Low temperature is an important factor restricting the cultivation of Brassica oleracea L.. At present, there are few studies on the cold tolerance mechanism of Brassica oleracea L.[17-20], and the differences of cold tolerance among different varieties have not been sufficiently analyzed and demonstrated. In this study, two varieties with different cold tolerance were used: low-temperature sensitive type (YN) and low-temperature tolerant type (ZG). Considering that it is common for Brassica oleracea L. to experience a temperature of about 4°C during winter overwintering cultivation, only one temperature treatment was carried out. In order to verify the results of RNA-seq data, 13 DEGs were randomly selected for qRT-PCR, and the results of these genes were consistent with the RNA-seq results.
Analysis of differentially expressed genes under low temperature stress
In this study, two varieties were treated at 4°C for 24h, and then several representative physiological indexes were determined[21-23]. These physiological indices indicated that ZG was more cold tolerant than YN. Through RNA-seq results, there were 9211 DEGs in response to low temperature in ZG, and 10191 DEGs in response to low temperature in YN. In the two varieties contain a large number of genes make response to low temperature. Interestingly, the number of DEGs in response to low temperature stress in YN was slightly higher than that in ZG, Therefore, it can be inferred that it is very important to analyze the cold-resistance mechanism of uniquely DEGs in ZG. All the DEGs of the two varieties were analyzed, and 6098 DEGs were found in both varieties. These genes were speculated to be involved in the common mechanism of response to low temperature stress in Brassica oleracea L.. In addition, 1844 DEGs were only specifically expressed in ZG, and these genes were speculated to be specific genes that could improve cold tolerance of Brassica oleracea L..
The GO enrichment of genes from biological processes, cellular components and molecular functions was carried out, and it was found that most of the DEGs, both common to the two varieties and specific to ZG, were concentrated in terms of biological processes, with little involvement in terms of the other two. In addition, among the top 30 enriched GO terms, 5 terms belonging to biological processes are involved in both genes. These 5 terms are GO: 0009719 (response to endogenous stimulus), GO: 0007165 (signal transduction), GO: 0023052 (signaling), GO: 0044700 (single organism signaling) and GO: 0001101 (response to acid chemical). These five terms are speculated to be involved in the basic mechanism of drought resistance of Brassica oleracea L. It is worth noting that 4 GO terms belonging to molecular functions only appear in ZG specific differentially expressed genes, they are respectively: GO: 0016740 (transferase activity), GO: 0019787 (ubiquitin-like protein transferase activity), GO: 0015399 (primary active transmembrane transporter activity) and GO: 0015405 (p-p-bond-bromo-driven transmembrane transporter activity), it can be speculated that these four terms are important functional terms to enhance the cold tolerance of Brassica oleracea L.. Both the same terms with different genes involved and the different terms with the same genes involved can reflect the response degree of varieties to chilling injury. On the other hand, after KEGG pathway enrichment of the two genes, it was found that among the top 20 enrichment pathways, there are two common enrichment pathways: ko04712 (Circadian rhythm-plant) and ko04075 (Plant hormone signal transduction), and all the genes that were commented into the transcription factor family were involved in these two pathways. It is speculated that these two pathways play an important role in the cold resistance mechanism of Brassica oleracea L..
Therefore, It is important to collate the focused pathways and analyze the differentially expressed genes involved in the pathways to explore the mechanism of cold resistance in Brassica oleracea L.. And the terms jointly enriched by these two varieties, the special terms of ZG with strong cold tolerance and the genes involved in these functional terms may be important basis for the improvement of Brassica oleracea L..
Plant hormone signal transduction
Under low temperature stress, plant growth hormone is involved in plant growth and development, signal transduction and other aspects of stress, such as ABA, GA and IAA[25, 26]. In this study, a large number of DEGs related to plant hormones appeared under low temperature stress.
In previous studies, the EIN3 is validated and its mutant EIN3-1 in ethylene signal transduction pathway, found three in Arabidopsis thaliana genome and EIN3 higher genetic sequence similarity, respectively named EIL1, EIL2 and EIL3. EIN3 and EIL1, EIL2 can be complementary EIN3-1 phenotype, that they are involved in the ethylene signal transduction[27, 28]. However, EIL1 has been proved to be the second transcriptional activator in regulating sulfur deficiency response in Arabidopsis thaliana, second only to SLIM1/EIL3, and EIL3 has also been shown to play an important role in resisting abiotic stress and regulating the expression of ethylene synthesis genes in mulberry, and is up-regulated in response to abiotic stress[29, 30]. In this study, EIF2 did not appear, EIF1 and EIF3 did appear in both varieties, but EIF1 showed a down-regulated trend while EIF3 showed an up-regulated trend. Therefore, it can be inferred that EIL3 also plays a positive regulatory role in the response of Brassica oleracea L. varieties to low temperature stress.
In this study, ABF3 and ABF1 appeared in both varieties, and both genes were up-regulated under low temperature stress while involved in regulating hormone signal transduction. The role of ABF1 and ABF3 in stress signal regulation network has been studied in Arabidopsis thaliana, ABF1 was mainly involved in response to low temperature and ABA stress. ABF3 was mainly induced by ABA, high salinity, low temperature, heat and oxidative stress [31-34].
