The different responses of tolerant and sensitive B. napus varieties to waterlogging stress

In our previous studies, it was discovered that 'Santana' is tolerant to flooding, while '23,651' is sensitive to flooding during the germination stage [30]. To examine the response of B. napus to waterlogging stress at the seedling stage, we selected 'Santana' as an extreme waterlogging-tolerant variety and '23,651' as an extreme waterlogging-sensitive variety for further study. Under normal conditions (CK), both varieties had similar root growth. However, after 7 days of waterlogging (WL), the growth inhibition in the sensitive variety was significantly more severe (Fig. 1a). Furthermore, the shoot fresh weight and root fresh weight of the sensitive variety were significantly reduced compared to the tolerant variety after the waterlogging treatment (Fig. 1b).

Fig. 1

The phenotypes of waterlogging tolerance and waterlogging sensitivity in B. napus varieties under waterlogging stress. a Growth of waterlogging tolerant (T) and sensitive (S) B. napus varieties under normal conditions (CK) and waterlogging (WL) for 7 days at 2-leaf stage. Bar = 5 cm. b Changes in shoot fresh weight and root fresh weight of waterlogged tolerant and sensitive B. napus varieties under CK and waterlogging conditions. Different letters indicate significant differences, while the same letters indicate no significant difference (n = 10, one-way ANOVA for multiple comparisons, P < 0.05). c Detection of cell membrane integrity by Evans Blue staining in root tips of waterlogging tolerant and sensitive B. napus varieties under CK and WL. Bars = 1 mm. d Measurement of O2.− content in root tips of waterlogging tolerant and sensitive B. napus varieties by Nitro Blue Tetrazolium (NBT) staining under CK and WL. Bars = 1 mm

The root of B. napus is the most susceptible organ to waterlogging stress and responds directly to the stress. Therefore, we focused on the changes in the root under waterlogging stress in B. napus. We investigated the degree of damage to the B. napus root tip caused by prolonged waterlogging stress. Evens Blue staining was then used to assess cell membrane integrity and the degree of damage in root tip cells. In the absence of waterlogging stress, the cell membrane was intact and staining was not prominent. However, after 10 to 12 h of waterlogging, the root tip of the sensitive variety showed obvious damage, and after 3 days of waterlogging, the cells began to collapse and dissociate. In contrast, the root of the tolerant variety showed damage after 1 day of waterlogging, and the cell membrane remained relatively intact even after 3 days of waterlogging (Fig. 1c). Additionally, Nitro Blue Tetrazolium (NBT) staining was performed on the root tip of both varieties. It was observed that under normal conditions, the root tips of both varieties were metabolically active and produced a certain level of O2.− (Fig. 1d). However, as the duration of waterlogging increased, the root cells gradually died and the level of O2.− decreased. After 12 h of waterlogging, the metabolic activity of root cells in the tolerant variety was normal and the level of O2.− remained relatively stable. In contrast, the sensitive variety exhibited impaired metabolic activity, resulting in a significant decrease in staining signals (Fig. 1d). Therefore, the results indicate that differential waterlogging tolerance between these two varieties may be attributed to the integrity and function of the root system.

Transcriptome analysis identified waterlogging-related metabolic pathways

To investigate the underlying genetic basis contributing to the contrasting root responses of the two materials under waterlogging conditions, transcriptome analysis was conducted to discern genetic differences between them. A total of three biological repetitions were performed at two treatment time points (12 h and 72 h) at the 2-leaf stage, corresponding to the roots of two B. napus lines (S, T), resulting in a total of 18 samples. Approximately ~ 60 Gb raw data were obtained. After quality control and filtering low-quality reads, high-quality reads were obtained, with an average of 95.57% of the reads being successfully mapped to the B. napus reference genome. Approximately 4.43% of the total reads were not matched due to stringent screening parameter settings, sequencing assembly errors, or an incomplete reference genome. Furthermore, both the heat map and PCA plot revealed that the gene expression correlations between biological repetitions of samples were above 90%, indicating a high level of reproducibility and confidence in the data (Additional file 1: Figure S1). For the transcriptome data, the differentially expressed genes (DEGs) in roots were identified based on the criteria of a P-value < 0.05 and |log2foldchange|> 1 (Additional file 2: Table S1, 2).

