BrrICE1.1 is associated with putrescine synthesis through regulation of the arginine decarboxylase gene in freezing tolerance of turnip (Brassica rapa var. rapa)

Abstract

Background: In the agricultural areas of Qinghai-Tibet Plateau, temperature varies widely from day to night during the growing season, which makes the extreme temperature become one of the limiting factors of crop yield. Turnip (Brassica rapa var. rapa) is a traditional crop of Tibet grown in the Tibet Plateau, but its molecular and metabolic mechanisms of freezing tolerance are unclear.

Results: Here, based on the changes in transcriptional and metabolic levels of Tibetan turnip under freezing treatment, the expression of the arginine decarboxylase gene BrrADC2.2 exhibited an accumulative pattern in accordance with putrescine content. Moreover, we demonstrated that BrrICE1.1 (Inducer of CBF Expression 1) could directly bind to the BrrADC2.2 promoter, activating BrrADC2.2 to promote the accumulation of putrescine, which was verified by RNAi and overexpression analyses for both BrrADC2.2 and BrrICE1.1 using transgenic hair root. The function of putrescine in turnip was further analyzed by exogenous application putrescine and its inhibitor DL-α-(Difluoromethyl) arginine (DFMA) under freezing tolerance. In addition, the BrrICE1.1 was found to be involved in the ICE1-CBF pathway to increase the freezing stress of turnip.

Conclusions: BrrICE1.1 could bind the promoter of BrrADC2.2 or CBFs to participate in freezing tolerance of turnip by transcriptomics and targeted metabolomics analyses. This study revealed the regulatory network of the freezing tolerance process in turnip and increased our understanding of the plateau crops response to extreme environments in Tibet.

Keywords: Brassica rapa var. rapa; Freezing tolerance; Metabolome; Putrescine; Transcriptome.

Fig. 1

Freezing treatment and physiological index determination of turnip KTRG-B49. a Freezing phenotypes. b Electrolyte leakage (%). Values are the mean of 5–8 biological replicates. Bars indicate SD. Different symbols indicate significant differences between treatments (P < 0.05) according to Tukey’s test. CK, C1, C4, C6, F2, F4, and R12 represent the samples from the untreated control; 4 °C at 1 h, 3 h, and 6 h; − 2 °C at 2 h; − 4 °C 1 h; and recovery for 12 h, respectively. c Survival rates. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, Student’s t test)

Fig. 2

Transcriptome and qRT-PCR analysis of key genes involved in the polyamine biosynthesis pathway in turnip. a Principal component analysis (PCA) of transcriptome data. The scale of the axis is the relative distance. Different colors or shapes represent different groups of samples under freezing treatments. b Different trend (subcluster_1) analysis of DEGs. c Scatterplot of KEGG pathways enriched for differentially expressed genes (DEGs) in subcluster_1. The top 5 enriched pathway terms in the KEGG database are listed. d Polyamine biosynthetic pathway (right) in plants. e qPCR analysis of key gene in polyamines biosynthesis, with three biological and technical replicates. The data are analyzed by one-way ANOVA (Tukey’s test). Different letters indicate significant difference (P < 0.05). CK, C1, C4, C6, F2, F4, and R12 represent the samples from the untreated control; 4 °C at 1 h, 3 h, and 6 h; − 2 °C at 2 h; − 4 °C 1 h; and recovery for 12 h, respectively

Fig. 3

Determination of polyamine content after freezing treatments and weighted gene coexpression network analysis (WGCNA) analysis of module eigengenes and polyamine metabolic profiles. a The contents of agmatine, putrescine, cadaverine, and spermidine under freezing treatment in turnip. Values are the mean of five biological replicates. Bars indicate SD. Different symbols indicate significant differences between treatments (P  <  0.05) according to Tukey’s test. b Clustering dendrogram of expressed genes. Gene modules were identified by dynamic hierarchical tree cut and shown in different colors. c For each module, the heatmap showed module eigengene (ME) correlations to traits. Numbers in each rectangular indicate the correlation coefficients and Student’s asymptotic P value for significant ME-trait relationships. The scale bar, bottom, indicates the range of possible correlations from positive (red, 1) to negative (blue, -1)

