Effects of exogenous calcium and calcium inhibitor on physiological characteristics of winter rape (Brassica rapa) under low temperature stress

Effects of exogenous calcium and calcium inhibitor on physiological characteristics of winter rape (Brassica rapa) under low temperature stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of exogenous calcium and calcium inhibitor on physiological characteristics of winter rape (Brassica rapa) under low temperature stress Junyan Wu, Qiaowen Pan, Fahim Abbas Muhammad, Lulu Zhang, Lijun Liu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4186636/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Oct, 2024 Read the published version in BMC Plant Biology → Version 1 posted 9 You are reading this latest preprint version Abstract Low temperature is one of the environmental factors that restrict the growth and geographical distribution of brassica. To investigate the effects of exogenous calcium and calcium inhibitors on the ability of winter rapeseed ( Brassica rapa L.) to withstand low temperatures, and its effect on physiological characteristics we used a strong cold-resistant Longyou 7 (L7) and a weak cold-resistant Longyou 99 (L99) cultivar. The seedlings were treated with CaCl 2 (20 mmol·L -1 ) and the calcium inhibitor LaCl 3 (10 mmol·L -1 ) at 0 h, 6 h, 12 h, 24 h and 48 h, the Ca 2+ flux and Ca 2+ concentration in the roots after 12 h and 24 h of treatment were analyzed, and results after low-temperature treatment, L99 showed Ca 2+ efflux with a rate of 30.21 pmol‧cm -2 ‧s -1 , whereas L7 briefly showed efflux then returned to influx. Moreover. our findings illustrate that under low-temperature conditions, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased by both CK and exogenous CaCl 2 treatments. The contents of soluble protein (SP) and proline (Pro) were increased, while the contents of malondialdehyde (MDA) were decreased, resulting in reduced membrane lipid peroxidation. But enzyme activity decreased and MDA content increased following treatment with exogenous LaCl 3 . The rate of Ca 2+ flow showed a higher uptake in L7 roots compared with L99. Calcium ion content in root showed a decrease in ion content in both cultivars after CaCl 2 treatment. The results of RNA-seq data revealed that the genes that are differentially expressed in response to low temperatures, hormones, photosystem II, chloroplasts, DNA replication, ribosomal RNA processing, and translation were significantly enriched. This study found significant expression of genes related to cellular signal transduction (MAPK signaling pathway) and material metabolism (nitrogen metabolism, glycerol ester metabolism). The analysis of MAPK signaling pathway and genes in two modules led to the screening of 8 candidate genes related to the regulation of root growth, development and signal transduction. Winter rapeseed Low temperature Calcium chloride Lanthanum chloride Antioxidant enzyme activity. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 1. Background Winter rapeseed ( Brassica rapa L.) is an important oilseed crop globally. In China, it contributes 5.2 million tons of high-quality edible oil per year, which represents over 50% of the country's total oil crop production [ 1 ] . Nevertheless, China is still lacking in the production and edible oil of rapeseed. It has always been important for us to produce more rapeseed. Winter rapeseed is the main cultivation type of rapeseed in China, and it is cultivated in both north and south [ 2 ] . But in north, freezing temperature in winter and chilling in spring has negatively impacted on the yield, growth, and quality of oil in China [ 3 ] . Therefore, it is crucial to look for the strong cold-resistant genes of winter rapeseed and enhance its cold resistance to ensure the safe overwintering in north China. This is of great significance for the development of the rapeseed industry and the protection of China grain and oil security. The application of exogenous substances is one of the simplest and most effective methods. Calcium chloride (CaCl 2 ), an inorganic salt, is used to help plants withstand environmental stress [ 4 ] . Previous studies have shown that calcium ions regulate enzyme activity in plants, including synthesis, secretion, and promotion of metabolic reactions which helps maintain normal plant metabolism, growth, and development, as well as aiding in resistance to environmental stresses such as drought, salinity, and low temperatures [ 5 ][ 6 ][ 7 ][ 8 ][ 9 ] . The use of exogenous Ca 2+ to treat rapeseed seedlings under low-temperature stress conditions is practically significant for studying rapeseed resistance [ 10 ] . Lanthanum chloride (LaCl 3 ) is a calcium channel inhibitor that binds to calcium channels and blocks the entry of calcium ions, reducing the intracellular calcium ion concentration and inhibiting calcium signaling [ 11 ] . Numerous studies have shown that lanthanum chloride can enhance the antioxidant enzyme system and osmoregulatory substances in plants at low concentrations while inhibiting them at high concentrations [ 12 ][ 13 ][ 14 ] . The impact of lanthanum chloride on plants is influenced by both treatment time and concentration [ 14 ] . Winter rapeseed ( Brassica rapa L ., AA) is a kind of oil crop that can survive in Northwest China where the extreme temperature is about 25℃ [ 15 ] . Under low-temperature stress, rapeseed undergoes morphological changes such as wilting and yellowing of leaves [ 16 ][ 3 ] . At the physiological level, low temperature triggers the production of a large amount of reactive oxygen species (ROS) in the plant body [ 3 ] . This affects the activity of antioxidant enzyme systems, leads to the accumulation of osmoregulatory substances and metabolic abnormalities, and destroys the integrity of cell membranes. Simultaneously, in vivo, there is rapid and extensive regulation at both physiological and molecular levels, involving multiple stages of low-temperature signal perception, conduction, and regulation [ 17 ] . This results in a reduction of various physiological activities, which affects the normal growth and development of plants [ 18 ] . However, at present some studies have not been clear, e.g. what is the hub gene affecting winter rapeseed strong cold resistance, and what is its mechanism. In this study we analyzed changes in osmoregulatory substances and antioxidant enzyme systems using exogenous CaCl 2 and LaCl 3 . The objective was to research the mechanism of the effects of these two treatments on rapeseed seedlings under low temperature stress. Then transcriptome sequencing was used to study the differences in expression patterns of genes in winter rapeseed varieties with varying cold-resistance under low-temperature conditions and different exogenous substance treatments. This is to identify some hub genes involved in low-temperature regulation and calcium signaling. These results will provide a theoretical basis and genetic resources for further study of the cold-resistant mechanism and the selection and breeding of new varieties with strong cold-resistance in winter rapeseed. 2. Materials and Methods 2.1 Plant material and treatment In present study we used winter rapeseed variety Longyou 7 (L7), a strong cold-resistant variety, and Longyou 99 (L99), a weak cold-resistant variety bred by Gansu Agricultural University [ 15 ] . Uniform-sized rape seeds were sterilized by immersing them in a 5% sodium hypochlorite solution for a duration of 10 minutes, subsequently washed 5 times with distilled water for one minute each time. The seeds were placed in petri dishes with a diameter of 15 cm, covered with filter paper to facilitate germination. After germination, seedlings were transferred to pots with a diameter of 15 cm and a height of 18 cm, which were filled with a nutrient-rich substrate. A total of 54 pots were used to plant each variety, with 3 seedlings kept in each pot. Seedlings were subjected to stress treatment at the five-leaf stage [ 19 ] . Samples were collected at different time points (0 h, 6 h, 12 h, 24 h, and 48 h) following treatments with externally applied distilled water (CK), 20 mmol·L -1 CaCl 2 , and 10 mmol·L -1 LaCl 3 , all at a temperature of 4°C. The leaves and roots were collected individually and preserved by quick-freezing by using liquid nitrogen. Subsequently, the collected samples were stored at -80°C for further analysis [ 20 ] . 2.2 Analysis of physiological Index A fresh leaf sample weighing 0.5 g was finely crushed in a phosphate buffer solution while being kept in a container filled with ice to maintain a low temperature. The crude enzyme solution was prepared through the process of freezing and centrifugation. The activity of superoxide dismutase (SOD) was determined using the nitrogen blue tetrazolium (NBT) photoreduction technique [ 21 ] . The activity of peroxidase (POD) was determined using the guaiacol method [ 22 ] . Catalase (CAT) activity was determined using the UV absorption method [ 23 ] . The level of MDA was determined using the thiobarbituric acid (TBA) method [ 24 ] . Soluble protein (SP) content was measured using the Coomassie brilliant blue method [ 25 ] . The acid ninhydrin mixing method was used to determine the proline (Pro) content [ 26 ] . The non-invasive microtomography (NMT) technique was used to determine the flow of calcium ions at the root surface site located 300µm away from the apex of the root tip [ 27 ] . Calcium ion content was determined using inductively coupled plasma emission spectroscopy [ 28 ] . 2.3 RNA isolation and transcriptomics analysis The transcriptome sequencer was provided by Shanghai Ouyi Biomedical Technology Company( https://cloud.oebiotech.com/task/ ). Differentially expressed genes were found by comparing gene expression levels among different samples. After differentially expressed genes were obtained, GO function analysis, KEGG enrichment analysis and co-expression network construction were carried out by relying on the online platform provided by Shanghai Ouyi Biomedical Technology Co., LTD. All groups were named for ease of analysis in Table 1 (Table 1 ). Table 1 Description of the sample groups and codes for the experimental treatment Sample group code L7-CK A L7-12 h B L7-12 h-Ca C L7-12 h-La D L7-24 h E L7-24 h-Ca F L7-24 h-La G L99-CK H L99-12 h I L99-12 h-Ca J L99-12 h-La K L99-24 h L L99-24 h-Ca M L99-24 h-La N 2.4 Weighted gene co-expression network Analysis (WGCNA) Transcriptome data were combined with physiological indicators and calcium ion content, and core genes were screened in strict compliance with the requirements of ensuring sample size ≥ 15 using WGCNA and gene data filtering and normalization of low-expression genes [ 29 ][ 30 ] . A total of 42 winter rape transcriptomes were identified, and 40578 genes were identified. Genes with low expression fluctuation (standard deviation ≤ 0.5) were filtered and left 11118 genes for WGCNA network construction. 2.5 Statistical Analysis Excel 2010 was used for data processing and graphing, and SPSS 22.0 software was used for ANOVA. The graphs were analyzed using Microsoft Excel 2010, TBtools, and the online platform provided by Shanghai Ouyi Biological Company ( https://cloud.oebiotech.com/task/ ). 