DISCUSSION
At present, many MIRNA genes functioning in stress response have been identified by small RNA profiling and RNA-seq (Ahmed et al., 2020; Ma et al., 2022; Zhu et al., 2022). However, the MIRNA genes that function in the response to cold stress in crops remain largely unexplored. In this study, we used small RNA profiling and GWAS to identify cold-responsive candidate miRNAs or MIRNA genes associated with low-temperature resistance. The results from these two methods provide mutual support for the roles of miR1885 in the cold response of Brassica crops.
We identified up-regulated or down-regulated miRNAs in small RNA sequencing libraries of B. rapa and candidate MIRNA genes from GWAS loci associated with low-temperature resistance in B. napus . The small RNA profiling suggested that miR1885 was drastically triggered by cold stress. Using GWAS, we found MIR1885 gene was in the loci associated with low-temperature resistance. We further confirmed that miR1885 was induced by cold stress in both B. rapa and B. napus . We used the newest version of B. rapa and B. napusgenome reference to analyze their genomic synteny. As expected, theB. napus miR1885 (ChrA06) is inherited from the genome ofB. rapa (ChrA06 genome) through genome merging (Fig. S5). Additionally, For the cold responsive miRNAs in this study, we found some miRNAs have been reported to regulate flowering, such as miR156/7, miR168 and miR158. This suggested that some the cold-responsive miRNAs also play roles in vernalization.
Mature miR1885 is a Brassica -specific miRNA that targets theR gene family. In A. thaliana , the TIR-NB-LRR protein RPP4 and CHS1 were involved in plant resistance to low temperature stress (Huang et al., 2010; Zbierzak et al., 2013). Here, two R genes,Bn.TIR.A09 and Bn.TNL.A03 , were predicted as targets for cleavage by miR1885 in B. napus . These R genes belonged to two different subgroups: Bn.TIR.A09 encoded a protein with the TIR motif lacking NBS-LRR domain; while Bn.TNL.A03 encoded a protein with the entire TIR-NBS-LRR structure. We found that these two targeted genes were down-regulated in wild-type B. napus under cold treatment, opposite to the expression pattern of miR1885 (Figure 6). These results demonstrated that miR1885 negatively regulated theseR genes under cold temperatures.
Overexpression of miR1885 in semi-winter type B. napus attenuated rapeseed sensitivity to low temperature at the seedling stage. While knocking down of miR1885 in Spring type B. napus (STTM1885lines) improved plant tolerance to low temperature (Figure 7). Additionally, we did not find obvious differences between knock-down (STTM1885) lines and control lines at the early seeding stage. ButSTTM1885 did delayed the flowering time and increased the plant height at a later stage. The RNA levels of miR1885-cleaved R genesBn.TIR.A09 and Bn.TNL.A03, which increased inSTTM1885 rapeseed and decreased in miR1885-OE lines, may contribute to rapeseed response to cold stress. Although a link between miR1885 and cold response has been revealed, the hiding mechanism is still uncovered. In addition, we have checked some CBF-dependent genes in miR1885-OE lines, including CBFs and CORs , and found that the transcript levels of these genes in miR1885-OE in response to cold stress were no different with that of wild type, suggesting that miR1885-targets regulated plant resistance to low temperature independent on CBFs pathway.
In this study, we revealed a link between miR1885 and cold response in Brassica through its targeted R genes. However, how miR1885 and these R genes involve in CBF-independent cold response in rapeseed requires further examination. In Arabidopsis, 22-nt miRNAs can trigger the production of 21-nt phased secondary siRNAs (Cuperus et al., 2010; Li et al., 2012). 22-nt miR482/2118, which widely present in seed plants, triggered phasiRNA biogenesis from its targeted R genes (NBS-LRR), forming another layer of regulation to reinforce its silencing effect (Fei et al., 2013; Liu et al.,2020). Therefore, 22-nt miR1885 in rapeseed possibly triggers phasiRNAs biogenesis from its targeted R genes, and these phasiRNAs may target unknown downstream genes to regulate plant response to low temperature stress. Additionally, co-immunoprecipitation (co-IP) followed by mass spectrometry can be performed to identify proteins interacting with miR1885-regulated R proteins under cold stress, which may play role in cold signal transfer and resistance to cold stress.
In rice, OsmiR1320 was repressed by cold stress. However, overexpressing OsmiR1320 enhanced plant tolerance to cold stress, indicating that OsmiR1320 played positive role in cold response (Sun et al., 2022). On the contrary, we found that, as a negative regulator of cold response, miR1885 was induced under cold stress potentially via its low-temperature response (LTR) cis-element in MIR1885 promoter. Therefore, MIRNA transcription could be regulated incoherently by cold stress. The genetic mutation in and close to this LTR in the rapeseed population may contribute to their diversity of cold response (Figure 3C and Figure S3E). Genetic editing in this LTR could be used for detaching the cold response of MIR1885 , and therefore enhance rapeseed resistance to cold stress. Previous works showed that over-expression of miR1885 affected development traits and biotic response, including flowering time and disease resistance. The abundance of miR1885 may regulated by different signals through potentially different promoter cis elements and played a very important role in regulating both plant growth and development.
In summary, using multi-omics data, we identified miR1885 involved in plant response to low temperature stress by small RNA sequencing and GWAS. Overexpression of miR1885 in B. napus not only increased plant sensitivity to low temperature, while knockdown of miR1885 improved plant tolerance to low temperature. Our findings suggested thatMIR1885 and its target genes can be used as genetic resource for improving resistance to low temperature in the breeding ofBrassica crops.
METHODS AND MATERIALS