complex detection (MCODE), was used to analyze densely connected regions. The circle size represents the numbers of DFASPs; red indicates increased and blue indicates decreased DFASPs. Further details are in Supplementary Table 10.
Figure 9
Figure 9
Protein–protein interaction (PPI) network of succinylated proteins in response to Pi recovery. Same as Figure 8.
Figure 10
Figure 10
Major succinylation-mediated metabolic processes and proteins involved in short-term Pi starvation and recovery, as depicted by succinyl-proteome analyses. For details of the proteins and their abbreviations, see Supplementary Table 14.
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