3. Results 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 CaCl 2 and LaCl 3 ). Following the application of calcium chloride treatment (CaCl 2 ), 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 LaCl 3 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 CaCl 2 . However, it decreased by 49.83% compared to the CK after being treated with LaCl 3 (Fig. 1 A 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 LaCl 3 was comparable to that of the CK. Throughout all treatment periods, the activity of SOD was consistently lower in the LaCl 3 treatment group compared to both the CK and CaCl 2 treatment. The most notable change was detected after a period of 24 hours (Fig. 1 C 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 CaCl 2 resulted in an initial rise in POD activity, which was subsequently followed by a decline as the treatment duration increased. When LaCl 3 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 CaCl 2 treatments. However, after the application of CaCl 2 , the activity of POD was dramatically increased compared to the CK after 24 h. Over time, the application of LaCl 3 resulted in a decrease in POD activity relative to the CK (Fig. 2 A and B ). The activity of POD in the roots of L7 increased gradually over time in the CaCl 2 treatments. However, it was suppressed by LaCl 3 . The POD activity in the root of L99 exhibited a progressive increase over time in both the CK and CaCl 2 groups, peaking at 12 h (Fig. 2 C 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 CaCl 2 , 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 LaCl 3 , 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 CaCl 2 . Nevertheless, it experienced a reduction of 30.83% in comparison to the CK following treatment with LaCl 3 (Fig. 3 A 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 LaCl 3 . These findings indicate that the application of LaCl 3 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 LaCl 3 treatment group compared to both the CK and CaCl 2 treatments. The greatest significant alteration was observed after 24 h period (Fig. 3 C 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 LaCl 3 . However, treatment with CaCl 2 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 LaCl 3 over a 24h. On the other hand, after being treated with CaCl 2 , the MDA content initially decreased and then increased over time in both cultivars' leaves (Fig. 4 A 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 LaCl 3 , as compared to the CK. The utilization of CaCl 2 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. 4 C and D . The SP content in the L7 leaf increased substantially following CaCl 2 and LaCl 3 treatment at 12 h, 24 h, and 48 h, whereas it remained lower than CK in the case of LaCl 3 treatment. While no significant difference was observed between the CK and treatment CaCl 2 and LaCl 3 in L99 leaves (Fig. 5 A and B ). The root exhibited an upward trend in SP content when subjected to CaCl 2 treatments in both cultivars. It increased significantly after 6h of CaCl 2 treatment compared to CK, while following the application of LaCl 3 , the SP content exhibited a decreasing trend compared to CK (Fig. 5 C and D ). Additionally, the SP content was increased significantly in the both cultivars after CaCl 2 treatment compared to LaCl 3 treatment at different treatment times of 6 h, 12 h, 24 h, and 48 h. More precisely, in L99 CaCl 2 treatment did not show any significant changes at any treatment time compared to CK. However, the findings illustrate that LaCl 3 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 CaCl 2 it reached it maximum at 12h, Conversely, it decreased following the LaCl 3 treatment in L7. In the leaves of L99 Proline content was significantly higher after CaCl 2 compared to CK at 12h and 24h. While after the treatment of LaCl 3 it drops down significantly at 12h and 24h compared to CaCl 2 and CK (Fig. 6 A and B ). Th proline content in the roots of L7 increased both in CK, and CaCl 2 treatment, at 12h and then drops significantly. There was no significant difference between LaCl 3 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 CaCl 2 treatment, reaching its maximum at 48 h, which was significantly increased by 149.19% com-pared to CK (Fig. 6 C and D ). 3.3 Effects of exogenous calcium and calcium inhibitors on Ca 2+ content of rapeseed roots under low-temperature stress The Ca 2+ 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 CaCl 2 treatment it drops significantly at 12h compared to CK, and after the application of LaCl 3 it increases significantly at 12h and then drops at 24h compared to CK (Fig. 7 A). 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 CaCl 2 and LaCl 3 the Ca 2+ contents drop significantly compared to CK. Both varieties showed obvious different trend of Ca 2+ contents change in CK while slightly similar trend was observed after the application of CaCl 2 and LaCl 3 (Fig. 7 A and B ) 3.4 Analysis of changes in Ca 2+ flow rate at low-temperature Both L7 and L99 roots showed Ca 2+ influx at normal temperature, while the influx rate was higher in L7 than in L99 (Fig. 7 C). After low-temperature treatment, L99 showed Ca 2+ 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. 8 A and B . The samples exhibited notable disparities, and the replication within each group was satisfactory (Fig. 8 C). 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. 9 A). 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. 9 B). 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. 9 C), 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. 10 A). 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. 10 B), 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. 10 C). 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. 11 A. 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 Ca 2+ content with the highest correlation coefficient (Fig. 11 B). The number of genes contained in each module shown in Fig. 11 C, 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. 12 A). The lightblue4 module contains a total of 38 genes. The majority of genes exhibited up-regulation following 12 hours of CaCl 2 treatment in L7, whereas the majority of genes displayed down-regulation following 24 hours of CaCl 2 treatment. Only a small number of genes showed up-regulation (Fig. 12 B). 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. 12 C 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. 12 C and 13 ). The expression levels of WRKY22 and LOC103837288 in CK were very low, but the expression increased significantly when it was treated CaCl 2 and LaCl 3 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 CaCl 2 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. 4. Discussion Low temperatures may result in the excessive accumulation of reactive oxygen species, leading to lipid peroxidation and damage to cell membranes, which can ultimately affect seedling growth [ 31 ] . The increase in antioxidant enzyme activity is commonly regarded as a crucial mechanism for inducing resistance to oxidative stress in plants [ 32 ] . The increase in antioxidant enzyme activity following exposure to low temperatures aids in the removal of reactive oxygen species and reduces oxidative damage caused by stress [ 33 ] . Pu [ 34 ] discovered that cold-tolerant rapeseed varieties maintained higher levels of superoxide dismutase and catalase activity, as well as higher soluble protein content while exhibiting lower malondialdehyde content when subjected to freezing treatment at low temperatures. When plants are exposed to low-temperature stress, changes occur not only in antioxidant enzyme activities but also in osmoregulatory substances in the body, which slow down the damage sustained [ 35 ] . Li [ 36 ] concluded, through the study of cotton seedlings, that the increase in soluble protein content was positively correlated with the cold resistance of varieties under different low-temperature conditions. The study showed that at low temperatures, the activities of superoxide dismutase, catalase, and peroxidase increased, as did the soluble protein and proline contents. Enzyme activities in leaves and roots of L7, which was more cold-resistant, increased more than those of L99, which was weak cold-resistant. MDA content also increased, but the increase was greater in L99. These findings are consistent with the results of previous studies by Tahmasebi [ 37 ] and Wei [ 38 ] . Some calcium ions can keep cell membranes stable, reduce malondialdehyde levels, keep cell membranes functioning normally, and reduce membrane lipid peroxidation [ 39 ] . Exogenous Ca 2+ slows down adversity stress, but the required concentration varies among different plants [ 40 ] (Shi et al., 2022). Zhou [ 41 ] found that the addition of 20 mmol-L-1 CaCl 2 significantly increased the activities of antioxidant enzymes (SOD, APX, CAT) in tobacco leaves. In this study, compared with distilled water treatment, CaCl 2 treatment decreased malondialdehyde content and increased antioxidant enzyme activities, soluble protein, and proline content in both leaves and roots of rapeseed, and reduced chilling injury to leaves and roots of rapeseed. These results agree with those of Pu [ 34 ] . La 3+ can enter the cell and compete with Ca 2+ for binding sites, it can replace Ca 2+ and affect Ca 2+ transport by binding to Ca 2+ binding sites on the outside of the membrane [ 42 ] . Chen [ 43 ] found that the optimal concentration of lanthanum chloride can increase the height and root growth of mauve bean seedlings, as well as root vigor and dry matter accumulation. In their study of Cymbidium macrorrhizum , Wang [ 44 ] found that lanthanum chloride had a dual effect on plant growth: at low concentrations, it increased the activity of antioxidant enzymes, the content of physiological regulators and plant resistance, but at high concentrations, it inhibited plant growth. This study found that treatment of rapeseed seedlings with LaCl 3 resulted in a decrease in superoxide dismutase, peroxidase, and catalase activities, as well as a decrease in soluble protein and proline content in both leaves and roots. In addition, MDA content increased significantly, with a higher concentration observed in leaves compared with in root. Notably, malondialdehyde content was significantly higher in roots treated with LaCl 3 compared with CK. These findings are consistent with those of Wang [ 44 ] . The inhibitory effects may be due to the high concentration of LaCl 3 , which increased the level of membrane peroxidation in rapeseed, suggesting that using lower concentrations of LaCl 3 may result in less peroxidation and therefore less inhibition. Calcium plays a direct role in increasing plant resistance to various stresses by stabilizing cell walls and membranes, facilitating ion uptake by the plant root system, and participating in several signal transduction processes within the plant [ 45 ] (Jones and Lunt, 1967). Even though the root system absorbs calcium ions, plants maintain the concentration of calcium ions in their body through various mechanisms [ 32 ] (Kong et al., 2023). If plants do not use calcium ions efficiently after absorption, they