iTRAQ-based quantitative proteome analysis insights into cold stress of Winter Rapeseed (Brassica rapa L.) grown in the field

Abstract

Winter rapeseed (Brassica rapa L.) is a major oilseed crop in Northern China, where its production was severely affected by chilling and freezing stress. However, not much is known about the role of differentially accumulated proteins (DAPs) during the chilling and freezing stress. In this study, isobaric tag for relative and absolute quantification (iTRAQ) technology was performed to identify DAPs under freezing stress. To explore the molecular mechanisms of cold stress tolerance at the cellular and protein levels, the morphological and physiological differences in the shoot apical meristem (SAM) of two winter rapeseed varieties, Longyou 7 (cold-tolerant) and Lenox (cold-sensitive), were explored in field-grown plants. Compared to Lenox, Longyou 7 had a lower SAM height and higher collar diameter. The level of malondialdehyde (MDA) and indole-3-acetic acid (IAA) content was also decreased. Simultaneously, the soluble sugars (SS) content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, soluble protein (SP) content, and collar diameter were increased in Longyou 7 as compared to Lenox. A total of 6330 proteins were identified. Among this, 98, 107, 183 and 111 DAPs were expressed in L7 CK/Le CK, L7 d/Le d, Le d/Le CK and L7 d/L7 CK, respectively. Quantitative real-time PCR (RT-qPCR) analysis of the coding genes for seventeen randomly selected DAPs was performed for validation. These DAPs were identified based on gene ontology enrichment analysis, which revealed that glutathione transferase activity, carbohydrate-binding, glutathione binding, metabolic process, and IAA response were closely associated with the cold stress response. In addition, some cold-induced proteins, such as glutathione S-transferase phi 2(GSTF2), might play an essential role during cold acclimation in the SAM of Brassica rapa. The present study provides valuable information on the involvement of DAPs during cold stress responses in Brassica rapa L, and hence could be used for breeding experiments.

Figure 1

The growth characteristics of (A) Longyou 7 and (B) Lenox varieties. The SAM and collar morphology of (C) Longyou 7 and (D) Lenox. Longyou 7 and Lenox varieties (E) IAA content, (F) MDA content, (G) SP content, (H) SS content, (I) POD activity, (J) SOD activity, (K) Collar diameter, (L) Height of SAM. Error bars denote the standard error of the mean. An letter denotes significant differences between varieties at p ≤ 0.05. SOD superoxide, POD peroxidase, IAA indole-3-acetic acid, SP soluble protein, MDA malondialdehyde, SS soluble sugar.

Figure 2

Venn diagrams of DAPs identified by iTRAQ between (A) L7 d/ L7 CK and Le d/ Le CK, (B) L7 d/ Le d and L7 CK/ Le CK. L7 d/ L7 CK is the protein abundance ratio of L7 d compared with L7 CK, Le d/ Le CK is the protein abundance ratio of Le d compared with Le CK, L7 CK/ Le CK is the protein abundance ratio of L7 CK compared with Le CK, and L7 d/ L7 CK is the protein abundance ratio of L7 d compared with L7 CK. (C) Number of up- and down-accumulated proteins among different comparison groups.

Figure 3

(A) Gene ontology (GO) enrichment analysis of up-regulated DAPs in L7 CK/Le CK. (B) GO enrichment analysis of down-regulated DAPs in L7 CK/Le CK.

Figure 4

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of (A) down-regulated DAPs and (B) up-regulated DAPs in L7 d/Le d.

Figure 5

qRT-PCR analysis of genes related to DAPs. Transcript abundance was calculated according to the difference in cycle threshold values between the target gene and β-actin transcripts normalized by the 2 − ΔΔCT method.