3.1 Effects of exogenous Calcium and Calcium inhibitors on SOD, POD, and CAT activities in B. rapa leaves and roots under low-temperature Stress
The SOD activity in the leaves of L7 and L99 increased gradually over time at a temperature 4°C in the CK (without application of CaCl2 and LaCl3). Following the application of calcium chloride treatment (CaCl2), there was a substantial rise of SOD activity in both cultivars compared to the CK. Furthermore, the increase in treatment time was directly proportional to the increase in the SOD activity. When LaCl3 was applied, it gradually decreased over time. The findings illustrate that the SOD activity in the leaves of L7 and L99 increased by 71.53% compared to the CK after being treated with CaCl2. However, it decreased by 49.83% compared to the CK after being treated with LaCl3 (Fig. 1A and B). The SOD activity in the roots of L7 and L99 increased gradually over time at a temperature of 4°C in the CK. It reached its peak at 24 hours and subsequently decreased at 48 hours. The trend seen after treatment with LaCl3 was comparable to that of the CK. Throughout all treatment periods, the activity of SOD was consistently lower in the LaCl3 treatment group compared to both the CK and CaCl2 treatment. The most notable change was detected after a period of 24 hours (Fig. 1C and D).
The POD activity in the leaves of L7 showed a progressive increase over time when exposed to a temperature of 4°C in the CK. The application of CaCl2 resulted in an initial rise in POD activity, which was subsequently followed by a decline as the treatment duration increased. When LaCl3 was applied, it caused a progressive decrease in the activity of POD compared to the CK over time. During the course of the experiment, the activity of POD in the leaves of L99 initially declined and then increased in response to the CK and CaCl2 treatments. However, after the application of CaCl2, the activity of POD was dramatically increased compared to the CK after 24 h. Over time, the application of LaCl3 resulted in a decrease in POD activity relative to the CK (Fig. 2A and B). The activity of POD in the roots of L7 increased gradually over time in the CaCl2 treatments. However, it was suppressed by LaCl3. The POD activity in the root of L99 exhibited a progressive increase over time in both the CK and CaCl2 groups, peaking at 12 h (Fig. 2C and D).
The CAT activity in the leaves of L7 and L99 increased gradually over time at a temperature of 4°C in the CK. After applying CaCl2, there was a significant increase in CAT activity in both cultivars compared to CK. Moreover, the duration of treatment showed a direct correlation with the rise in CAT activity. Upon the application of LaCl3, the activity of CAT exhibited a progressive decline over time in comparison to the CK. The results demonstrate that the CAT activity in the leaves of L7 and L99 showed a significant increase of 79.53% and 50.36% respectively, compared to the CK, after being treated with CaCl2. Nevertheless, it experienced a reduction of 30.83% in comparison to the CK following treatment with LaCl3 (Fig. 3A and B).
The CAT activity in the roots of L7 and L99 exhibited a progressive increase over time at a temperature of 4°C. In the CK, the CAT activity reached its maximum level after 24 hours. The activity of CAT was consistently lower than that of CK after treatment with LaCl3. These findings indicate that the application of LaCl3 worsened the oxidative damage to the root system induced by low temperature compared to that of the CK. During all treatment periods, the level of SOD activity was consistently lower in the LaCl3 treatment group compared to both the CK and CaCl2 treatments. The greatest significant alteration was observed after 24 h period (Fig. 3C and D).
3.2 Effects of exogenous calcium and calcium inhibitors on MDA, SP, and Proline contents in B. rapa leaves and roots under low-temperature Stress
Both cultivars of B. rapa exhibited an increasing trend of MDA content in their leaves after treatment with LaCl3. However, treatment with CaCl2 resulted in a decrease in MDA content compared to CK at the same treatment time. The L7 leaves showed a significant increase of 93.68% after being treated with LaCl3 over a 24h. On the other hand, after being treated with CaCl2, the MDA content initially decreased and then increased over time in both cultivars' leaves (Fig. 4A and B). The roots of both cultivars displayed an increasing trend in MDA level, the L99 cultivar showing more pronounced changes following a 12-hour treatment with LaCl3, as compared to the CK. The utilization of CaCl2 led to significant decreases in MDA content at 12 h, 24 h, and 48 h as compared to CK in both cultivars, as depicted in Fig. 4C and D.