may accumulate in plant body [ 46 ] . Secondly, there are a large number of transporter proteins in plants that are involved in the uptake and use of calcium ions. Insufficient transporter proteins or compromised function can cause the accumulation of absorbed calcium ions [ 47 ] . Furthermore, plants possess a range of genes and enzymes that are involved in calcium ion metabolism. The expression and activity of these genes and enzymes can be influenced by external factors [ 48 ] . Environmental factors also affect the efficiency of calcium ion absorption and utilization [ 49 ] . In conclusion, the influence of various factors such as physiological regulation and environmental factors may explain the high rate but low content of calcium ion uptake in plants. A significant difference in the ion flow rate was observed between the L7 and the L99. Research has shown that there is a correlation between the height of the calcium ion uptake peak and cold hardiness of plants at low temperatures [ 50 ] . The higher the calcium ion uptake peak t, the higher its cold hardiness [ 50 ] . Contrary to the results of the present study, which show that the calcium ion uptake peak does not solely indicate the strength of the plant's cold resistance, the calcium ion uptake peak of L99, which has weak cold resistance, was higher than that of L7, which has strong cold resistance. The amount of increase in the uptake and efflux of calcium ions during low-temperature stress plays a key role to a certain extent. Upon analyzing the genes expressed differentially, it was discovered that the calcium chloride treatment exhibited the highest number of such genes among the three treatment groups in low-temperature conditions. Calcium chloride is a Ca 2+ signaling molecule that is believed to contribute to this process. These modifications involve the stimulation of the antioxidant enzyme system and the accumulation of proline. Furthermore, a significant number of genes, particularly those connected to cold tolerance, are expressed in the plant. GO enrichment analysis showed that genes related to low-temperature response, hormone response, photosystem II, chloroplasts, DNA replication, ribosomal RNA processing, and translation were significantly enriched in different exogenous substance treatments under low-temperature conditions. Photosystem II and chloroplasts play a crucial role in plant photosynthesis, facilitating the chemical processes that provide energy for growth [ 51 ] . DNA replication is a crucial process that organisms undergo in preparation for cell division and proliferation. Meanwhile, the processing of ribosomal RNA and translation are essential steps in protein synthesis and construction [ 52 ] . All of these processes are fundamental to the cell's life activities and are essential for its proper functioning. Calmodulin is an important intracellular signaling protein that binds to calcium ions in response to cellular stimuli, thereby regulating cellular biological responses [ 53 ] . Changes in the expression levels of these genes can have significant effects on plant growth, cell signaling, and transmembrane transport, and may represent cellular responses to maintain normal life activities in the face of environmental stress or challenge [ 54 ] . It is important to maintain objectivity and avoid subjective assessments of the response of winter rapeseed to the low-temperature environment. KEGG pathway analysis in this study revealed a significant enrichment of differentially expressed genes in MAPK signaling pathway, both in the two varieties. MAPK signaling pathway is a key pathway in plants that responds to abiotic stress involved in many cellular activities, such as growth, proliferation, cell differentiation, movement, and death [ 55 ] . Calcium ions can regulate MAPK activity, affecting cellular signaling processes in the interaction with MAPK signaling pathway [ 55 ] . It has been suggested that lanthanide ions reduce the activity level of MAPK pathway by inhibiting MAPK kinase activity [ 56 ] . Furthermore, lanthanide ions can influence the expression of plant genes, specifically those associated with cell division, cell wall synthesis, and photosynthesis. Upregulation of these genes can facilitate plant growth and development [ 57 ] . However, further studies are required to confirm the mechanism of lanthanide ion regulation of MAPK signaling pathway in plants. Abbreviations Abbreviation English name B. Brassica CAT Catalase SOD superoxide dismutase POD peroxidase SP soluble protein Pro proline MDA Malondialdehyde CaCl 2 Calcium chloride LaCl 3 Lanthanum chloride ROS reactive oxygen species NBT nitrogen blue tetrazolium TBA thiobarbituric acid NMT non-invasive microtomography WGCNA weighted gene co-expression network analysis PCA principal component analysis Declarations Ethics approval and consent to participate The authors declared that experimental research works on the plants described in this paper comply with institutional, national and international guidelines. Consent for publication Not applicable. Availability of data and materials Data and materials will be made available on request. Competing Interests The authors declare no competing interests. Funding This research was funded by the Gansu Agricultural University youth mentor support fund project (GAU-QDFC-2021-13), Gansu Province college industry support plan project (2023CYZC-51), the China Agriculture Research System of MOF and MARA (CARS-12-09). Authors' contributions Junyan Wu conceived and designed the study. Qiaowen Pan and Lulu Zhang analyzed the data. Fahim Abbas Muhammad revised and polished the paper. Lijun Liu, Gang Yang, Wangtian Wang, Yuanyuan Pu and Yan Fang contributed materials and analysis tools. Junyan Wu wrote the paper. Li Ma and Wancang Sun revised the manuscript. All authors contributed to the article and approved the submitted version. Acknowledgements We thank Yahong Zhang, Qixian Chen and Feng Li for identification of the cold tolerance of materials. Reviewers are acknowledged for their contribution to the improvement of the manuscript in the revision process. Corresponding author Correspondence to Li Ma( [email protected] ). 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Cite Share Download PDF Status: Published Journal Publication published 09 Oct, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 08 May, 2024 Reviews received at journal 08 May, 2024 Reviews received at journal 28 Apr, 2024 Reviewers agreed at journal 24 Apr, 2024 Reviewers agreed at journal 11 Apr, 2024 Reviewers invited by journal 06 Apr, 2024 Editor assigned by journal 04 Apr, 2024 Submission checks completed at journal 04 Apr, 2024 First submitted to journal 29 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4186636","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288947742,"identity":"2fffac7f-60c8-48c4-bc8e-d9154e39be71","order_by":0,"name":"Junyan Wu","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Junyan","middleName":"","lastName":"Wu","suffix":""},{"id":288947744,"identity":"d262fe06-a741-45f1-ad69-048a5de12741","order_by":1,"name":"Qiaowen Pan","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qiaowen","middleName":"","lastName":"Pan","suffix":""},{"id":288947745,"identity":"2583daa9-e74c-4a54-b9be-af53815f544a","order_by":2,"name":"Fahim Abbas Muhammad","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fahim","middleName":"Abbas","lastName":"Muhammad","suffix":""},{"id":288947747,"identity":"2b178fbd-3c4d-4252-84f4-ff7efa24bea4","order_by":3,"name":"Lulu Zhang","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lulu","middleName":"","lastName":"Zhang","suffix":""},{"id":288947749,"identity":"b6d408ad-5293-448c-a574-92274789882a","order_by":4,"name":"Lijun Liu","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lijun","middleName":"","lastName":"Liu","suffix":""},{"id":288947751,"identity":"94277ca9-dcc6-47a6-b362-65048ba46638","order_by":5,"name":"Gang Yang","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Yang","suffix":""},{"id":288947752,"identity":"f833c8c0-46ac-43d3-a852-ac6bc4a34173","order_by":6,"name":"Wangtian Wang","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wangtian","middleName":"","lastName":"Wang","suffix":""},{"id":288947753,"identity":"52150b80-640b-4e05-a629-be75dc2dd92f","order_by":7,"name":"Yuanyuan Pu","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Pu","suffix":""},{"id":288947754,"identity":"33281b4e-12d6-465e-a921-669c30cdbfcf","order_by":8,"name":"Yan Fang","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Fang","suffix":""},{"id":288947755,"identity":"c8bbdbab-46e6-4ad3-959a-703f8b125573","order_by":9,"name":"Li Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACNvbGxgcf/9jw8LO3H3yQUFFDWAsfz+Fmw5kNaXKSPWeSDR6cOUZYi5xEeps0b8MhY4MZCWaSD1uYiXAYz8FmY94dBxI3MCSkVSQ2sDHwt3cnEPTLw7ln7iRuZzh47EbiDhkGiTNnNxC0xeAN27PEnY0NaTcSz7AxGEjkEtAikdgmwcN2OHHDYQazgsQ2ZuK0SPK2HTY2OMZgxkCcFqDDDGecAQUyT7JEwpljPAT9It/e/vDBhwpgVMo/P/jxR0WNHH97L34tGICHNOWjYBSMglEwCrACAB/XUnU6eFCLAAAAAElFTkSuQmCC","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Ma","suffix":""},{"id":288947756,"identity":"c34af4eb-ed41-4318-b8fb-794258a15f19","order_by":10,"name":"Wancang Sun","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wancang","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-03-29 08:29:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4186636/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4186636/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-05556-w","type":"published","date":"2024-10-09T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54386401,"identity":"3d97f49d-8cc3-4062-9a82-38e6e827b54f","added_by":"auto","created_at":"2024-04-09 17:49:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206608,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of low temperature on SOD activity of winter rape leaves and roots (A) SOD activity of L7 leaves; (B) SOD activity of L99 leaves; (C) SOD activity of L7 roots; (D) SOD activity of L99 roots. Note: CK is the distilled water control group, La is the lanthanum chloride treatment group, and Ca is the calcium chloride treatment group; different lowercase letters indicate that the differences between different treatments at the same time were significant (p \u0026lt; 0.05), the same below.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/744c18b7c75e533b813f2588.jpg"},{"id":54386403,"identity":"2b31de40-6e92-457d-a096-2bac2c288298","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":191540,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of low temperature on POD activity of winter rapeseed leaves and roots (A) graph of POD activity of L7 leaves; (B) graph of POD activity of L99 leaves; (C) graph of POD activity of L7 roots; (D) graph of POD activity of L99 roots.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/491cc1352b15ded55cbc109c.jpg"},{"id":54386402,"identity":"72acef72-07ec-4e17-b4fb-c49c4b71fc02","added_by":"auto","created_at":"2024-04-09 17:49:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":168354,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of low temperature on CAT activity of winter rapeseed leaves and roots (A) L7 leaf CAT activity graph; (B) L99 leaf CAT activity graph; (C) L7 root CAT activity graph; (D) L99 root CAT activity graph.