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 .
MiR1885 is a Brassica -specific miRNA that targets the Rgene 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 B. napus attenuated rapeseed sensitivity to low temperature at the seedling stage. While knocking down of miR1885 in B. napus (STTM1885 lines) improved plant tolerance to low temperature (Figure 7). The RNA levels of miR1885-cleaved R genes Bn.TIR.A09 and Bn.TNL.A03, which increased in STTM1885 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 andCORs , and found that the transcript levels of these genes in miR1885-OE in response to cold stress were not different with that of wild type, suggesting that miR1885-targets regulated plant resistance to low temperature independent on CBFs pathway. How miR1885 and its cascade target genes modulated plant resistance to low temperature remains to be further investigated.
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.
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 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.