The results of transcriptome analysis revealed a significant increase in the number of DEGs in response to waterlogging stress, which was correlated with the duration of the waterlogging treatment (Fig. 2a; Additional file 2: Table S1-2). This could be attributed to the fact that the root is the most direct and sensitive organ to waterlogging damage in B. napus. In order to obtain a comprehensive expression profile of DEGs in B. napus roots under waterlogging stress, we conducted mclust analysis on 19,587 DEGs and divided them into 20 clusters. Additionally, we performed GO enrichment analysis on the biological processes of gene expression in each cluster. The pathways (FDR < 0.05) that were related to waterlogging stress or deemed significant in the GO enrichment analysis were marked for each cluster (Fig. 2b). Notably, clusters 2, 3, 9, and 14 exhibited biological processes that correspond to waterlogging stress in B. napus roots (Fig. 2b). For instance, clusters 2 and 14 were enriched in polysaccharide metabolic processes, disaccharide transport, and sucrose synthesis pathways (Additional file 1: Figure S2a-b; Additional file 2: Table S3, 4), while clusters 3 and 9 were enriched in singlet oxygen, oxygen-containing compounds, carbohydrate metabolism, and multiple pathways responsive to waterlogging injury (Additional file 1: Figure S2c-d; Additional file 2: Table S5, 6).

Fig. 2

Identification of differentially expressed genes (DEGs) and module division by transcriptome analysis. a Volcano map of DEGs compared with normal conditions (CK) and under waterlogging (WL) of B. napus roots. Red dots represent up-regulated genes, green dots represent down-regulated genes, and blue dots represent non-differentially expressed genes. The X-axis represents the fold change of difference after conversion to log2, and the Y-axis represents the significance value after conversion to -log10. b Mclust clustering heatmap of DEGs in the root. Heat map of DEGs clustered under different time points (12 h, 72 h) of waterlogged root, and the pathways with FDR < 0.05 and associated with waterlogged metabolism or the most significantly enriched pathway in each cluster are indicated. c Venn diagram of up-regulated and down-regulated DEGs in the roots of sensitive (S) and tolerant (T) B. napus varieties under WL at different time points. Each circle represents a set of gene sets. The overlapping areas of different circles represent DEGs common to the gene sets. Non-overlapping parts represent uniquely DEGs. The numbers in the figure represent the number of DEGs in the corresponding regions. d Mfuzz time clusters of DEGs in the root. Time clusters of DEGs in B. napus root at different time points (12 h, 72 h) under WL. The pathways with FDR < 0.05 and associated with waterlogged metabolism or the most significantly enriched pathway for each cluster gene in GO enrichment analysis are indicated

Furthermore, in both the tolerant and sensitive varieties, more genes were specifically down-regulated in the root. However, when compared to the control (CK), there were more genes regulated after 72 h of waterlogging in the tolerant variety, with the number of down-regulated genes being lower than that of up-regulated genes in the root (Fig. 2c). To obtain transcriptome time series characteristic expression profiles, we clustered genes with similar expression patterns through mfuzz clustering analysis to understand the biological dynamic patterns and functions of these genes. The gene expression matrices of the root were divided into 12 clusters, each representing a different gene expression pattern (Fig. 2d). All clusters in the root exhibited similar change trends in expression patterns between the tolerant and sensitive varieties under the same treatment, although the expression of genes in some modules differed slightly between the two varieties (Fig. 2d). Moreover, GO enrichment analysis was conducted on the biological processes of gene expression in each cluster. Under waterlogging conditions, many physiological and metabolic activities tend to be slowed down, such as oxidoreductase activity, organo nitrogen, catalytic activity, cell wall biogenesis, tubulin binding, phenylpropanoid metabolic, and amide biosynthetic processes (Fig. 2d). Conversely, metabolic processes associated with abiotic stress resistance were up-regulated, including zinc ion, response to abiotic stimulus, sucrose starvation, response to chemical stimulus, and binding (Fig. 2d). Cluster 2 and cluster 4 were primarily enriched in metabolic processes such as oxidoreductase activity, ATPase activator activity, and sugar starvation, which could be attributed to the anaerobic or anoxic condition induced by waterlogging stress, forcing the plants to limit aerobic respiration and energy production to sustain vital activity (Additional file 1: Figure S3a, b; Additional file 2: Table S7, 8). Genes in cluster 3 and cluster 9 predominantly responded to abiotic stimuli, and the expression patterns of genes in cluster 9 showed significant differences between the sensitive and tolerant varieties (Fig. 2d). Specifically, the sensitive variety exhibited consistent and smooth growth trends after 12 and 72 h of treatment, whereas no sudden increase in gene expression was observed in the tolerant variety after 72 h (Additional file 1: Figure S3c, d; Additional file 2: Table S9, 10). Furthermore, it was observed that genes in cluster 7 and cluster 10 were mainly enriched in cell wall synthesis, hemicellulose metabolic processes, secondary metabolic genes and phenylpropanoid metabolic processes (Fig. 2d; Additional file 1: Figure S3e, f). Additionally, the activity of cellulases, pectinases, and xylanases, may be involved in root cell wall biosynthesis under waterlogging stress (Additional file 1: Figure S3e, f; Additional file 2: Table S11, 12). Based on the transcriptome analysis, it is likely that cell wall polysaccharide metabolism plays a role in the response of B. napus to waterlogging stress.