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/71237a63b3e17f835e8a37fd.jpg"},{"id":54386404,"identity":"a6f9631b-320b-4277-ab24-ba3f3637c340","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":192541,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of low temperature on MDA content of leaves and roots of winter rapeseed (A) graph of MDA content of L7 leaves; (B) graph of MDA content of L99 leaves; (C) graph of MDA content of L7 roots; (D) graph of MDA content of L99 roots.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/37ba2a4e0f25391158d1df5e.jpg"},{"id":54386407,"identity":"88bc2909-8712-46c6-bd3c-8ba9ddb5159a","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":204258,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of low temperature on SP content of winter rape leaves and roots (A) graph of SP content of L7 leaves; (B) graph of SP content of L99 leaves; (C) graph of SP amount of L7 roots; (D) graph of SP content of L99 roots.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/f57730aa8b5c9296d3308434.jpg"},{"id":54386405,"identity":"2a0e4754-d8ad-4888-baad-0b98344237b8","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":210477,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of low temperature on the Pro content of winter rapeseed leaves and roots (A) graph of Pro content of L7 leaves; (B) graph of Pro content of L99 leaves; (C) graph of Pro amount of L7 roots; (D) graph of Pro content of L99 roots.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/8f472248d78c32084a6ea68a.jpg"},{"id":54386410,"identity":"3240bc30-f630-4296-85f8-1df08cef1bc4","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":136245,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Comparison of calcium ion content in root system under different treatments of L7; (B) Comparison of calcium ion content in root system under different treatments of L99; (C) Comparison of Ca\u003csup\u003e2+\u003c/sup\u003e flow rate in rapeseed root system before and after low-temperature treatment.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/41e44c4c786736d0e2a87b92.jpg"},{"id":54386412,"identity":"ed6300da-da11-4ef8-bb05-e8b3c6c2b95d","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":296186,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis of all samples (A: 2 D, B: 3 D); (C)Analysis of the number of differentially expressed genes in each comparison group. Note: Each point represents one sample (one biological replicate) and each color represents one treatment\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/33a1dfb5eb7df5801b7ddae7.jpg"},{"id":54386414,"identity":"37cd835c-1fd9-4253-ba7b-206e0b08784a","added_by":"auto","created_at":"2024-04-09 17:49:17","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":395674,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of GO function enrichment results of differentially expressed genes (A) Analysis of GO function enrichment results of differentially expressed genes of L7; (B) Analysis of GO function enrichment results of differentially expressed genes of L99; (C) Analysis of GO function enrichment results of differentially expressed genes comparing between two varieties. Note: The horizontal axis Enrichment Score in the figure is the enrichment score, the bigger the bubble the more the number of differential protein-coding genes contained in the entry, the color of the bubble changes from blue-white-yellow-red, and the smaller the enrichment p-value value, the bigger the degree of significance, the bigger the bubble, the more the number of genes. Same as below.\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/e829361f17d4bffe409b5374.jpg"},{"id":54386409,"identity":"c865093d-d023-4e7f-b714-ea257f4d6913","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":406077,"visible":true,"origin":"","legend":"\u003cp\u003eBubble diagram of KEGG functional enrichment of differentially expressed genes (A) analysis of KEGG functional enrichment of differentially expressed genes of L7; (B) Analysis of KEGG functional enrichment of differentially expressed genes of L99; (C) analysis of KEGG functional enrichment of differentially expressed genes for the comparison of the two varieties.\u003c/p\u003e","description":"","filename":"Picture10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/8f7e7e5e6abde564d005e102.jpg"},{"id":54386408,"identity":"bcc38566-ca64-4653-ae40-ef488f5730a5","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":532903,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Gene clustering tree and distribution of modules; (B) Heat map of trait module associations; (C) Number of genes in each module. Note:The figure shows the gene clustering tree (top) and the distribution of genes in each module (bottom). The same color indicates the same module and the Dynamic Tree Cut color indicates the module obtained by using the Dynamic Tree Cut method to identify the modules. Certain modules were merged based on their correlation. The final module obtained is Merged Dynamic in the lower part. These modules were used for subsequent analysis. The correlation between the corresponding module and the trait is indicated by the number (r-value) in each cell. The number in the parentheses stands for the correlation (P-value), and the strength of the correlation is represented by the color of the cell\u003c/p\u003e","description":"","filename":"Picture11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/7d4569212eddca66f7a5239e.jpg"},{"id":54386411,"identity":"e432e622-3927-4b6e-aad1-dac58c15980e","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":528961,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Darkslateblue module gene expression (B) Lightblue4 module gene expression (C) Heatmap of 24 candidate genes expression\u003c/p\u003e","description":"","filename":"Picture12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/790b5c10feb837be8eb5d72e.jpg"},{"id":54386413,"identity":"9e83739b-30fa-4081-b9bb-8533cd64e3ad","added_by":"auto","created_at":"2024-04-09 17:49:16","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":342652,"visible":true,"origin":"","legend":"\u003cp\u003eExpression analysis of 8 candidate genes. Note: CK is room temperature control, 12 h is distilled water treatment for 12 h, 12 h-Ca is CaCl2 treatment for 12 h, 12 h-La is LaCl3 treatment for 12 h, 24 h is distilled water treatment for 24 h, 24 h-Ca is CaCl2 treatment for 24 h, 24 h-La is LaCl3 treatment for 24 h.\u003c/p\u003e","description":"","filename":"Picture13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/76b7774cd592f26b49686ef5.jpg"},{"id":66597185,"identity":"5226126a-57b0-4850-b19f-20f4463443fb","added_by":"auto","created_at":"2024-10-14 16:08:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4723164,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4186636/v1/3dceb3c9-a0ff-430e-9b0a-a3e1304956ea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of exogenous calcium and calcium inhibitor on physiological characteristics of winter rape (Brassica rapa) under low temperature stress","fulltext":[{"header":"1. Background","content":"\u003cp\u003eWinter rapeseed (\u003cem\u003eBrassica rapa\u003c/em\u003e L.) is an important oilseed crop globally. In China, it contributes 5.2\u0026nbsp;million tons of high-quality edible oil per year, which represents over 50% of the country's total oil crop production\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, China is still lacking in the production and edible oil of rapeseed. It has always been important for us to produce more rapeseed. Winter rapeseed is the main cultivation type of rapeseed in China, and it is cultivated in both north and south \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. But in north, freezing temperature in winter and chilling in spring has negatively impacted on the yield, growth, and quality of oil in China\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is crucial to look for the strong cold-resistant genes of winter rapeseed and enhance its cold resistance to ensure the safe overwintering in north China. This is of great significance for the development of the rapeseed industry and the protection of China grain and oil security. The application of exogenous substances is one of the simplest and most effective methods.\u003c/p\u003e \u003cp\u003eCalcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e), an inorganic salt, is used to help plants withstand environmental stress \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that calcium ions regulate enzyme activity in plants, including synthesis, secretion, and promotion of metabolic reactions which helps maintain normal plant metabolism, growth, and development, as well as aiding in resistance to environmental stresses such as drought, salinity, and low temperatures \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The use of exogenous Ca\u003csup\u003e2+\u003c/sup\u003e to treat rapeseed seedlings under low-temperature stress conditions is practically significant for studying rapeseed resistance\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Lanthanum chloride (LaCl\u003csub\u003e3\u003c/sub\u003e) is a calcium channel inhibitor that binds to calcium channels and blocks the entry of calcium ions, reducing the intracellular calcium ion concentration and inhibiting calcium signaling\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Numerous studies have shown that lanthanum chloride can enhance the antioxidant enzyme system and osmoregulatory substances in plants at low concentrations while inhibiting them at high concentrations\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e][\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e][\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The impact of lanthanum chloride on plants is influenced by both treatment time and concentration\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWinter rapeseed (\u003cem\u003eBrassica rapa L\u003c/em\u003e., AA) is a kind of oil crop that can survive in Northwest China where the extreme temperature is about 25℃\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Under low-temperature stress, rapeseed undergoes morphological changes such as wilting and yellowing of leaves\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. At the physiological level, low temperature triggers the production of a large amount of reactive oxygen species (ROS) in the plant body\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. This affects the activity of antioxidant enzyme systems, leads to the accumulation of osmoregulatory substances and metabolic abnormalities, and destroys the integrity of cell membranes. Simultaneously, in vivo, there is rapid and extensive regulation at both physiological and molecular levels, involving multiple stages of low-temperature signal perception, conduction, and regulation\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. This results in a reduction of various physiological activities, which affects the normal growth and development of plants\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. However, at present some studies have not been clear, e.g. what is the hub gene affecting winter rapeseed strong cold resistance, and what is its mechanism.