Unraveling the Root Proteome Changes and Its Relationship to Molecular Mechanism Underlying Salt Stress Response in Radish (Raphanus sativus L.)

Abstract

To understand the molecular mechanism underlying salt stress response in radish, iTRAQ-based proteomic analysis was conducted to investigate the differences in protein species abundance under different salt treatments. In total, 851, 706, and 685 differential abundance protein species (DAPS) were identified between CK vs. Na100, CK vs. Na200, and Na100 vs. Na200, respectively. Functional annotation analysis revealed that salt stress elicited complex proteomic alterations in radish roots involved in carbohydrate and energy metabolism, protein metabolism, signal transduction, transcription regulation, stress and defense and transport. Additionally, the expression levels of nine genes encoding DAPS were further verified using RT-qPCR. The integrative analysis of transcriptomic and proteomic data in conjunction with miRNAs was further performed to strengthen the understanding of radish response to salinity. The genes responsible for signal transduction, ROS scavenging and transport activities as well as several key miRNAs including miR171, miR395, and miR398 played crucial roles in salt stress response in radish. Based on these findings, a schematic genetic regulatory network of salt stress response was proposed. This study provided valuable insights into the molecular mechanism underlying salt stress response in radish roots and would facilitate developing effective strategies toward genetically engineered salt-tolerant radish and other root vegetable crops.

Keywords: association analysis; iTRAQ; proteomics; radish; salt stress.

Figure 1

Primary data analysis and protein identification. (A) Basic information statistics. (B) Number of peptides that match proteins using MASCOT. (C) Distribution of protein's sequence coverage. (D) Protein mass distribution.

Figure 2

GO classification of the identified protein species.

Figure 3

COG classification of the identified protein species.

Figure 4

Identification and statistics of differential abundance protein species (DAPS) under salt stress. (A) Number of up- or down-accumulated protein species between any two different salt treatments. (B) Venn diagram analysis of up-accumulated protein species. (C) Venn diagram analysis of down-accumulated protein species.

Figure 5

Classification of differential abundance protein species (DAPS) into cellular and molecular categories.

Figure 6

The most significantly-enriched GO terms of differential abundance protein species (DAPS) between any two different salt treatments (p-value ≤ 0.001).

Figure 7

Relative mRNA expression analysis using RT-qPCR on nine protein species under diverse salt treatments. The expression level in the untreated samples (0 mM) was set to a value of 1. Each bar shows the mean ± SE (n = 3). Letters above the columns indicate significant differences at p < 0.05 according to Duncan's multiple range test.

Figure 8

A schematic genetic regulatory network model of salt stress response in radish. The miRNAs and transcription factors (TFs) were identified in previous studies (Sun et al., 2015, 2016). ROS, Reactive oxygen species; CAM, calmodulin; CML, calmodulin-like protein; CDPK, calcium-dependent protein kinase; PLC, phospholipase C; MYB, myeloblastosis protein; NAC, (No Apical Meristem) domain-containing protein; bHLH, basic helix-loop-helix; PAQ, plasma membrane aquaporin; eIF, eukaryotic initiation factor; EF, elongation factor; PFK3, 6-phosphofructokinase 3; HXK, hexokinase; PK, pyruvate kinase; ENO1, enolase 1; FBA, fructose-bisphosphate aldolase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; PDH, pyruvate dehydrogenase; SDH, succinate dehydrogenase.