Comparative Proteomic Analysis of Tolerant and Sensitive Varieties Reveals That Phenylpropanoid Biosynthesis Contributes to Salt Tolerance in Mulberry

Abstract

Mulberry, an important woody tree, has strong tolerance to environmental stresses, including salinity, drought, and heavy metal stress. However, the current research on mulberry resistance focuses mainly on the selection of resistant resources and the determination of physiological indicators. In order to clarify the molecular mechanism of salt tolerance in mulberry, the physiological changes and proteomic profiles were comprehensively analyzed in salt-tolerant (Jisang3) and salt-sensitive (Guisangyou12) mulberry varieties. After salt treatment, the malondialdehyde (MDA) content and proline content were significantly increased compared to control, and the MDA and proline content in G12 was significantly lower than in Jisang3 under salt stress. The calcium content was significantly reduced in the salt-sensitive mulberry varieties Guisangyou12 (G12), while sodium content was significantly increased in both mulberry varieties. Although the Jisang3 is salt-tolerant, salt stress caused more reductions of photosynthetic rate in Jisang3 than Guisangyou12. Using tandem mass tags (TMT)-based proteomics, the changes of mulberry proteome levels were analyzed in salt-tolerant and salt-sensitive mulberry varieties under salt stress. Combined with GO and KEGG databases, the differentially expressed proteins were significantly enriched in the GO terms of amino acid transport and metabolism and posttranslational modification, protein turnover up-classified in Guisangyou12 while down-classified in Jisang3. Through the comparison of proteomic level, we identified the phenylpropanoid biosynthesis may play an important role in salt tolerance of mulberry. We clarified the molecular mechanism of mulberry salt tolerance, which is of great significance for the selection of excellent candidate genes for saline-alkali soil management and mulberry stress resistance genetic engineering.

Keywords: TMT proteomics; mulberry; phenylpropanoid metabolism; salt stress.

Figure 1

Phenotypic and physiological characteristics of mulberry varieties Jisang3 and Guisangyou12 under control and saline conditions. (A) Performance of Jisang3 and Guisangyou12 plants under control and saline conditions. (B) Proline content of root in both J and G12 groups. (C) MDA content of root in both J and G12 groups. (D) Total antioxidant capacity of root of the two varieties in saline condition. (E–G) Ion content (potassium, calcium, sodium) of root of mulberry varieties Jisang3 and Guisangyou12 under control and saline conditions. Data are means ± SD with three biological replicates. Different letters represent statistically significant differences between control and salt treated plants by Duncan’s multiple range test (p < 0.05). Jck, JS, G12ck and G12S represent the two states of Jisang3 and Guisangyou12 under control and salt stress, respectively.

Figure 2

Physiological responses of mulberry to salt stress. (A) Net photosynthetic rate of mulberry leaves in both J and G12 groups. (B) Relative water content in both J and G12 groups. (C) Intercellular carbon dioxide concentration in both J and G12 groups. (D) Stomatal conductance to water vapor in both J and G12 groups. Data are means ± SD with three biological replicates. Different letters represent statistically significant differences between control and salt treated plants by Duncan’s multiple range test (p < 0.05). Jck, JS, G12ck and G12S represent the two states of Jisang3 and Guisangyou12 under control and salt stress, respectively.

Figure 3

Analysis of Venn diagram. (A,B) Venn diagram of differentially expressed proteins identified at different treatments. (C,D) Venn diagram of differentially expressed proteins identified between Jisang3 and Guisangyou12 in the saline condition.

Figure 4

Heatmap of differentially expressed proteins identified in mulberry roots after NaCl treatment. Fold change >1.5. Red represents a high gene expression level and green represents a low gene expression level.

Figure 5

Heatmap of proteins involved in phenylpropanoid biosynthesis pathway. (A) Heatmap of proteins identified in phenylpropanoid biosynthesis pathway. (B) Heatmap of DRPs involved in phenylpropanoid biosynthesis pathway. Red represents a high gene expression level and blue represents a low gene expression level.

Figure 6

Combined analysis of proteome and transcriptome. (A) Scatter plot of transcripts and their corresponding proteins. (B) GSEA analysis of KEGG pathway based on quantitative correlation coefficients of transcriptome and proteome.

Figure 7

A part scheme of phenylpropanoid metabolism. 4CL, 4-coumarate-CoA ligase; C3H, coumarate 3-hydroxylase; C3′H, p-coumaroyl shikimate 3′ hydroxylase; C4H, cinnamic acid 4-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; HCT, Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; CCR, cinnamoyl-CoA reductase; CHS, chalcone synthase; PAL, phenylalanine ammonia-lyase.