\u003c/p\u003e \u003cp\u003eIn this study we analyzed changes in osmoregulatory substances and antioxidant enzyme systems using exogenous CaCl\u003csub\u003e2\u003c/sub\u003e and LaCl\u003csub\u003e3\u003c/sub\u003e. The objective was to research the mechanism of the effects of these two treatments on rapeseed seedlings under low temperature stress. Then transcriptome sequencing was used to study the differences in expression patterns of genes in winter rapeseed varieties with varying cold-resistance under low-temperature conditions and different exogenous substance treatments. This is to identify some hub genes involved in low-temperature regulation and calcium signaling. These results will provide a theoretical basis and genetic resources for further study of the cold-resistant mechanism and the selection and breeding of new varieties with strong cold-resistance in winter rapeseed.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant material and treatment\u003c/h2\u003e \u003cp\u003eIn present study we used winter rapeseed variety Longyou 7 (L7), a strong cold-resistant variety, and Longyou 99 (L99), a weak cold-resistant variety bred by Gansu Agricultural University\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Uniform-sized rape seeds were sterilized by immersing them in a 5% sodium hypochlorite solution for a duration of 10 minutes, subsequently washed 5 times with distilled water for one minute each time. The seeds were placed in petri dishes with a diameter of 15 cm, covered with filter paper to facilitate germination. After germination, seedlings were transferred to pots with a diameter of 15 cm and a height of 18 cm, which were filled with a nutrient-rich substrate. A total of 54 pots were used to plant each variety, with 3 seedlings kept in each pot. Seedlings were subjected to stress treatment at the five-leaf stage \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Samples were collected at different time points (0 h, 6 h, 12 h, 24 h, and 48 h) following treatments with externally applied distilled water (CK), 20 mmol\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e CaCl\u003csub\u003e2\u003c/sub\u003e, and 10 mmol\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e LaCl\u003csub\u003e3\u003c/sub\u003e, all at a temperature of 4\u0026deg;C. The leaves and roots were collected individually and preserved by quick-freezing by using liquid nitrogen. Subsequently, the collected samples were stored at -80\u0026deg;C for further analysis\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Analysis of physiological Index\u003c/h2\u003e \u003cp\u003eA fresh leaf sample weighing 0.5 g was finely crushed in a phosphate buffer solution while being kept in a container filled with ice to maintain a low temperature. The crude enzyme solution was prepared through the process of freezing and centrifugation. The activity of superoxide dismutase (SOD) was determined using the nitrogen blue tetrazolium (NBT) photoreduction technique\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The activity of peroxidase (POD) was determined using the guaiacol method\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Catalase (CAT) activity was determined using the UV absorption method\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The level of MDA was determined using the thiobarbituric acid (TBA) method\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Soluble protein (SP) content was measured using the Coomassie brilliant blue method\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The acid ninhydrin mixing method was used to determine the proline (Pro) content\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The non-invasive microtomography (NMT) technique was used to determine the flow of calcium ions at the root surface site located 300\u0026micro;m away from the apex of the root tip\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Calcium ion content was determined using inductively coupled plasma emission spectroscopy\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 RNA isolation and transcriptomics analysis\u003c/h2\u003e \u003cp\u003eThe transcriptome sequencer was provided by Shanghai Ouyi Biomedical Technology Company(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.oebiotech.com/task/\u003c/span\u003e\u003cspan address=\"https://cloud.oebiotech.com/task/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Differentially expressed genes were found by comparing gene expression levels among different samples. After differentially expressed genes were obtained, GO function analysis, KEGG enrichment analysis and co-expression network construction were carried out by relying on the online platform provided by Shanghai Ouyi Biomedical Technology Co., LTD. All groups were named for ease of analysis in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescription of the sample groups and codes for the experimental treatment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ecode\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7-CK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7-12 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7-12 h-Ca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7-12 h-La\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7-24 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7-24 h-Ca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL7-24 h-La\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL99-CK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL99-12 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL99-12 h-Ca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL99-12 h-La\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL99-24 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL99-24 h-Ca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL99-24 h-La\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Weighted gene co-expression network Analysis (WGCNA)\u003c/h2\u003e \u003cp\u003eTranscriptome data were combined with physiological indicators and calcium ion content, and core genes were screened in strict compliance with the requirements of ensuring sample size\u0026thinsp;\u0026ge;\u0026thinsp;15 using WGCNA and gene data filtering and normalization of low-expression genes\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e][\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA total of 42 winter rape transcriptomes were identified, and 40578 genes were identified. Genes with low expression fluctuation (standard deviation\u0026thinsp;\u0026le;\u0026thinsp;0.5) were filtered and left 11118 genes for WGCNA network construction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e \u003cp\u003eExcel 2010 was used for data processing and graphing, and SPSS 22.0 software was used for ANOVA. The graphs were analyzed using Microsoft Excel 2010, TBtools, and the online platform provided by Shanghai Ouyi Biological Company (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.oebiotech.com/task/\u003c/span\u003e\u003cspan address=\"https://cloud.oebiotech.com/task/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003e3.1 Effects of exogenous Calcium and Calcium inhibitors on SOD, POD, and CAT activities in\u003c/b\u003e \u003cb\u003eB. rapa\u003c/b\u003e \u003cb\u003eleaves and roots under low-temperature Stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe SOD activity in the leaves of L7 and L99 increased gradually over time at a temperature 4\u0026deg;C in the CK (without application of CaCl\u003csub\u003e2\u003c/sub\u003e and LaCl\u003csub\u003e3\u003c/sub\u003e). Following the application of calcium chloride treatment (CaCl\u003csub\u003e2\u003c/sub\u003e), 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 LaCl\u003csub\u003e3\u003c/sub\u003e 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 CaCl\u003csub\u003e2\u003c/sub\u003e. However, it decreased by 49.83% compared to the CK after being treated with LaCl\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e). The SOD activity in the roots of L7 and L99 increased gradually over time at a temperature of 4\u0026deg;C in the CK. It reached its peak at 24 hours and subsequently decreased at 48 hours. The trend seen after treatment with LaCl\u003csub\u003e3\u003c/sub\u003e was comparable to that of the CK. Throughout all treatment periods, the activity of SOD was consistently lower in the LaCl\u003csub\u003e3\u003c/sub\u003e treatment group compared to both the CK and CaCl\u003csub\u003e2\u003c/sub\u003e treatment. The most notable change was detected after a period of 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe POD activity in the leaves of L7 showed a progressive increase over time when exposed to a temperature of 4\u0026deg;C in the CK. The application of CaCl\u003csub\u003e2\u003c/sub\u003e resulted in an initial rise in POD activity, which was subsequently followed by a decline as the treatment duration increased. When LaCl\u003csub\u003e3\u003c/sub\u003e 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 CaCl\u003csub\u003e2\u003c/sub\u003e treatments. However, after the application of CaCl\u003csub\u003e2\u003c/sub\u003e, the activity of POD was dramatically increased compared to the CK after 24 h. Over time, the application of LaCl\u003csub\u003e3\u003c/sub\u003e resulted in a decrease in POD activity relative to the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e). The activity of POD in the roots of L7 increased gradually over time in the CaCl\u003csub\u003e2\u003c/sub\u003e treatments. However, it was suppressed by LaCl\u003csub\u003e3\u003c/sub\u003e. The POD activity in the root of L99 exhibited a progressive increase over time in both the CK and CaCl\u003csub\u003e2\u003c/sub\u003e groups, peaking at 12 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe CAT activity in the leaves of L7 and L99 increased gradually over time at a temperature of 4\u0026deg;C in the CK. After applying CaCl\u003csub\u003e2\u003c/sub\u003e, 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 LaCl\u003csub\u003e3\u003c/sub\u003e, 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 CaCl\u003csub\u003e2\u003c/sub\u003e. Nevertheless, it experienced a reduction of 30.83% in comparison to the CK following treatment with LaCl\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe CAT activity in the roots of L7 and L99 exhibited a progressive increase over time at a temperature of 4\u0026deg;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 LaCl\u003csub\u003e3\u003c/sub\u003e. These findings indicate that the application of LaCl\u003csub\u003e3\u003c/sub\u003e 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 LaCl\u003csub\u003e3\u003c/sub\u003e treatment group compared to both the CK and CaCl\u003csub\u003e2\u003c/sub\u003e treatments. The greatest significant alteration was observed after 24 h period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Effects of exogenous calcium and calcium inhibitors on MDA, SP, and Proline contents in\u003c/b\u003e \u003cb\u003eB. rapa\u003c/b\u003e \u003cb\u003eleaves and roots under low-temperature Stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBoth cultivars of \u003cem\u003eB. rapa\u003c/em\u003e exhibited an increasing trend of MDA content in their leaves after treatment with LaCl\u003csub\u003e3\u003c/sub\u003e. However, treatment with CaCl\u003csub\u003e2\u003c/sub\u003e 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 LaCl\u003csub\u003e3\u003c/sub\u003e over a 24h. On the other hand, after being treated with CaCl\u003csub\u003e2\u003c/sub\u003e, the MDA content initially decreased and then increased over time in both cultivars' leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e). 