It is acknowledged that freezing stress causes serious injuries to the plant, which is general in many winter crop growing areas around China (Zhu, 2016), especially for winter rapeseed grown in Northern China (Xu et al. 2018). Some studies have suggested the effects of freezing stress on winter rapeseed resistance formation by the transcriptome, proteome and physiology (Zeng et al. 2018; Pu et al. 2019). However, the molecular mechanism and the formation of freezing tolerance in winter rapeseed are still poorly understood. The winter rapeseed cultivar NS can survive normally in winter in most northern areas from 38 degrees north latitude to 42 degrees north latitude, and its tolerance at low temperature environments is better than other types of winter rapeseed, which contains abundant cold-resistant genes. We therefore this study focus on levels of transcript, protein, and physiology and biochemistry to elucidate the molecular basis of freezing tolerance in leaves of winter rapeseed freezing-tolerant NS and sensitive NF. It will be greatly helpful to plant molecular breeding under freezing stress.
Comparison of the differences in freezing tolerance between both winter rapeseed cultivars
Numerous TFs have been reported in plants that monitor the expression of C-repeat binding transcription factor (CBF) by combining corresponding cis-elements in their promoters under cold stress. After exposed to freezing stress, CBF proteins rapidly recognize the promoter regions of downstream cold-regulated genes to activate their expression, thereby enhancing freezing tolerance (Jia et al. 2016; Zhao et al. 2016). For instance, bHLH transcription factor ICE1, calmodulin-binding transcription activator 3 (CAMTA3), brassinazole-resistant 1 (BZR1), heat shock transcription factor C1 (HSFC1), and MYB88 /MYB124 transcription factors positively regulate the CBF expression at the transcriptional level that contribute to cold stress (Ding et al. 2015; Park et al. 2015; Li et al. 2017; Xie et al. 2018). In contrast, MYB15, phytochrome interacting factor 3 (PIF3), PIF4, PIF7 and ethylene sensitivity 3 (EIN3) negatively modulate the transcriptional repression of CBF (Shi et al. 2012; Kim et al. 2017; Jiang et al. 2017). In the present study, most of AP2/ERF, WRKY, NAC, HSF, and TIFY transcription factors were up-regulated in both cultivars, while transcription factors bHLH, bZIP, ARF/GFP, OFP, and mTERF were down-regulated (Fig. 7AB; Table S6). In addition, we found that AP2/ERF and HSF genes have differences in DNA methylation levels (Table 1), indicating that these transcription factor genes may defend against freezing stress through epigenetic modifications. Interestingly, these differentially expressed TFs encoded by genes were not found at the protein level, implying that those transcriptional moderators might regulate the gene expression just at the transcription level in contribution to the freezing resistance of winter rapeseed under freezing stress.
Table 1
The list of DNA methylation levels of some DEGs identified in both cultivars (unpublished).
Gene Name
|
Meth.type
|
Meth.diff
|
Gene ID
|
log2(FC)
|
Position (start)
|
Position (end)
|
Cultivar
|
Ethylene-responsive transcription factor RAP2-4
|
CHH
|
-15.1
|
LOC106353462
|
2.513787538
|
66399101
|
66399300
|
NTS57
|
Ethylene-responsive transcription factor ERF114
|
CHH
|
-17.4
|
LOC106380120
|
3.264061601
|
11674501
|
11674700
|
NTS57
|
CHH
|
-29.3
|
LOC106380120
|
3.264061601
|
11675201
|
11675400
|
NTS57
|
Heat stress transcription factor B1
|
CHG
|
34.7
|
LOC106407137
|
3.106258687
|
59711401
|
59711600
|
NTS57
|
CHG
|
29.2
|
LOC106407137
|
3.106258687
|
59711601
|
59711800
|
NTS57
|
CHG
|
28.2
|
LOC106407137
|
3.106258687
|
59711801
|
59712000
|
NTS57
|
CBL-interacting serine/threonine-protein kinase 9
|
CHG
|
-51.9
|
LOC106401821
|
2.289092273
|
490801
|
491000
|
NTS57
|
Caffeic acid 3-O-methyltransferase
|
CHG
|
-27.7
|
LOC106417606
|
2.739228139
|
23901201
|
23901400
|
NTS57
|
CHG
|
-28.2
|
LOC106417606
|
2.739228139
|
23901301
|
23901500
|
NTS57
|
calcium-dependent protein kinase 28
|
CHG
|
25.5
|
LOC106392253
|
2.20728941
|
7242601
|
7242800
|
NQF24
|
Calcium-dependent protein kinase 6
|
CHH
|
20.9
|
LOC106400806
|
2.158244799
|
50391101
|
50391300
|
NQF24
|
Furthermore, some protein kinases including calcium-dependent protein kinases (CDPKs), CBL-interacting serine/threonine-protein kinases (CIPKs), mitogen-activated protein kinases (MAPKs), receptor-like kinases (RLKs), serine/threonine-protein kinases (SPK), and SNF1-related protein kinases (SnRKs) have been characterized to be crucial regulators of cold-stress responses in plant (Liu et al. 2017; Zhao et al. 2017; Ding et al. 2019b). In addition, a putative cold sensor chilling-tolerance divergence 1 (COLD1), was reported to mediate a cold-sensing calcium channel in rice, leading to the activation of cold-regulated genes (Ma et al. 2015; Shi et al. 2018). A similar result was obtained in the present study, a total of 7 CDPKs, 4 CIPKs, 13 MAPKs, 81 RLKs, 1 SnRK, and 42 SPKs were up-regulated in the cultivar NF under freezing stress, whereas 3 CDPKs, 3 CIPKs, 95 RLKs, 2 SnRKs, and 37 SPKs were down-regulated; a total of 9 CDPKs, 8 CIPKs, 9 MAPKs, 22 RLKs, 1 SnRK and 30 SPKs were up-regulated in the cultivar NS under freezing stress, whereas 9 CDPKs, 6 CIPKs, 11 MAPKs, 124 RLKs, 8 SnRKs and 49 SPKs were down-regulated (Table S5). Combined with the differences in DNA methylation levels of CDPK and CIPK genes (Table 1), a popular explanation is that freezing treatment might induce DNA methylation modification of these genes, thereby trigger the activation of other freezing-responsive genes, which needs further study.