The SP content in the L7 leaf increased substantially following CaCl2 and LaCl3 treatment at 12 h, 24 h, and 48 h, whereas it remained lower than CK in the case of LaCl3 treatment. While no significant difference was observed between the CK and treatment CaCl2 and LaCl3 in L99 leaves (Fig. 5A and B). The root exhibited an upward trend in SP content when subjected to CaCl2 treatments in both cultivars. It increased significantly after 6h of CaCl2 treatment compared to CK, while following the application of LaCl3, the SP content exhibited a decreasing trend compared to CK (Fig. 5C and D). Additionally, the SP content was increased significantly in the both cultivars after CaCl2 treatment compared to LaCl3 treatment at different treatment times of 6 h, 12 h, 24 h, and 48 h. More precisely, in L99 CaCl2 treatment did not show any significant changes at any treatment time compared to CK. However, the findings illustrate that LaCl3 treatment resulted in a significant reduction of SP content in leaves and roots of both cultivars, with an average of 33.96% compared to CK.
The content of proline in the CK reached its peak at 24h, while after the application of CaCl2 it reached it maximum at 12h, Conversely, it decreased following the LaCl3 treatment in L7. In the leaves of L99 Proline content was significantly higher after CaCl2 compared to CK at 12h and 24h. While after the treatment of LaCl3 it drops down significantly at 12h and 24h compared to CaCl2 and CK (Fig. 6A and B). Th proline content in the roots of L7 increased both in CK, and CaCl2 treatment, at 12h and then drops significantly. There was no significant difference between LaCl3 treatment and CK while the content of proline in L7 roots was 57.88% higher than CK at 48 h. In L99 Pro content of roots exhibited an increasing trend after CaCl2 treatment, reaching its maximum at 48 h, which was significantly increased by 149.19% com-pared to CK (Fig. 6C and D).
3.3 Effects of exogenous calcium and calcium inhibitors on Ca 2+ content of rapeseed roots under low-temperature stress
The Ca2+ contents in the roots of L7 maintained at lower level, and there were no significant changes occurs in the CK at 12h compared to 0h but the content increased at 24 h. while after CaCl2 treatment it drops significantly at 12h compared to CK, and after the application of LaCl3 it increases significantly at 12h and then drops at 24h compared to CK (Fig. 7A). In case of L99 a weak cold tolerant variety the Ca2 + contents in its roots maintained at higher level with a slight drop at 24h compared to 12h in CK. After the application of both CaCl2 and LaCl3 the Ca2+ contents drop significantly compared to CK. Both varieties showed obvious different trend of Ca2+ contents change in CK while slightly similar trend was observed after the application of CaCl2 and LaCl3 (Fig. 7A and B)
3.4 Analysis of changes in Ca2+ flow rate at low-temperature
Both L7 and L99 roots showed Ca2+ influx at normal temperature, while the influx rate was higher in L7 than in L99 (Fig. 7C). After low-temperature treatment, L99 showed Ca2+ efflux with a rate of 30.21 pmol‧cm-2‧s-1, whereas L7 briefly showed efflux then returned to influx.
3.5. Transcriptome analyses
A total of 289.78G of clean data was obtained from sequencing the referenced transcriptomes of 42 samples, with a range of 5.7G to 7.08G per sample. The distribution of Q30 bases ranged from 95.61–97.41%, with an average GC content of 46.61%. The percentage of reads that successfully matched the reference genome varied between 88.65% and 90.10%. The matching percentage to the unique position of the reference genome was 86.19–87.55%, and the matching percentage to multiple positions of the reference genome was 2.45–2.92%. A total of 42 samples were subjected to principal component analysis (PCA), as depicted in Fig. 8A and B. The samples exhibited notable disparities, and the replication within each group was satisfactory (Fig. 8C). These results satisfy the requirements for further analysis.