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 LaCl\u003csub\u003e3\u003c/sub\u003e, as compared to the CK. The utilization of CaCl\u003csub\u003e2\u003c/sub\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe SP content in the L7 leaf increased substantially following CaCl\u003csub\u003e2\u003c/sub\u003e and LaCl\u003csub\u003e3\u003c/sub\u003e treatment at 12 h, 24 h, and 48 h, whereas it remained lower than CK in the case of LaCl\u003csub\u003e3\u003c/sub\u003e treatment. While no significant difference was observed between the CK and treatment CaCl\u003csub\u003e2\u003c/sub\u003e and LaCl\u003csub\u003e3\u003c/sub\u003e in L99 leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e). The root exhibited an upward trend in SP content when subjected to CaCl\u003csub\u003e2\u003c/sub\u003e treatments in both cultivars. It increased significantly after 6h of CaCl\u003csub\u003e2\u003c/sub\u003e treatment compared to CK, while following the application of LaCl\u003csub\u003e3\u003c/sub\u003e, the SP content exhibited a decreasing trend compared to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e). Additionally, the SP content was increased significantly in the both cultivars after CaCl\u003csub\u003e2\u003c/sub\u003e treatment compared to LaCl\u003csub\u003e3\u003c/sub\u003e treatment at different treatment times of 6 h, 12 h, 24 h, and 48 h. More precisely, in L99 CaCl\u003csub\u003e2\u003c/sub\u003e treatment did not show any significant changes at any treatment time compared to CK. However, the findings illustrate that LaCl\u003csub\u003e3\u003c/sub\u003e 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe content of proline in the CK reached its peak at 24h, while after the application of CaCl\u003csub\u003e2\u003c/sub\u003e it reached it maximum at 12h, Conversely, it decreased following the LaCl\u003csub\u003e3\u003c/sub\u003e treatment in L7. In the leaves of L99 Proline content was significantly higher after CaCl\u003csub\u003e2\u003c/sub\u003e compared to CK at 12h and 24h. While after the treatment of LaCl\u003csub\u003e3\u003c/sub\u003e it drops down significantly at 12h and 24h compared to CaCl\u003csub\u003e2\u003c/sub\u003e and CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e). Th proline content in the roots of L7 increased both in CK, and CaCl\u003csub\u003e2\u003c/sub\u003e treatment, at 12h and then drops significantly. There was no significant difference between LaCl\u003csub\u003e3\u003c/sub\u003e 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 CaCl\u003csub\u003e2\u003c/sub\u003e treatment, reaching its maximum at 48 h, which was significantly increased by 149.19% com-pared to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Effects of exogenous calcium and calcium inhibitors on Ca\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003econtent of rapeseed roots under low-temperature stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe Ca\u003csup\u003e2+\u003c/sup\u003e 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 CaCl\u003csub\u003e2\u003c/sub\u003e treatment it drops significantly at 12h compared to CK, and after the application of LaCl\u003csub\u003e3\u003c/sub\u003e it increases significantly at 12h and then drops at 24h compared to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In case of L99 a weak cold tolerant variety the Ca2\u0026thinsp;+\u0026thinsp;contents in its roots maintained at higher level with a slight drop at 24h compared to 12h in CK. After the application of both CaCl\u003csub\u003e2\u003c/sub\u003e and LaCl\u003csub\u003e3\u003c/sub\u003e the Ca\u003csup\u003e2+\u003c/sup\u003e contents drop significantly compared to CK. Both varieties showed obvious different trend of Ca\u003csup\u003e2+\u003c/sup\u003e contents change in CK while slightly similar trend was observed after the application of CaCl\u003csub\u003e2\u003c/sub\u003e and LaCl\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Analysis of changes in Ca\u003csup\u003e2+\u003c/sup\u003e flow rate at low-temperature\u003c/h2\u003e \u003cp\u003eBoth L7 and L99 roots showed Ca\u003csup\u003e2+\u003c/sup\u003e influx at normal temperature, while the influx rate was higher in L7 than in L99 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). After low-temperature treatment, L99 showed Ca\u003csup\u003e2+\u003c/sup\u003e efflux with a rate of 30.21 pmol‧cm\u003csup\u003e-2\u003c/sup\u003e‧s\u003csup\u003e-1\u003c/sup\u003e, whereas L7 briefly showed efflux then returned to influx.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Transcriptome analyses\u003c/h2\u003e \u003cp\u003eA 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\u0026ndash;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\u0026ndash;87.55%, and the matching percentage to multiple positions of the reference genome was 2.45\u0026ndash;2.92%. A total of 42 samples were subjected to principal component analysis (PCA), as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e. The samples exhibited notable disparities, and the replication within each group was satisfactory (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). These results satisfy the requirements for further analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.6 GO and KEGG enrichment analysis of DEGs\u003c/h2\u003e \u003cp\u003eGO functional enrichment analysis was performed on the differentially expressed genes of L7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGO 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.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). 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.\u003c/p\u003e \u003cp\u003eThe results illustrate that DEGs were significantly enriched in DNA-binding transcription factor activity, membrane constituents (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC), 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.\u003c/p\u003e \u003cp\u003eIn 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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). 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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB), 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn 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.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC). Furthermore, transcriptome analysis revealed 220 genes that were differentially expressed and enriched in MAPK signaling pathway in which we identified 6 candidate genes (\u003cem\u003eLOC103873775, WRKY22, LOC103857966, LOC103866369, LOC103830167, LOC103837288\u003c/em\u003e) that were relevant to plant growth and development, as well as signal transduction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Weighted gene co-expression network analysis and identification of Hub genes\u003c/h2\u003e \u003cp\u003eA 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\u0026thinsp;\u0026le;\u0026thinsp;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.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA. 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 Ca\u003csup\u003e2+\u003c/sup\u003e content with the highest correlation coefficient (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eB). The number of genes contained in each module shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC, whereas the darkorange module contains the highest number of genes (3390) and the darkolivegreen2 module contains the lowest number of genes (29).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased 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.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA). The lightblue4 module contains a total of 38 genes. The majority of genes exhibited up-regulation following 12 hours of CaCl\u003csub\u003e2\u003c/sub\u003e treatment in L7, whereas the majority of genes displayed down-regulation following 24 hours of CaCl\u003csub\u003e2\u003c/sub\u003e treatment. Only a small number of genes showed up-regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eC \u003cb\u003eand\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFunctional annotation of all candidate genes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHub gene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene abbreviation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFunctional annotation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103828211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003euncharacterized\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC108871878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003euncharacterized protein At1g43920, Chloroplastic-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103839598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003euncharacterized\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103872839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4CLL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-coumarate\u0026ndash;CoA ligase-like 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103847303\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSPH9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epumilio homolog 15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103829158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOs03g0733400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ezinc finger BED domain-containing protein RICESLEEPER\u003c/p\u003e \u003cp\u003e2-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC108871342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003euncharacterized LOC108871342\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103847561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZFP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ezinc finger protein 8-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC108869010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAt3g58270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMATH domain and coiled-coil domain-containing protein\u003c/p\u003e \u003cp\u003eAt3g58270-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103828089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAt3g50520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ephosphoglycerate mutase-like protein 4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103856351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLTA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003euncharacterized LOC103856351\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103850180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003euncharacterized