Waterlogging stress altered cell wall structure and polysaccharide contents in root

The cell wall serves as an important barrier for plants to resist external adversity stress [18, 31]. Our transcriptome analysis revealed its involvement in the response of B. napus to waterlogging stress (Fig. 2b). Under normal conditions, cell structure remained intact, and cells were closely arranged in both tolerant and sensitive varieties. However, in the sensitive variety, the cell wall was broken and the intracellular structure was partially damaged in root cells after 12 h of waterlogging. The cell wall became thinner and almost no normal cell morphology or complete intracellular structure could be observed in the sensitive variety. Similar changes in the tolerant variety appeared after 24 h of waterlogging (Fig. 3a). The primary cell wall of plants is primarily composed of cellulose, hemicellulose, and pectin, which are crucial for plant cell morphogenesis [32]. By analyzing the contents of cellulose, hemicellulose, and pectin, we found that the pectin content significantly decreased in the sensitive variety after 1 and 3 days of waterlogging, while no significant difference was observed in the tolerant variety. Additionally, the hemicellulose content significantly decreased after 1 and 3 days of waterlogging compared to the control group in both the tolerant and sensitive varieties. However, the cellulose content did not significantly change in either the tolerant or sensitive varieties after 3 days of waterlogging, although it did significantly decrease in the sensitive variety after 1 day of waterlogging (Fig. 3b-d). These findings strongly suggest that the cell wall structure and polysaccharide contents undergo significant changes in root cells in response to waterlogging, and that pectin may play a vital role in the different responses of tolerant and sensitive B. napus varieties to waterlogging stress.

Fig. 3

Analysis of root cell wall structure and polysaccharides under waterlogging stress. a Ultrastructure of root tips of tolerant (T) and sensitive (S) B. napus varieties at 2-leaf stage under normal conditions (CK) and waterlogging (WL) for 12 h and 24 h observed by transmission electron microscopy (TEM). Bars = 2 μm. The red arrows point to the cell wall. b Determination of pectin content in waterlogging tolerant (T) and sensitive (S) B. napus varieties at 2-leaf stage under CK and WL for 1 day and 3 days. Different letters indicate significant differences, while the same letters indicate no significant differences (n = 6, one-way ANOVA for multiple comparisons, P < 0.05). c Determination of hemicellulose content in waterlogging tolerant and sensitive B. napus varieties at 2-leaf stage under CK and WL for 1 day and 3 days. d Determination of cellulose content in waterlogging tolerant and sensitive B. napus varieties at 2-leaf stage under CK and WL for 1 day and 3 days

BnaPGIP2s reduce pectin degradation by inhibiting the activity of polygalacturonases (PGs) in response to waterlogging stress

In B. napus, there are five homologous BnaPGIP2 genes, protein sequence analysis showed that there is a high similarity among BnaPGIP2s (Additional file 1: Figure S4a), sequence identities among BnaPGIP2s are more than 0.76 (Additional file 1: Figure S4b). The expression of three of the BnaPGIP2s (BnaA10g24080D, BnaA10g24090D, BnaC09g48700D) were up-regulated under waterlogging stress, and BnaA10g24090D showed a higher expression level in the roots of both tolerant and sensitive varieties after waterlogging for 72 h. The expression levels of BnaA10.PGIP2 (BnaA10g24090D) and BnaC09.PGIP2 (BnaC09g48700D) were significantly increased in the tolerant variety (Fig. 4a). On the other hand, phylogenetic analysis revealed that BnaA10.PGIP2 closely clustered with BnaC09.PGIP2 (Additional file 1: Figure S4c). To study the subcellular localization of BnaA10.PGIP2 and BnaC09.PGIP2, a plasma-wall separation experiment was conducted, and it was found that both BnaA10.PGIP2 and BnaC09.PGIP2 were localized to the cell wall (Fig. 4b). These results were consistent with the function of PGIP2s, which participate in pectin catabolism.