3.6 GO and KEGG enrichment analysis of DEGs
GO functional enrichment analysis was performed on the differentially expressed genes of L7 (Fig. 9A). At the 12-hour treatment time, the biological processes mainly annotated for the response to hydrogen peroxide, response to low temperature, response to salicylic acid, Jasmonic acid, and response to injury. In the cellular component category, annotations were made for photosystem II, chloroplast-like vesicle members, vesicle-like vesicles, chloroplasts, and others. The molecular function category showed significant enrichment in DNA-binding transcription factor activity, calmodulin binding, and phosphatase activity. DNA-binding transcription factor activity was the most enriched among the DEGs in all three group comparisons. In the three group comparisons after 24 hours of treatment, the biological processes were annotated to ribosomal RNA processing, translation, and DNA replication initiation. Cellular component category showed enrichment mainly in the nucleus and cytoplasmic ribosomes. Nucleus was the most enriched in the three group comparisons. Molecular function category showed significant enrichment on ribosomal structural components, photosystem II binding, and DNA replication initiation binding.
GO functional enrichment analysis of DEGs of L99 revealed significant enrichment in biological processes related to light intensity response, photosynthesis, low-temperature response, salicylic acid response, and response to Jasmonic acid in all three treatment groups at 12 h (Fig. 9B). Additionally, cellular components such as chloroplast-like vesicle members, chloroplasts, vesicles, and photosystems I and II were enriched. Molecular function categories were mainly annotated with DNA-binding transcription factor activity and sequence-specific DNA binding. Under 24-hour treatment, biological processes such as translation, rRNA processing, and photosynthesis were significantly enriched. The cellular components that were enriched include the nucleus, cytoplasmic ribosomes, cytosolic vesicles, and chloroplast-like vesicles. Molecular function categories that were enriched include ribosomal structural components, chlorophyll-binding, and mRNA binding.
The results illustrate that DEGs were significantly enriched in DNA-binding transcription factor activity, membrane constituents (Fig. 9C), and sequence-specific DNA binding in CK comparison (H-vs-A) of the two varieties. At treatment time of 12 h, the three comparisons (I vs B, J vs C, K vs D) showed enrichment in the following biological process categories: DNA binding transcription factor activity, positive transcriptional regulation, and DNA templates. A large number of genes were differentially expressed and enriched in response to chitin, wounding, water deficit, salicylic acid, and defense at 24 h treatment.
In L7 variety, MAPK signaling pathway, glycerol ester metabolism, nitrogen metabolism, photosynthesis antennae proteins, and phytopathogen interactions were significantly enriched in B-vs-A, C-vs-A, and D-vs-A groups (Fig. 10A). And MAPK signaling pathway, eukaryotic ribosome biogenesis, ribosomes, and photosynthesis were significantly enriched in E-vs-A, F-vs-A and G-vs-A groups. Meanwhile, In L99 variety (Fig. 10B), MAPK signaling pathway, phytohormone signaling, photosynthesis, and photosynthesis antenna proteins were significantly enriched in different groups (I-vs-H, J-vs-H, and K-vs-H). MAPK signaling pathway, ribosomes, eukaryotic ribosome biogenesis, photosynthesis, and photosynthesis antenna proteins were significantly enriched in L-vs-H, M-vs-H, and N-vs-H.
In comparison group of both varieties, significant enrichments in phytohormone signaling, tryptophan metabolism, starch, and sucrose metabolism, photosynthesis, MAPK signaling pathway, plant-pathogen interactions, and phenylpropanoid biosynthesis were observed (Fig. 10C). Furthermore, transcriptome analysis revealed 220 genes that were differentially expressed and enriched in MAPK signaling pathway in which we identified 6 candidate genes (LOC103873775, WRKY22, LOC103857966, LOC103866369, LOC103830167, LOC103837288) that were relevant to plant growth and development, as well as signal transduction.
3.7. Weighted gene co-expression network analysis and identification of Hub genes
A total of 42 samples were taken from the winter rapeseed root transcriptome, identifying 40,578 genes. Genes with low fluctuation of expression changes (standard deviation ≤ 0.5) were filtered, leaving 11,118 genes for WGCNA analysis. Based on pairwise correlations analysis of gene expression 26 merged co-expression modules marked with different colors are shown in Fig. 11A. The analysis of module-trait relationships for the 42 samples revealed that darkslateblue and lightblue4 were significantly positively correlated with POD, CAT, SP, and Pro, and negatively correlated with MDA, and significantly negatively correlated with Ca2+ content with the highest correlation coefficient (Fig. 11B). The number of genes contained in each module shown in Fig. 11C, whereas the darkorange module contains the highest number of genes (3390) and the darkolivegreen2 module contains the lowest number of genes (29).