Fig. 4

Binding motif analysis in the target regions of turnip BrrICE1.1 and interaction analysis of BrrICE1.1 with the promoter of the differentially expressed genes in the polyamine pathway in vivo and in vitro. a The potential MYC-binding site (CANNTG) of BrrICE1.1. The binding sequences of the BrrICE1.1 with BrrAIH1.1, BrrAIH1.2, BrrADC2.1, and BrrADC2.2 are shown in red box. b Yeast one-hybrid assays showed that the MYC element mediates BrrICE1.1 binding to the BrrAIH1.1, BrrAIH1.2, BrrADC2.1, and BrrADC2.2 promoters, and the BrrAIH1.1, BrrAIH1.2, BrrADC2.1, and BrrADC2.2 promoters were mutated (deleted MYC element) to abolish the MYC element alone. The experiments were repeated three times with the same results. c BrrICE1.1 activated the activity of BrrAIH1.1, BrrAIH1.2, BrrADC2.1, and BrrADC2.2 in vivo. N. benthamiana leaves. Representative images of N. benthamiana leaves 72 h after infiltration are shown. d ChIP experiment using BrrICE1.1-6flag transgenic hair root. The structure of the BrrADC2.2 gene promoter. The primer sequence regions used for ChIP assays are marked with a horizontal line to the left of the TSS. The control primer sequence (GD) was on the left side of TSS. ChIP-qPCR showing binding of BrrICE1.1 to BrrADC2.2 promoters in vivo. WT and BrrADC2.2-GD were used as negative controls. The data are the mean of three replicates ± SD, and the asterisks indicate significant differences compared with IgG (*P < 0.05, **P < 0.01, Student’s t test)

Fig. 5

Detection of expression and putrescine contents in transgenic and RNAi hairy roots. a The phenotype of the hair root (GFP, control; BrrADC2.2-OE, overexpression of BrrADC2.2 in hair root; BrrICE1.1-OE, overexpression of BrrICE1.1 in hair root). b Laser confocal detection of overexpressed GFP, BrrADC2.2-OE and BrrICE1.1-OE in the hairy roots. CN: cell nucleus, CM: cell membrane. Bar = 20 μm. c d The expression levels of BrrADC2.2 and putrescine contents in BrrADC2.2-OE transgenic and BrrADC2.2-RNAi hairy roots compared with those of the CK. e Laser confocal detection of overexpressed BrrICE1.1 and its amino acid sequence mutant (BrrICE1.1-m) protein localization in N. benthamiana leaves. Bar = 20 μm. f The structure of the AtICE1 and BrrICE1.1 protein at the N-terminus (1–30). The red line represents a single peptide in the BrrICE1.1 protein. g The expression levels of BrrICE1.1 and BrrADC2.2 in BrrICE1.1-OE transgenic hair roots (left) and BrrICE1.1 and BrrADC2.2 in BrrICE1.1-RNAi transgenic hair roots (right). h Putrescine contents in BrrICE1.1-OE transgenic and BrrICE1.1-RNAi hairy roots. In c, d, g and h, the data are the mean of three replicates ± SD, and the asterisks indicate significant differences compared with the CK (*P < 0.05, **P < 0.01, Student’s t test)

Fig. 6

Effects of exogenous putrescine and its inhibitor on freezing tolerance of KTRG-B49. a Representative plants of KTRG-B49 with and without 1.5 mM putrescine and 0.5 mM DFMA before (Control) and after freezing treatment (Freezing), respectively. b Electrolyte leakage of leaves from control and freezing treated plants. Values are means of 5–8 biological replicates. Bars indicate SD. Different symbols indicate significant differences between treatments (P < 0.05) according to Tukey’s test

Fig. 7

Expression of the BrrICE1.1, BrrCBF3 and BrrCOR15A genes upon freezing treatment with three biological and technical replicates. Different symbols indicate significant differences between treatments (P < 0.05) according to Tukey’s test

Fig. 8

BrrICE1.1 bound to the BrrCBF3 promoter. a The potential binding site of BrrICE1.1 is the MYC element (CATTTG) in the BrrCBF3 promoter. b Yeast one-hybrid assays showed that the MYC element mediates BrrICE1.1 binding to the BrrCBF3 promoter and that the BrrCBF3 promoters were mutated (deleted MYC element) to abolish the MYC element alone. The experiments were repeated three times with the same results. c BrrICE1.1 activated the binding activity of BrrCBF3 in vivo; N. benthamiana leaves were transformed with the positive control (35S::LUC) and negative control (35S::BrrICE1.1, BrrCBF3pro::LUC), and the interaction was detected. Representative images of N. benthamiana leaves 72 h after infiltration are shown. d ChIP experiment using BrrICE1.1-6flag transgenic hair root. The structure of the BrrCBF3 gene promoter. The primer sequence regions used for ChIP assays are marked with a horizontal line to the left of the TSS. The control primer sequence (GD) is on the left side of the TSS. ChIP-qPCR showing binding of BrrICE1.1 to the BrrCBF3 promoters in vivo. WT and BrrCBF3-GD were used as negative controls. e The expression of BrrCBF3 in BrrICE1.1-OE and BrrICE1.1-RNAi transgenic hair roots. f BrrICE1.1 regulatory network under freezing stress in turnip. In d and e, the data are the mean of three replicates ± SD, and the asterisks indicate significant differences compared with the control (*P < 0.05, **P < 0.01, Student’s t test)