Integrative Proteomic and Phosphoproteomic Analyses Revealed the Regulatory Mechanism of the Response to Ultraviolet B Stress in Clematis terniflora DC

Abstract

Clematis terniflora DC. (C. terniflora) has been used as an ancient Chinese traditional herbal medicine. The active substances in C. terniflora have been confirmed to be effective in treating diseases such as prostatitis. UV light radiation is a common environmental factor that damages plants and influences primary and secondary metabolism. Previous studies showed that ultraviolet B (UV-B) radiation followed by dark stress resulted in the accumulation of secondary metabolites in C. terniflora leaves. An in-depth understanding of how C. terniflora leaves respond to UV-B stress is crucial for improving C. terniflora value. Here, we conducted label-free proteomic and phosphoproteomic analyses to explore the protein changes under UV-B and UV-B combined with dark treatment. A total of 2839 proteins and 1638 phosphorylated proteins were identified. Integrative omics revealed that the photosynthetic system and carbohydrate balance were modulated under both stresses. The phosphoproteomic data indicated that the mitogen-activated protein kinase signaling pathway was triggered, while the abundance of phosphorylated proteins related to osmotic stress was increased under UV-B stress. Differentially abundant phosphoproteins from UV-B followed by dark treatment were mainly enriched in response to stimulus including calcium-mediated proteins. This study provides new insight into the impact of UV-B stress on C. terniflora and plant molecular resistance mechanisms through proteomic and phosphoproteomic analyses.

Figure 1

Differentially abundant proteins (DAPs) identified in response to UVB and UVD stresses. Volcano maps of DAPs in (A) UVB/CK and (B) UVD/CK. Red dots refer to increased DAPs, and blue dots refer to decreased DAPs. (C) Venn diagram shows the overlap of DAPs under two different treatments. Bar chart of significantly enriched KEGG pathways of DAPs in (D) UVB/CK and (E) UVD/CK. The x axis of (D) and (E) represents −log10 (p value) of KEGG enrichment significance.

Figure 2

Clustering heatmap of DAPs related to photosynthesis and photosynthesis–antenna protein pathways. (A) DAPs in UVB/CK. (B) DAPs in UVD/CK.

Figure 3

Statistical information of phosphorylation sites and subcellular localization of differentially phosphorylated proteins. Distribution of (A) phosphorylated sites (serine, Ser; threonine, Thr; tyrosine, Tyr) in identified phosphoproteins. (B) Proportion of phosphorylation site numbers per identified phosphoprotein. The subcellular localization of DPPs of (C) UVB/CK and (D) UVD/CK predicted by WoLF PSORT.

Figure 4

Bubble plot of significantly enriched KEGG pathways of DPPs in (A) UVB/CK and (B) UVD/CK. The color of the bubble represents the −log10 (p value) of KEGG enrichment significance. The size of the bubble represents the number of DPPs.

Figure 5

Levels of (A) total chlorophyll, (B) chlorophyll a, and (C) chlorophyll b of C. terniflora leaves from CK, UVB, and UVD groups. The different letters indicate significant differences according to one-way ANOVA.

Figure 6

(A) Soluble sugar content and (B) T-AOC content change in different groups of C. terniflora (CK, UVB, and UVD). Letters a, b, and c indicated that the content changed significantly according to one-way ANOVA.

Figure 7

Identified DAPs from UVD/CK and UVB/CK mapped to carbohydrate metabolism. The red ellipses represent increased DAPs. The blocks with the letter “B” inside indicate that the DAP comes from UVB/CK, and those with the letter “D” mean that the DAP comes from UVD/CK. G3P, glyceraldehyde 3 phosphate; G1P, glucose 1 phosphate; F6P, fructose 6-phosphate; S6P, sucrose 6 phosphate; GP, glycogen phosphorylase; SS, starch synthase; SUS, sucrose synthase; SPS, sucrose phosphate synthase; and SPP, sucrose phosphatase.

Figure 8

DPPs of UVB/CK mapped to the MAPK signaling pathway. The red blocks represent increased phosphorylated proteins, while the green blocks represent decreased phosphorylated protein.