Ribosomal proteins (RPs) are well-known for their role in maintaining the stability of the ribosomal complex, which performs mRNA directed protein synthesis (Moin et al. 2016). A drastic lowering of the ambient temperature entails extensive reprogramming of the gene expression pattern (He et al. 2021). In this study, compared to the control, all DAPs enriched in the ribosome pathway were accumulated in abundance in NF under freezing stress (Fig. 5B; Table S5), implying that the protein synthesis was enhanced, which might be beneficial to cope with freezing stress in NF.
Photosynthesis is main driving force for the growth and biomass production in plants, it can supply the energy and carbon for the biosynthesis of necessary organic compounds (Nowicka et al. 2018). In this study, the photosynthesis pathway was significantly enriched by DEGs and DAPs in NS, and all ferredoxins (FDX) and chlorophyll a/b-binding proteins (CBPs) enriched in this pathway were reduced in transcription and abundance (Fig. 6; Table S5), which can recruit photons and transfer the absorbed energy towards the photosynthetic reaction centre (Palm et al. 2018), suggesting that the photosynthesis activity was crippled under freezing stress. Consistent with transcriptome and proteome results, the measured net photosynthetic rates were decreased in NS and NF under freezing stress, but it was significantly higher in the former than that in later (Fig. 1B), which might attribute to more stable chloroplasts ultrastructure in the leaves of NS.
Plant hormones, including abscisic acid (ABA), auxin, brassinosteroid (BR), cytokinin, ethylene, gibberellin, jasmonic acid (JA) and salicylic acid, combine endogenous substances with environmental signals to regulate plant growth, development and defense (Huang et al. 2017). In many instances, plants respond to environmental stresses by producing amount of ABA, ethylene and JA (Huang et al. 2016; Hu et al. 2017). Similar results were obtained in this study, a total of 115 and 104 unique DEGs in NF and NS, respectively, were involved in plant hormone signal transduction pathway (Table S5), among them, 10 and 7 DEGs were up-regulated in NF and NS, respectively, and participated in ABA signaling, encoding abscisic acid-insensitive protein (ABI), SPK and protein phosphatase 2C (PP2C), whereas 6 and 10 DEGs encoding ABI, SPK and abscisic acid receptor (ABR) were down-regulated in NF and NS, respectively. 5 out of 48 DEGs and 15 out of 39 DEGs were up-regulated in NF and NS, respectively, and involved in auxin signaling, encoding auxin-induced protein (AIP), auxin-responsive protein (ARP), auxin transporter protein (ATP), and GH3 auxin-responsive promoter (GH3). Therefore, we surmised that there may be more than one ABA and auxin metabolic pathway related to freezing tolerance, and one of them was enhanced. Furthermore, 14 up-regulated DEGs and 7 down-regulated DEGs were found in NF and NS, respectively, and gone in for ethylene signaling, encoding ethylene insensitive protein (EIN), EIN3-binding F-box protein (EBF), ethylene-responsive transcription factor (ERF) and ethylene receptor (ER). Correspondingly, we found that two key enzymes of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate synthases (ACS) and 1-aminocyclopropane-1-carboxylate oxidases (ACO), were up-regulated in NF and down-regulated in NS, this may be due to feedback regulation in NS, resulting in the decreasing of ethylene metabolism. 5 DEGs were up-regulated in NF and engaged in JA signaling, encoding TIFY proteins and transcription factor MYC2, which was a key regulator for the activation of JA under cold stress (Kazan, 2015). These are consistent with what has been found in the previous research, which indicated that high levels of plant hormones could improve the freezing resistance (Pu et al. 2019; Wei et al. 2021). In addition, one DEG was down-regulated in NF, encoding protein BZR, which is plant-specific positive regulator of BR signaling (Li et al. 2017). BRs can be perceived by the brassinosteroid insensitive1 (BRI1; She et al. 2011), and BRI1 interacted with BRI1-associated receptor kinase and positively regulated BZR1 (Fang et al. 2019). Interestingly, in this study, 2 BRI1 genes were found and they were down-regulated in NF, however, we found only one down-regulated BZR2 gene in NS. Therefore, we speculate that BZR1 could combine with BRI1 to negatively regulate BR signaling in NF under freezing stress. Apart from that, numerous studies have reported that BRs could improve photosynthesis and ROS scavenging enzymes activities (Xia et al. 2018; Yan et al. 2020). Overall these findings are in accordance with the results of photosynthesis and antioxidant enzymes (Fig. 1B; Fig. 2). Simultaneously, other DEGs identified in both cultivars in our study mainly represent some TFs, which are essential for plants under abiotic stresses (Li et al. 2019; Frank et al. 2020). Taken together, these results suggested that plant hormones play profound roles in coping with the freezing stress of rapeseed.