In addition, ERF2 overexpression has been proved to improve the cold tolerance of tomato and rice, and the expression of ERF15 and ERF16 is regulated by the JA signaling pathway in tomato plants[35, 36]. In this experiment, ERF2 and ERF15 appeared in the two varieties. ERF15 showed an up-regulation trend in response to low temperature stress in the two varieties, while ERF2 showed the opposite trend.
In addition to the above contents, some other genes only appeared in ZG under low temperature stress and showed an up-regulated trend: ARF7, TGA1, TGA3, and TGA7. At present, there are few research results on ARF7, and it is basically confirmed that ARF7 is involved in the growth and development of flower buds and pericarp of plants[37, 38]. However, in this study, ARF7 was annotated as an auxin response factor. For TGA1, TGA3 and TGA7, it has only been confirmed that they are widely involved in the disease resistance response of plants, and there is no direct connection with plant hormone signal transduction [39-42].
Photoperiod changes and day-night alternation in nature play an important and complex role in plant growth and development. Studies have shown that circadian rhythm is involved in the response of plants to low temperature stress[43, 44]. The effect of biological clocks on plants has also been demonstrated.
In down-regulated genes present in two cultivars, only PIF3 was involved in both plant circadian subtitles and plant hormone signal transduction. Recent studies have shown that PIF3 is involved in regulating plant hormone synthesis and response to low temperature stress[46, 47]. It is necessary to further study the role of this gene in the cold tolerance mechanism of Brassica oleracea L..
In Arabidopsis thaliana, loss of the central clock genes, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELON-GATED HYPOCOTYL (LHY), results in both reduced feuptake and photosynthetic efﬁciency, whereas CCA1 overexpression confers the opposite effects[48, 49]. In this study, LHY gene and CCA1 gene appeared in the differential genes in response to low temperature stress, which participated in the process of plant circadian rhythm and belong to the MYB related family. The difference is that LHY plays a role in both cultivars, while CCA1 plays a role in ZG, and both of them showed an up-regulated trend after cold injury. Its suggesting that the chilling tolerance of LHY gene on Brassica oleracea L. is more basic, while CCA1 is more targeted to improve the chilling tolerance of Brassica oleracea L..
Also worth mentioning is COL2 gene, which shows an up-regulated trend in ZG but a down-regulated trend in YN under low temperature stress. In addition, CO and COL1 genes were found in both cultivars and belonged to the same gene family as the above COL2 gene. Studies have confirmed that CO gene is involved in the photoperiodic regulation pathway of plants, and plays an important role in it [50, 51]. And the function of COL1 and COL2 genes, which are homologous to the CO gene in Arabidopsis, was also preliminarily identified. COL1 gene was proved to shorten the circadian rhythm, but the function of the COL2 gene is not yet understood. CO and COL1 genes are also involved in the circadian rhythm of the two varieties of plants, and both of them show an up-regulated trend under low temperature stress. There have been some studies on CO-like gene family[53, 54], and it is speculated that this gene or this gene family may be one of the reasons for the strong cold tolerance of ZG. How COL2 gene and CO-like gene family participate in the cold tolerance mechanism of Brassica oleracea L. can be studied in the future.
Now it has been proved that low temperature induced anthocyanin accumulation under light requires HY5 and HYH, and it has also been found that the reduction of endogenous gibberellin is conducive to low temperature induced anthocyanin accumulation. The gibberellin-degrading enzyme encoding gene GA2ox1 was also up-regulated at low temperature and was dependent on HY5/HYH [55-57]. In this study, HY5 gene appeared in the circadian rhythm pathways of both varieties. HYH gene and HY5 gene showed an up-regulation trend in response to low temperature stress in the two varieties. However, CDF1, which is only specific in ZG, was studied in tea tree, non-heading Chinese cabbage, tomato and other crops [58-60], and it was found that CDF1 may interact with FKF1 in non-heading Chinese cabbage, and then participate in the photoperiod flowering pathway. However, CDF1 did not play an obvious role in regulating photoperiod flowering pathway in tea and tomato. In this study, CDF1 gene only appears in ZG and participates in the circadian rhythm pathway, which also presents an up-regulated trend in response to low temperature stress. So, this gene may be an important gene of ZG with strong cold tolerance.
Transcription factors and gene family classification involved in Brassica olerace L. responses to cold stress
At present, a series of transcription factors have been identified to participate in the regulation of plant response to low temperature stress, including AP2/ERF, MYB, BHLH, NAC, WRKY and VOZ, etc. In this study, transcription factors that are up-regulated in both hormone signal transduction and plant circadian rhythm pathways were identified and noted into 9 gene families, namely, bZIP, MYB-related, CO-like, EIL, ERF, WRKY, DOF, and ARF. In particular, most of the up-regulated genes were concentrated in the bZIP gene family, nowadays, the study of bZIP gene families are many, in rapeseed in bZIP transcription factors of mining under the low temperature induction, identified 108 response induced by low temperature rapeseed bZIP transcription factors, cold resistant and drought tolerant, salt resistance performance and participate in ABA response features[18, 61].