iTRAQ Proteomic Analysis of Continuously Cropped Soybean Root Inoculated With Funneliformis mosseae

Abstract

Soybean (Glycine max) is susceptible to root rot when subjected to continuous cropping, and this disease can seriously diminish the crop yield. Proteomics analyses can show the difference of protein expression in different treatment samples. Herein, isobaric tag for relative and absolute quantitation (iTRAQ) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed for proteomic analysis of continuously cropped soybean inoculated with the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae. The AMF can reduce the incidence of root rot and increase plant height, biomass index in 1, 2, and 4 year of continuous cropping. Differential expression of proteins in soybean roots was determined following 1 year of continuous cropping. A total of 131 differentially expressed proteins (DEPs) were identified in F. mosseae-treated samples, of which 49 and 82 were up- and down-regulated, respectively. The DEPs were annotated with 117 gene ontology (GO) terms, with 48 involved in biological processes, 31 linked to molecular functions, and 39 associated with cell components. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis mapped the DEPs to 113 mainly metabolic pathways including oxidative phosphorylation, glycolysis, and amino acid metabolism. Expression of glucan 1,3-beta-glucosidase, chalcone isomerase, calcium-dependent phospholipid binding and other defense-related proteins was up-regulated by F. mosseae, suggesting inoculation promotes the growth and development of soybean and increases disease resistance. The findings provide an experimental basis for further research on the molecular mechanisms of AMF in resolving problems associated with continuous soybean cropping.

Keywords: Funneliformis mosseae; LC-MS/MS; continuous soybean cropping; differentially expressed proteins; iTRAQ proteomic analysis; root rot.

FIGURE 1

Hyphae and vesicle structures in soybean roots (10 × 40). (A–C) infection by Funneliformis mosseae in treatment groups at 40, 50, and 60 days after infection, respectively.

FIGURE 2

Soybean root rot index At 60 days after planting. C, control group; T, treatment group; x-axis 1, 2, and 4 represents the years of continuous cropping. Vertical bars represent the standard deviation of the means. Lowercase indicate significant differences at 0.05 level.

FIGURE 3

Plant height in continuous cropping soil at different growth stages. C, control group; T, treatment group. 1, 2, and 4 represents the years of continuous cropping. The small letters denote the significant differences among the different treatments at the same growth stages; the capital letters denote the significant differences among the same treatment at different growth stages. The differences in each parameter were detected at P < 0.05 level.

FIGURE 4

Summary of iTRAQ results. (A) Molecular weight. (B) Isoelectric point. (C) Sequence coverage. (D) The degree of up- and down-regulation of differentially expressed proteins (DEPs), colored red, and green, respectively.

FIGURE 5

Volcano plot of treatment and control groups. The x-axis represents log2 fold-change values, and the y-axis indicates significant differences in log10 fold-change values.

FIGURE 6

Gene ontology (GO) enrichment analysis of DEPs. DEPs in each group are sorted into three categories; biological process (BP), molecular function (MF), and cellular component (CC). Different GO terms are indicated on the x-axis, and the number of proteins in the indicated categories is indicated on the y-axis.

FIGURE 7

(A) KEGG enrichment analysis of the identified DEPs. The x-axis indicates the degree of enrichment (rich factor), and the y-axis represents enriched KEGG pathways. (B) Pathways linked to the identified DEPs. The x-axis indicates different KEGG pathways, and the y-axis represents the number of proteins.

FIGURE 8

Cluster analysis of DEPs at 60 days after planting in continuous cropping for 1 year. C, control group; T, treatment group. 1, 2, and 3, parallel samples.

FIGURE 9

Phenylalanine ammonia-lyase activity of continuously cropped soybean roots at different growth stages. C, control group; T, treatment group. 1, 2, and 4 represents the years of continuous cropping. The small letters denote the significant differences among the different treatments at the same growth stages; the capital letters denote the significant differences among the same treatment at different growth stages. The differences in each parameter were detected at P < 0.05 level.