Comprehensive Phosphoproteomic Analysis of Nostoc flagelliforme in Response to Dehydration Provides Insights into Plant ROS Signaling Transduction

Abstract

Terrestrial cyanobacteria, originated from aquatic cyanobacteria, exhibit a unique mechanism for drought adaptation during long-term evolution. To elucidate this diverse adaptive mechanism exhibited by terrestrial cyanobacteria from the post-translation modification aspect, we performed a global phosphoproteome analysis on the abundance of phosphoproteins in response to dehydration using Nostoc flagelliforme, a kind of terrestrial cyanobacteria having strong ecological adaptability to xeric environments. A total of 329 phosphopeptides from 271 phosphoproteins with 1168 phosphorylation sites were identified. Among these, 76 differentially expressed phosphorylated proteins (DEPPs) were identified for each dehydration treatment (30, 75, and 100% water loss), compared to control. The identified DEPPs were functionally categorized to be mainly involved in a two-component signaling pathway, photosynthesis, energy and carbohydrate metabolism, and an antioxidant system. We concluded that protein phosphorylation modifications related to the reactive oxygen species (ROS) signaling pathway might play an important role in coordinating enzyme activity involved in the antioxidant system in N. flagelliforme to adapt to dehydration stress. This study provides deep insights into the extensive modification of phosphorylation in terrestrial cyanobacteria using a phosphoproteomic approach, which may help to better understand the role of protein phosphorylation in key cellular mechanisms in terrestrial cyanobacteria in response to dehydration.

Figure 1

Workflow of the experiment to analyze the phosphoproteome, a representative MS/MS spectrum, and general description of the phosphopeptides identified. (A) Overview of the analytical workflow used in this study. Proteins were prefractionated using gel-free methods, followed by trypsin digestion and TiO₂ enrichment of phosphopeptides. Phosphopeptides were separated by nano-LC–MS/MS and mass measured and fragmented using the mass spectrometer. (B) Example of an MS/MS spectrum assigned to VSSKIGVIETLLEK from phosphoglycerate kinase (PGK) (WP_100901575.1). The b and y ions including loss of ammonia and water were considered when we calculated the PTM score. (C) Distribution of singly, doubly, and triply phosphorylated peptides. The photos in panel (A) were taken by Wenyu Liang, an author listed in this manuscript.

Figure 2

Characterization of differentially expressed phosphopeptides (DEPs) in N. flagelliforme exposed to different dehydration treatments. (A) Comparison of differentially expressed phosphopeptides (DEPs) among the group samples with different dehydration treatments (30, 75, and 100% water loss) and control (0%) based on one-way analysis of variance (ANOVA) analysis. Significant change in abundance >1.2 fold, p value < 0.05. (B) Heatmap representing the results of DEPs; hierarchical clustering results are represented by a treelike thermograph in which each row represents a phosphopeptide and each column represents a set of samples. The abundance represents the logarithmic values of expression amounts of the DEPs in different samples (log₂ expression). Different colors are shown in the thermogram, with red representing significantly upregulated DEPs, green representing significantly downregulated DEPs, and gray representing no quantitative information for DEP. (C) Cluster analysis of differential expression pattern for the 60 DEPs. (D) Subcellular location of DEPs.

Figure 3

GO and KEGG pathway analysis of phosphoproteins in N. flagelliforme between 75% water loss dehydration treatment and control. (A) Functional enrichment analysis of GO in group one-way ANOVA. The abscissa indicates the GO functional classification, which is divided into three major categories, biological processes (BPs), molecular function (MFs), and cell components (CCs); ordinates denote the number of differentially expressed proteins under each functional classification; the color bar denotes the significance of enriched GO functional classification based on the P-value calculated from Fisher’s exact test; the color gradient from orange to red represents the size of the P value: the closer the red, the smaller the P value and the higher the corresponding GO functional category richness level. The label above the bar chart shows the enrichment factor (richFactor ≤1), which indicates that the number of differentially expressed proteins annotated to a GO functional class account for all of the identified proteins annotated to that GO functional class. (B) Ordinates in the graph represent the significantly enriched KEGG pathway; the abscissa represents the number of differentially expressed proteins contained in each KEGG pathway, and the bar color represents the significance of the enriched KEGG pathway, i.e., the P value is calculated based on Fisher’s exact test. The color gradient represents the size of the P value, and the color changes from orange to red. The closer the red, the smaller the P value and the higher the significant level of the corresponding KEGG pathway enrichment.

Figure 4

Reverse transcription real-time polymerase chain reaction (RT-qPCR) testing for differential phosphoproteins related to carbon metabolism in N. flagelliforme on dehydration. Each bar data represents the average of three independent replicates (± standard error (SE)). (A–F) Dehydration-induced relative expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 6-phosphoglueonate dehydrogenase (6PGDH), glucose 6-phosphate dehydrogenase (G6PDH), phosphoribulosekinase (PRK), fructose-1,6-bisphosphatase (FBP), and phosphoglycerate kinase (PGK), respectively. The adjacent alphabetic letters to the bars reflect the significant levels based on one-way ANOVA (P < 0.05).

Figure 5

Accumulation of the antioxidant system and activities of antioxidant enzymes in N. flagelliforme on dehydration. (A) Heatmap of differently expressed phosphorylated proteins (DEPPs) related to the antioxidant system. (B–H) Enzymatic activities and substrates related to ROS signaling pathway involved in dehydration response, including catalase (CAT) activities, peroxidase (POD), superoxide dismutase (SOD) activities, and glutathione-S-transferase (GST) activities and glutathione reductase (GR), oxygen free radical (OFR), and H₂O₂ contents. For panels (B–H), each bar data represents the average of three independent replicates (±SE). The alphabetic letters adjacent to the bars reflect the significant levels based on one-way ANOVA (P < 0.05).

Figure 6

Phosphorylation motif analysis on all phosphorylated sites in N. flagelliforme across dehydration treatments and control. (A) Number of identified peptides containing phosphorylation sites in each motif. (B) Sequence motif analysis of phosphorylation sites. (C) Relative abundance of amino acid residues flanking the phosphorylation sites represented by an intensity map. The intensity map shows the relative abundance of six amino acids from the phosphorylation site. The colors in the intensity map represent the log₁₀ of the ratio of frequencies (red shows enrichment, green shows depletion). Default: occurrences = 10, significance = 0.00018, background = P17036_NCBI_Nostoc_flagelliforme_18909_20171228.

Figure 7

Schematic presentation of phosphoproteins related to photosynthesis, carbohydrate metabolism, energy conversion, and ROS scavenging of N. flagelliforme in response to drought stress. Dehydration stress induced a number of signaling molecules (SMs) that were accumulated in the cytoplasm. These SMs are then involved in the two-component signaling system, activating some protein kinases, such as histidine kinase (HistK) and downstream transcription factors (TFs). Under extremely server dehydration stress, some defensive proteins related to ROS scavenging were impaired and degraded by phosphorylation modification and some unknown pathways. The increased and decreased phosphorylation levels of proteins were depicted in red and blue, respectively. The dotted arrow was used to represent the potential cross talk between the photosynthetic light reaction and ROS scavenging through supplying reducing power by NADPH.