Based on the values of WGCNA edge weight and node scores, the top 18 genes were identified in the darkslateblue and lightblue4 module, 9 genes from each module. In the darkslateblue module, it was observed that 50 genes exhibited up-regulation in L7, however the opposite trend was observed in L99 (Fig. 12A). The lightblue4 module contains a total of 38 genes. The majority of genes exhibited up-regulation following 12 hours of CaCl2 treatment in L7, whereas the majority of genes displayed down-regulation following 24 hours of CaCl2 treatment. Only a small number of genes showed up-regulation (Fig. 12B). These selected 18 genes from the two modules were subjected to functional annotation and expression analysis, while an additional 6 potential genes were screened within the MAPK signaling pathway (Fig. 12C and Table 2).
Table 2
Functional annotation of all candidate genes
Hub gene ID | Gene abbreviation | Functional annotation |
LOC103828211 | | uncharacterized |
LOC108871878 | | uncharacterized protein At1g43920, Chloroplastic-like |
LOC103839598 | | uncharacterized |
LOC103872839 | 4CLL2 | 4-coumarate–CoA ligase-like 2 |
LOC103847303 | SPH9 | pumilio homolog 15 |
LOC103829158 | Os03g0733400 | zinc finger BED domain-containing protein RICESLEEPER 2-like |
LOC108871342 | | uncharacterized LOC108871342 |
LOC103847561 | ZFP2 | zinc finger protein 8-like |
LOC108869010 | At3g58270 | MATH domain and coiled-coil domain-containing protein At3g58270-like |
LOC103828089 | At3g50520 | phosphoglycerate mutase-like protein 4 |
LOC103856351 | LTA2 | uncharacterized LOC103856351 |
LOC103850180 | | uncharacterized LOC103850180 |
LOC103863198 | ATHB-15 | homeobox-leucine zipper protein ATHB-15-like |
LOC103872950 | DPMS1 | probable dolichol-phosphate mannosyltransferase |
LOC108870222 | | uncharacterized LOC108870222 |
LOC103853785 | BI-1 | Bax inhibitor 1 |
LOC103858143 | WRKY12 | probable WRKY transcription factor 12 |
LOC103830648 | FIB1 | probable mediator of RNA polymerase II transcription subunit 36b |
LOC103857966 | CAM5 | calmodulin-5 |
WRKY22 | WRKY22 | WRKY transcription factor 22 |
LOC103837288 | CP1 | calmodulin |
LOC103873775 | SRK2H | serine/threonine-protein kinase SRK2H |
LOC103830167 | SRK2A | serine/threonine-protein kinase SRK2A |
LOC103866369 | ERS1 | ethylene response sensor 1 |
Furthermore, among these 24 candidate genes, 8 core candidate genes with special expression patterns were screened (Fig. 12C and 13). The expression levels of WRKY22 and LOC103837288 in CK were very low, but the expression increased significantly when it was treated CaCl2 and LaCl3 for 12 h (12h-Ca, 12h-La), and decreased significantly at 24 h (24h-Ca, 24h-La). LOC103873775 gene expression was significantly higher in L7 than in L99, and it was increased by CaCl2 treatment (12h-Ca, 24h-Ca,). The expression of LOC103857966 in L7was higher than that of L99 after treatment for 12 h (12h, 12H-CA, 12H-LA). It was higher in L99 than that of L7 at 24 h (24h, 24H-CA, 24H-LA), and the expression level of L7 was significantly decreased compared with that of 12 h. The expression of LOC103830167 was the highest in CK, but it was significantly lower than that of CK after adding exogenous substances at low temperature. The expression level of LOC103866369 in L99 (CK, 12h, 24h) was significantly increased with time, and the expression level of 24h was the highest, which was 358.91% higher than CK and 140.49% higher than that of 12h. LOC103863198 and LOC103858143 were highly expressed in L7 and low in L99, and the expression levels of LOC103863198 and LOC103858143 genes showed a decreasing trend with time in each treatment.