Plant lipoxygenases (LOXs) are a kind of multifunctional fatty acid dioxygenases that catalyze the peroxidation of linolenic acid and linoleic acid. The synthesis of jasmonic acid begins with the peroxidation of α-linolenic acid by lipoxygenase. It was reported that biotic and abiotic stresses could cause the liberation of α-linolenic acid, which is an important precursor for the biosynthesis of jasmonic acid (Yan et al. 2013; Zhao et al. 2014), which interacted with other hormones signaling to regulate leaf tolerance to cold stress (Hu et al. 2017). In this study, all unique LOXs participated in the alpha-linolenic acid metabolism were found to be down-regulated in NF, whereas they were up-regulated in NS (Fig. 5A; Table S5). An interesting finding was that the alpha-linolenic acid metabolism was enriched by DAPs in NF, and all DAPs were accumulated in abundance (Fig. 5B; Table S5). These results suggested that there are differences in temporal and spatial between the expression of gene and protein. Beyond those, combined with JA signaling resulting from DEGs, implying that increased jasmonic acid signal might regulate the expression of downstream freezing-responsive genes.
Plant secondary metabolites often have no fundamental role in the maintenance of plant life processes, but they are crucial for the plant to interrelate to its environment for adaptation and defense (Ramakrishna et al. 2011). In the present study, there were 7 and 6 unique up-regulated DEGs in NF and NS, respectively, encoding caffeic acid O-methyltransferase (CCMT), shikimate O-hydroxycinnamoyltransferase (SHCT) and S-adenosylmethionine-dependent methyltransferase (SAMTs), implicated in the secondary metabolism (Fig. 5B; Table S5), which were proclaimed in many plant species (Ranjan et al. 2020). It is in agreement with those reported by Zeng et al. (2018) and Pu et al. (2019). It is interesting to note that some DNA methylation differences in the CCMT gene were found under freezing inducement (Table 1), which is particularly important when investigating the epigenetic freezing-reponsive mechanism of winter rapeseed. A further novel finding was that the plant-pathogen interaction pathway was significantly enriched by DEGs in NS, and most of DEGs enriched in this pathway were up-regulated (Fig. 6A; Table S5), implying that its defense system was strengthen after freezing treatment. These results suggested that accumulated secondary metabolites and enhanced plant-pathogen interaction together contribute to the formation of freezing tolerance of rapeseed cultivar NS under freezing stress.
It is acknowledged that heat shock proteins (HSPs) regarded as the target genes of HSFs, are beneficial to defense against cold stress in grasses (Wang et al. 2016). Similarly, Lin et al. (2019) reported that some HSPs and chaperone proteins (CPs) were induced by cold stress. Scarpeci et al. (2008) indicated that ROS through direct interacted with HSFs to offer abiotic stress tolerance in plants. In the present study, some up-regulated HSFs, CPs and HSPs were concomitant with high level of POD activity (Fig. 2C; Table S5), showing that the HSFs might interact with ROS signals to regulate the expression of CPs and HSPs under freezing stress. Furthermore, several up-regulated DEGs encoding protein disulfide isomerase (PDI) associated with protein synthesis were found in NS, whereas they were down-regulated in NF (Table S5). In addition, we discovered 10 unique down-regulated DEGs in NF, which encode mitochondria-localized ATP synthase subunit (ASS) and cytochrome c oxidase (CCO), and 2 unique up-regulated CCOs in NS (Table S5). As we all know, ASS is the structural basis of proton transportation and energy generation in mitochondrial (Klusch et al. 2017). CCO is the last respiratory complex of the electron transmit chain in mitochondria and is responsible for transferring electrons to the final acceptor oxygen in the respiratory metabolism (Dahan et al. 2014). The down-regulated ASS and CCO indicated the decrease of energy requirement in the developing leaf of NF under freezing stress, which might be one of the major reasons why it was more freezing-sensitive than the cultivar NS.