LOC103850180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103863198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATHB-15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehomeobox-leucine zipper protein ATHB-15-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103872950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDPMS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eprobable dolichol-phosphate mannosyltransferase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC108870222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003euncharacterized LOC108870222\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103853785\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBI-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBax inhibitor 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103858143\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWRKY12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eprobable WRKY transcription factor 12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103830648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFIB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eprobable mediator of RNA polymerase II transcription subunit 36b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103857966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecalmodulin-5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWRKY22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWRKY22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWRKY transcription factor 22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103837288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecalmodulin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103873775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSRK2H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eserine/threonine-protein kinase SRK2H\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103830167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSRK2A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eserine/threonine-protein kinase SRK2A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103866369\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eERS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eethylene response sensor 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFurthermore, among these 24 candidate genes, 8 core candidate genes with special expression patterns were screened (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eC and \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e). The expression levels of \u003cem\u003eWRKY22\u003c/em\u003e and \u003cem\u003eLOC103837288\u003c/em\u003e in CK were very low, but the expression increased significantly when it was treated CaCl\u003csub\u003e2\u003c/sub\u003e and LaCl\u003csub\u003e3\u003c/sub\u003e for 12 h (12h-Ca, 12h-La), and decreased significantly at 24 h (24h-Ca, 24h-La). \u003cem\u003eLOC103873775\u003c/em\u003e gene expression was significantly higher in L7 than in L99, and it was increased by CaCl\u003csub\u003e2\u003c/sub\u003e treatment (12h-Ca, 24h-Ca,). The expression of \u003cem\u003eLOC103857966\u003c/em\u003e 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 \u003cem\u003eLOC103830167\u003c/em\u003e 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 \u003cem\u003eLOC103866369\u003c/em\u003e 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. \u003cem\u003eLOC103863198\u003c/em\u003e and \u003cem\u003eLOC103858143\u003c/em\u003e were highly expressed in L7 and low in L99, and the expression levels of \u003cem\u003eLOC103863198\u003c/em\u003e and \u003cem\u003eLOC103858143\u003c/em\u003e genes showed a decreasing trend with time in each treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eLow temperatures may result in the excessive accumulation of reactive oxygen species, leading to lipid peroxidation and damage to cell membranes, which can ultimately affect seedling growth\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The increase in antioxidant enzyme activity is commonly regarded as a crucial mechanism for inducing resistance to oxidative stress in plants\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. The increase in antioxidant enzyme activity following exposure to low temperatures aids in the removal of reactive oxygen species and reduces oxidative damage caused by stress\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Pu \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e discovered that cold-tolerant rapeseed varieties maintained higher levels of superoxide dismutase and catalase activity, as well as higher soluble protein content while exhibiting lower malondialdehyde content when subjected to freezing treatment at low temperatures. When plants are exposed to low-temperature stress, changes occur not only in antioxidant enzyme activities but also in osmoregulatory substances in the body, which slow down the damage sustained\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Li \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e concluded, through the study of cotton seedlings, that the increase in soluble protein content was positively correlated with the cold resistance of varieties under different low-temperature conditions. The study showed that at low temperatures, the activities of superoxide dismutase, catalase, and peroxidase increased, as did the soluble protein and proline contents. Enzyme activities in leaves and roots of L7, which was more cold-resistant, increased more than those of L99, which was weak cold-resistant. MDA content also increased, but the increase was greater in L99. These findings are consistent with the results of previous studies by Tahmasebi \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e and Wei \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Some calcium ions can keep cell membranes stable, reduce malondialdehyde levels, keep cell membranes functioning normally, and reduce membrane lipid peroxidation\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Exogenous Ca\u003csup\u003e2+\u003c/sup\u003e slows down adversity stress, but the required concentration varies among different plants\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e(Shi et al., 2022). Zhou \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e found that the addition of 20 mmol-L-1 CaCl\u003csub\u003e2\u003c/sub\u003e significantly increased the activities of antioxidant enzymes (SOD, APX, CAT) in tobacco leaves. In this study, compared with distilled water treatment, CaCl\u003csub\u003e2\u003c/sub\u003e treatment decreased malondialdehyde content and increased antioxidant enzyme activities, soluble protein, and proline content in both leaves and roots of rapeseed, and reduced chilling injury to leaves and roots of rapeseed. These results agree with those of Pu\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLa\u003csup\u003e3+\u003c/sup\u003e can enter the cell and compete with Ca\u003csup\u003e2+\u003c/sup\u003e for binding sites, it can replace Ca\u003csup\u003e2+\u003c/sup\u003e and affect Ca\u003csup\u003e2+\u003c/sup\u003e transport by binding to Ca\u003csup\u003e2+\u003c/sup\u003e binding sites on the outside of the membrane\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Chen \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e found that the optimal concentration of lanthanum chloride can increase the height and root growth of mauve bean seedlings, as well as root vigor and dry matter accumulation. In their study of \u003cem\u003eCymbidium macrorrhizum\u003c/em\u003e, Wang \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e found that lanthanum chloride had a dual effect on plant growth: at low concentrations, it increased the activity of antioxidant enzymes, the content of physiological regulators and plant resistance, but at high concentrations, it inhibited plant growth. This study found that treatment of rapeseed seedlings with LaCl\u003csub\u003e3\u003c/sub\u003e resulted in a decrease in superoxide dismutase, peroxidase, and catalase activities, as well as a decrease in soluble protein and proline content in both leaves and roots. In addition, MDA content increased significantly, with a higher concentration observed in leaves compared with in root. Notably, malondialdehyde content was significantly higher in roots treated with LaCl\u003csub\u003e3\u003c/sub\u003e compared with CK. These findings are consistent with those of Wang\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. The inhibitory effects may be due to the high concentration of LaCl\u003csub\u003e3\u003c/sub\u003e, which increased the level of membrane peroxidation in rapeseed, suggesting that using lower concentrations of LaCl\u003csub\u003e3\u003c/sub\u003e may result in less peroxidation and therefore less inhibition.\u003c/p\u003e \u003cp\u003eCalcium plays a direct role in increasing plant resistance to various stresses by stabilizing cell walls and membranes, facilitating ion uptake by the plant root system, and participating in several signal transduction processes within the plant\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e(Jones and Lunt, 1967). Even though the root system absorbs calcium ions, plants maintain the concentration of calcium ions in their body through various mechanisms\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e(Kong et al., 2023). If plants do not use calcium ions efficiently after absorption, they may accumulate in plant body\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Secondly, there are a large number of transporter proteins in plants that are involved in the uptake and use of calcium ions. Insufficient transporter proteins or compromised function can cause the accumulation of absorbed calcium ions\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Furthermore, plants possess a range of genes and enzymes that are involved in calcium ion metabolism. The expression and activity of these genes and enzymes can be influenced by external factors\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Environmental factors also affect the efficiency of calcium ion absorption and utilization\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. In conclusion, the influence of various factors such as physiological regulation and environmental factors may explain the high rate but low content of calcium ion uptake in plants.\u003c/p\u003e \u003cp\u003eA significant difference in the ion flow rate was observed between the L7 and the L99. Research has shown that there is a correlation between the height of the calcium ion uptake peak and cold hardiness of plants at low temperatures\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. The higher the calcium ion uptake peak t, the higher its cold hardiness\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Contrary to the results of the present study, which show that the calcium ion uptake peak does not solely indicate the strength of the plant's cold resistance, the calcium ion uptake peak of L99, which has weak cold resistance, was higher than that of L7, which has strong cold resistance. The amount of increase in the uptake and efflux of calcium ions during low-temperature stress plays a key role to a certain extent.