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.