Fig. 4

BnaPGIP2s promote pectin accumulation in the root under waterlogging stress. a Heat map displays the expression pattern of BnaPGIP2s in response to waterlogging (WL) in tolerant (T) and sensitive (S) B. napus varieties. The redder the color bar, the higher the gene expression. It represents the expression level (TPM value) of BnaPGIP2s at 0 h, 12 h, and 72 h after root waterlogging. b Subcellular localization of BnaA10.PGIP2 and BnaC09.PGIP2 expressed in tobacco leaves. Cells were plasmolyzed by treatment with 0.75 mol L−1 mannitol for 15–20 min. Green fluorescence derived from BnaA10.PGIP2::GFP or BnaC09.PGIP2::GFP, orange fluorescence derived from membrane marker PM::OFP. The red arrowheads show BnaA10.PGIP2::GFP and BnaC09.PGIP2::GFP localized at the cell wall. Bars = 25 μm. c Determination of polygalacturonases (PGs) activity in BnaPGIP2s mutants under CK and WL for 1 day at 2-leaf stage (n = 4). d Determination of pectin content in BnaPGIP2s-overexpressing lines at 2-leaf stage under CK and WL for 1 day (n = 4). e Pectin level in the root tip of BnaPGIP2s mutants and overexpression lines at 2-leaf stage detected by ruthenium red staining under normal condition (CK) and WL for 3 days. Bars = 1 mm. f Determination of pectin content in BnaPGIP2s mutants at 2-leaf stage under CK and WL for 1 day and 3 days (n = 6). g Determination of pectin content in BnaPGIP2s-overexpressing lines at 2-leaf stage under CK and WL for 1 day and 3 days (n = 6). h Determination of hemicellulose content in BnaPGIP2s mutants under CK and WL for 1 day and 3 days (n = 6). i Determination of hemicellulose content in BnaPGIP2s-overexpressing lines at 2-leaf stage under CK and WL for 1 day and 3 days (n = 6). Different letters in the same chart indicate significant differences, while the same letters indicate no significant difference. (one-way ANOVA for multiple comparisons, P < 0.05)

To analyze the function of BnaA10.PGIP2 and BnaC09.PGIP2 in pectin catabolism under waterlogging stress, the CRISPR/Cas9 gene editing method was used to mutate the BnaA10.PGIP2 and BnaC09.PGIP2 genes in B. napus. Two sgRNA sites were designed for gene editing, with the sgRNA2 site being the off target. Based on the sgRNA1 site, single and double mutants were finally identified (Additional file 1: Figure S5a). CR-1 is the single mutant for BnaA10.PGIP2, while CR-2, CR-3, and CR-4 are the double mutants. BnaA10.PGIP2 and BnaC09.PGIP2 overexpression transgenic lines were also constructed in B. napus, respectively. qRT-PCR analysis showed that the expression of both BnaA10.PGIP2 and BnaC09.PGIP2 genes was significantly increased compared to the wild-type (WT) (Additional file 1: Figure S5b-c). Under normal conditions, the root PGs activity of all mutants was stronger than that in the WT, and the differences between the double mutants (CR-2, CR-3, and CR-4) and WT were significant (Fig. 4c). After 3 days of waterlogging, the differences between mutants and WT increased, and all mutants showed significantly higher root PGs activity than WT (Fig. 4c). Under normal conditions, the overexpression lines and WT had similar root PGs activity, except for the OE-A10-8 line, which showed weaker root PGs activity compared to WT (Fig. 4d). After 3 days of waterlogging, the root PGs activity of all overexpression lines was significantly lower than that in WT (Fig. 4d). To determine whether BnaPGIP2 affects polysaccharide pectin metabolism in B. napus, ruthenium red stain was used to directly stain the root tip. It was found that under normal conditions, the red signals in the overexpression lines were stronger, while the red signals were weaker in the mutants compared to WT, and this difference became even greater after 3 days of waterlogging (Fig. 4e). The pectin content of the single mutant (CR-1) roots showed a significant difference compared to WT after 1 day of waterlogging, and the double mutant (CR-2, CR-3, CR-4) showed significantly lower pectin content than WT after 1 day of waterlogging (Fig. 4f). The pectin content was the opposite in the overexpression lines. All overexpression lines showed an increasing trend in pectin content compared to WT under 3 days of waterlogging (Fig. 4g). The hemicellulose content showed a significant reduction after 1 day and 3 days of waterlogging compared to normal conditions in all lines, while there was no significant difference in hemicellulose content among all lines (Fig. 4h-i). These results suggest that both BnaA10.PGIP2 and BnaC09.PGIP2 confer a reduction in pectin degradation in B. napus roots by inhibiting the activity of PGs in response to waterlogging stress.