\u003c/p\u003e \u003cp\u003eUpon analyzing the genes expressed differentially, it was discovered that the calcium chloride treatment exhibited the highest number of such genes among the three treatment groups in low-temperature conditions. Calcium chloride is a Ca\u003csup\u003e2+\u003c/sup\u003e signaling molecule that is believed to contribute to this process. These modifications involve the stimulation of the antioxidant enzyme system and the accumulation of proline. Furthermore, a significant number of genes, particularly those connected to cold tolerance, are expressed in the plant.\u003c/p\u003e \u003cp\u003eGO enrichment analysis showed that genes related to low-temperature response, hormone response, photosystem II, chloroplasts, DNA replication, ribosomal RNA processing, and translation were significantly enriched in different exogenous substance treatments under low-temperature conditions. Photosystem II and chloroplasts play a crucial role in plant photosynthesis, facilitating the chemical processes that provide energy for growth\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. DNA replication is a crucial process that organisms undergo in preparation for cell division and proliferation. Meanwhile, the processing of ribosomal RNA and translation are essential steps in protein synthesis and construction\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. All of these processes are fundamental to the cell's life activities and are essential for its proper functioning. Calmodulin is an important intracellular signaling protein that binds to calcium ions in response to cellular stimuli, thereby regulating cellular biological responses\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Changes in the expression levels of these genes can have significant effects on plant growth, cell signaling, and transmembrane transport, and may represent cellular responses to maintain normal life activities in the face of environmental stress or challenge\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. It is important to maintain objectivity and avoid subjective assessments of the response of winter rapeseed to the low-temperature environment.\u003c/p\u003e \u003cp\u003eKEGG pathway analysis in this study revealed a significant enrichment of differentially expressed genes in MAPK signaling pathway, both in the two varieties. MAPK signaling pathway is a key pathway in plants that responds to abiotic stress involved in many cellular activities, such as growth, proliferation, cell differentiation, movement, and death\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Calcium ions can regulate MAPK activity, affecting cellular signaling processes in the interaction with MAPK signaling pathway\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. It has been suggested that lanthanide ions reduce the activity level of MAPK pathway by inhibiting MAPK kinase activity\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. Furthermore, lanthanide ions can influence the expression of plant genes, specifically those associated with cell division, cell wall synthesis, and photosynthesis. Upregulation of these genes can facilitate plant growth and development\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. However, further studies are required to confirm the mechanism of lanthanide ion regulation of MAPK signaling pathway in plants.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnglish name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBrassica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCAT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSOD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003esuperoxide dismutase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePOD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eperoxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003esoluble protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePro\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eproline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMDA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMalondialdehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCaCl\u003csub\u003e2\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCalcium chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLaCl\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLanthanum chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eROS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ereactive oxygen species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNBT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003enitrogen blue tetrazolium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTBA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ethiobarbituric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNMT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003enon-invasive microtomography\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWGCNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eweighted gene co-expression network\u0026nbsp;analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePCA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eprincipal component analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that experimental research works on the plants described in this paper comply with institutional, national and international guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData and materials will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Gansu Agricultural University youth mentor support fund project (GAU-QDFC-2021-13), Gansu Province college industry support plan project (2023CYZC-51), the China Agriculture Research System of MOF and MARA (CARS-12-09).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJunyan Wu conceived and designed the study. Qiaowen Pan and Lulu Zhang analyzed the data. Fahim Abbas Muhammad revised and polished the paper. Lijun Liu, Gang Yang, Wangtian Wang, Yuanyuan Pu and Yan Fang contributed materials and analysis tools. Junyan Wu wrote the paper. Li Ma and Wancang Sun revised the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Yahong Zhang, Qixian Chen and Feng Li for identification of the cold tolerance of materials. Reviewers are acknowledged for their contribution to the improvement of the manuscript in the revision process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Li Ma([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhu, J., Dai, W., Chen, B., Cai, G., Wu, X., and Yan, G. (2023). 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Journal of Plant Nutrition and Fertilizers, 29, 1543-1555.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Winter rapeseed, Low temperature, Calcium chloride, Lanthanum chloride, Antioxidant enzyme activity.","lastPublishedDoi":"10.21203/rs.3.rs-4186636/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4186636/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLow temperature is one of the environmental factors that restrict the growth and geographical distribution of brassica. To investigate the effects of exogenous calcium and calcium inhibitors on the ability of winter rapeseed (\u003cem\u003eBrassica rapa\u003c/em\u003e L.) to withstand low temperatures, and its effect on physiological characteristics we used a strong cold-resistant Longyou 7 (L7) and a weak cold-resistant Longyou 99 (L99) cultivar. The seedlings were treated with CaCl\u003csub\u003e2\u003c/sub\u003e (20 mmol·L\u003csup\u003e-1\u003c/sup\u003e) and the calcium inhibitor LaCl\u003csub\u003e3\u003c/sub\u003e (10 mmol·L\u003csup\u003e-1\u003c/sup\u003e) at 0 h, 6 h, 12 h, 24 h and 48 h, the Ca\u003csup\u003e2+ \u003c/sup\u003eflux and Ca\u003csup\u003e2+ \u003c/sup\u003econcentration in the roots after 12 h and 24 h of treatment were analyzed, and results after low-temperature treatment, L99 showed Ca\u003csup\u003e2+\u003c/sup\u003e efflux with a rate of 30.21 pmol‧cm\u003csup\u003e-2\u003c/sup\u003e‧s\u003csup\u003e-1\u003c/sup\u003e, whereas L7 briefly showed efflux then returned to influx. Moreover. our findings illustrate that under low-temperature conditions, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased by both CK and exogenous CaCl\u003csub\u003e2\u003c/sub\u003e treatments. The contents of soluble protein (SP) and proline (Pro) were increased, while the contents of malondialdehyde (MDA) were decreased, resulting in reduced membrane lipid peroxidation. But enzyme activity decreased and MDA content increased following treatment with exogenous LaCl\u003csub\u003e3\u003c/sub\u003e. The rate of Ca\u003csup\u003e2+\u003c/sup\u003e flow showed a higher uptake in L7 roots compared with L99. Calcium ion content in root showed a decrease in ion content in both cultivars after CaCl\u003csub\u003e2\u003c/sub\u003e treatment. The results of RNA-seq data revealed that the genes that are differentially expressed in response to low temperatures, hormones, photosystem II, chloroplasts, DNA replication, ribosomal RNA processing, and translation were significantly enriched. This study found significant expression of genes related to cellular signal transduction (MAPK signaling pathway) and material metabolism (nitrogen metabolism, glycerol ester metabolism). The analysis of MAPK signaling pathway and genes in two modules led to the screening of 8 candidate genes related to the regulation of root growth, development and signal transduction.\u003c/p\u003e","manuscriptTitle":"Effects of exogenous calcium and calcium inhibitor on physiological characteristics of winter rape (Brassica rapa) under low temperature stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-09 17:49:10","doi":"10.21203/rs.3.rs-4186636/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-08T10:40:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-08T05:33:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-28T10:51:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5f0940a1-9fc3-4cc1-b045-925bf48f5a19","date":"2024-04-24T11:27:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0ec58f9b-d1b5-45ea-931a-4b47a485c917","date":"2024-04-11T12:01:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-06T16:36:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-04T11:41:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-04T11:41:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-03-29T08:24:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ce8d288-5e36-405f-bc5c-3ff7e04bab84","owner":[],"postedDate":"April 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:01:43+00:00","versionOfRecord":{"articleIdentity":"rs-4186636","link":"https://doi.org/10.1186/s12870-024-05556-w","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-10-09 15:57:32","publishedOnDateReadable":"October 9th, 2024"},"versionCreatedAt":"2024-04-09 17:49:10","video":"","vorDoi":"10.1186/s12870-024-05556-w","vorDoiUrl":"https://doi.org/10.1186/s12870-024-05556-w","workflowStages":[]},"version":"v1","identity":"rs-4186636","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4186636","identity":"rs-4186636","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}