Physiological and comparative transcriptome analyses reveal the mechanisms underlying waterlogging tolerance in a rapeseed anthocyanin-more mutant

Abstract

Background: Rapeseed (Brassica napus) is the second largest oil crop worldwide. It is widely used in food, energy production and the chemical industry, as well as being an ornamental. Consequently, it has a large economic value and developmental potential. Waterlogging is an important abiotic stress that restricts plant growth and development. However, little is known about the molecular mechanisms underlying waterlogging tolerance in B. napus.

Results: In the present study, the physiological changes and transcriptomes of germination-stage rapeseed in response to waterlogging stress were investigated in the B. napus cultivar 'Zhongshuang 11' (ZS11) and its anthocyanin-more (am) mutant, which was identified in our previous study. The mutant showed stronger waterlogging tolerance compared with ZS11, and waterlogging stress significantly increased anthocyanin, soluble sugar and malondialdehyde contents and decreased chlorophyll contents in the mutant after 12 days of waterlogging. An RNA-seq analysis identified 1370 and 2336 differently expressed genes (DEGs) responding to waterlogging stress in ZS11 and am, respectively. An enrichment analysis revealed that the DEGs in ZS11 were predominately involved in carbohydrate metabolism, whereas those in the am mutant were particularly enriched in plant hormone signal transduction and response to endogenous stimulation. In total, 299 DEGs were identified as anthocyanin biosynthesis-related structural genes (24) and regulatory genes encoding transcription factors (275), which may explain the increased anthocyanin content in the am mutant. A total of 110 genes clustered in the plant hormone signal transduction pathway were also identified as DEGs, including 70 involved in auxin and ethylene signal transduction that were significantly changed in the mutant. Furthermore, the expression levels of 16 DEGs with putative roles in anthocyanin accumulation and biotic/abiotic stress responses were validated by quantitative real-time PCR as being consistent with the transcriptome profiles.

Conclusion: This study provides new insights into the molecular mechanisms of increased anthocyanin contents in rapeseed in response to waterlogging stress, which should be useful for reducing the damage caused by waterlogging stress and for further breeding new rapeseed varieties with high waterlogging tolerance.

Keywords: Anthocyanin; Brassica napus; Candidate gene; Physiological; Transcriptome; Waterlogging stress.

Fig. 1

Phenotypic differences between the wild type (WT) and am mutant at different periods after waterlogging. a The seeds germinated for 36 h were waterlogged for 0, 12, 24 and 36 h. b Effects of waterlogging on root lengths, stem lengths and fresh weights of the WT and am mutant. Error bars represent the SDs from three replicated experiments. Significant differences between the am mutant and WT at different time points are indicated (Student’s t-test) as follows: ***P < 0.001; **P < 0.01; *P < 0.05. c The young seedlings of the WT ZS11 and am mutant were waterlogged for 5, 7 and 12 d. d The morphological changes in the ZS11 and am mutant seedlings after waterlogging for 12 d. Bars = 1.0 cm

Fig. 2

Waterlogging stress had different effects on the physiological indicators of WT and the am mutant. a–f The chlorophyll a (a), chlorophyll b (b), total chlorophyll (c), anthocyanin (d), MDA (e) and soluble sugar (f) contents were determined in the WT and am mutant after 12 d of waterlogging stress. Error bars represent the SDs from three replicated experiments. Significant differences between the am mutant and WT with and without (CK) waterlogging stress are indicated (Student’s t-test) as follows: ***P < 0.001; **P < 0.01; *P < 0.05

Fig. 3

The expression profiles of waterlogging-regulated DEGs in the WT and am mutant. a Column diagram representing the numbers of DEGs in the WT and am mutant after the waterlogging treatment. b Venn diagrams showing DEGs and the overlaps of sets obtained across comparisons. c Heatmap clustering showing expression patterns of waterlogging-regulated DEGs based on FPKM values in the two different rapeseed cultivars

Fig. 4

Functional classifications of DEGs based on the a, b GO annotation and c, d KEGG pathway enrichments in the WT and am mutant

Fig. 5

Identification of DEGs involved in anthocyanin biosynthesis in the two different rapeseed cultivars. a Schematic diagram of anthocyanin biosynthetic pathways and key enzymes involved in the pathways. Anthocyanin early biosynthesis-related structural genes includes genes encoding phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumaroyl:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H) and flavonoid 3′-hydroxylase (F3′H). Anthocyanin late biosynthesis-related structural genes includes dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), UDP-flavonoid 3-O-glucosyltransferase (UF3GT) and anthocyanin acyltransferase (AAT). b Expression profiles of DEGs related to anthocyanin biosynthetic structural genes. Transcript level changes were normalized using log₂ FC transformed counts. The blue and yellow represent up- and downregulated expression, respectively. The red and green words represent DEGs and their FCs in the mutant and WT, respectively

Fig. 6

The distributions of differentially expressed TFs under waterlogging stress in a WT and b the mutant. The numbers in the histograms represent the number of up- or downregulated TFs in each TF family

Fig. 7

The distributions of DEGs related to plant hormone signal transduction pathways under waterlogging-stress conditions in a, b the mutant and c, d WT

Fig. 8

The qRT-PCR analysis of selected DEGs in B. napus leaves of the two different rapeseed cultivars during the waterlogging treatment. Data are presented as means ± SDs from three independent experiments. Student’s t-test was used for the statistical analysis between treated and untreated (control) samples in each rapeseed cultivar (*P < 0.05; **P < 0.01; ***P < 0.001)