BnaPGIP2s improve physiological condition and enhance resistance to waterlogging stress in B. napus

To determine the effects of these genes, Evens Blue staining and NBT staining were performed on root tips of mutants and overexpression lines. Under normal conditions, both mutants and overexpression lines showed similar cell membrane integrity and O2.− levels as the wild type (Fig. 5a-b). However, after 12 h of waterlogging, the mutants exhibited greater damage to cell membranes, resulting in reduced O2.− production, while the overexpression lines showed the opposite phenotype (Fig. 5a-b). In terms of other physiological parameters, under normal conditions, the mutants and overexpression lines displayed similar levels of proline, malondialdehyde (MAD), and leaf water content as the wild type (Fig. 5c-h). However, after 7 days of waterlogging treatment, the proline levels in all mutants (CR-1, CR-2, CR-3, and CR-4) were higher than in the wild type, while the proline levels in all overexpression lines were lower (Fig. 5c-d). The MDA content in all mutants was higher than in the wild type, while the overexpression lines showed similar levels of proline after 7 days of waterlogging treatment (Fig. 5e-f). Leaf water content in all mutants was lower than in the wild type, with significant differences observed between the double mutants (CR-2, CR-3, and CR-4) and the wild type (Fig. 5g). Leaf water content in all overexpression lines was higher than in the wild type, whereas OE-A10-8 and OE-C09-4 showing significantly higher levels (Fig. 5h). In the absence of treatment, both mutants and overexpression lines had similar shoot fresh weight and root fresh weight compared to the control (Fig. 6a). However, after 7 days of waterlogging treatment, the shoot fresh weight and root fresh weight in the mutants tended to decrease, and the root fresh weight of the double mutants (CR-3, CR-4) significantly decreased (Fig. 6b-c). On the other hand, there was an overall increase in the overexpression lines compared to the wild type after 7 days of waterlogging treatment. OE-A10-7 and OE-A10-8 showed significant increases in shoot fresh weight, and OE-A10-8 and OE-C9-4 showed significant increases in root fresh weight (Fig. 6d-e). Taken together, these results suggest that both BnaA10.PGIP2 and BnaC09.PGIP2 confer waterlogging resistance to B. napus by reducing pectin degradation in the cell wall.

Fig. 5

BnaPGIP2s improve B. napus physiological condition under waterlogging stress. a Detection of cell membrane integrity by Evans Blue staining in the root tip of BnaPGIP2 mutants and overexpression lines under normal condition (CK) and waterlogging (WL) for 12 h at 2-leaf stage. Bars = 1 mm. b Measurement of O2.− in the root tip of BnaPGIP2s mutants and overexpression lines by NBT staining under CK and WL for 12 h at 2-leaf stage. Bars = 1 mm. c Measurement of proline content in the root of BnaPGIP2s mutants under CK and WL for 7 days at 2-leaf stage. d Measurement of proline content in the root of BnaPGIP2s-overexpressing lines under CK and WL for 7 days at 2-leaf stage. e Measurement of malondialdehyde (MDA) content in the root of BnaPGIP2s mutants under CK and WL for 7 days at 2-leaf stage. f Measurement of malondialdehyde (MDA) content in the root of BnaPGIP2s-overexpressing lines under CK and WL for 7 days at 2-leaf stage. g Measurement of leaf water content in the root of BnaPGIP2s mutants under CK and WL for 7 days at 2-leaf stage. h Measurement of leaf water content in the root of BnaPGIP2s-overexpressing lines under CK and WL for 7 days at 2-leaf stage. All statistical significance was determined by Student's t-test, n = 5, *P < 0.05, **P < 0.01

Fig. 6

BnaPGIP2s enhance B. napus resistance to waterlogging stress. a Identification of plant growth phenotype of BnaPGIP2s mutants and overexpression lines under normal condition (CK) and waterlogging (WL) for 7 days at 2-leaf stage. Bars = 5 cm. Measurement of shoot fresh weight (b) and root fresh weight (c) of BnaPGIP2s mutants under CK and WL at 2-leaf stage. Measurement of shoot fresh weight (d) and root fresh weight (e) of BnaPGIP2s-overexpressing lines under CK and WL at 2-leaf stage. Different letters in the same chart indicate significant differences, while the same letters indicate no significant difference (n = 6, one-way ANOVA for multiple comparisons, P < 0.05)