INTRODUCTION
Brassica crop species include Brassica napus (rapeseed),Brassica rapa , Brassica oleracea , Brassica juncea ,
and Brassica nigra . Among them, B. napus arose
approximately 7500 years ago by allopolyploidy between its ancestors,B. rapa and B. oleracea (Chalhoub et al., 2014). B.
rapa and Arabidopsis thaliana share a common ancestor, butB. rapa has a larger genome than that of A. thaliana due
to genome triplication (Wang et al., 2011). Both B. napus andB. rapa are important food sources. B. napus mainly
provides edible plant oil and B. rapa is a valuable oil and
vegetable crop. However, Brassica crops are negatively affected
by low temperature, especially those that over-winter in frigid
environments. Worldwide, extensive economic losses of Brassicacrops are attributed to low temperature, often in combination with other
abiotic or biotic stresses (Sanghera et al., 2011).
Previous studies have identified many genes involved in the plant
response to low temperature (Ding et., 2019). Cold induced the
expression of the transcription factor gene CBF (encoding
C-repeat binding factor), and CBF triggered the expression of COR(COLD RESPONSIVE) genes (Shi et al., 2018). CBF/COR-dependent factors
allowed plants to withstand subsequent freezing stress (McClung and
Davis, 2010). In addition, many important genes, which contributed to
low-temperature response in CBF -independent manner, were also
found (Bolt et al., 2017; Liu et al., 2018). Although breeding for
low-temperature resistance is possible, the genetic improvement of crops
is still hindered by the lack of elite genetic resources related to
low-temperature resistance.
MicroRNAs (miRNAs) are regulators of plant adaptation to abiotic or
biotic stress (Biggar and Storey, 2015). They are approximately 20–24
nucleotides (nt) in length and originate from pre-miRNAs with stem-loop
structures (Song et. al., 2019). The mature miRNAs are loaded into the
RNA-induced silencing complex that regulate target transcripts involved
in various aspects of plant growth, development, and stress responses,
including the response to cold or freezing stress (Chen, 2009; Anjali
and Sabu, 2020). For instance, overexpression of miR156 increased cold
tolerance in species including rice, A. thaliana, and pine (Zhou
and Tang, 2019). In rice, OsmiR319b down-regulated OsPCF6 andOsTCP21 , resulting in enhanced cold tolerance (Wang et al.,
2014), and overexpressing OsmiR1320 improved cold tolerance, although
OsmiR1320 was repressed under cold stress (Sun et al., 2022). InA. thaliana, plants with knocked-down miR165/166 displayed a
drought- and cold-resistant phenotype (Yan et al., 2016). Other known
cold-responsive miRNAs included miR172, miR396, miR845, and miR168
(Gupta et al., 2014; Zeng et al., 2018). However, miRNAs in response to
low temperature stress have not been well studied in Brassicacrops.
The plant R (RESISTANCE) gene encodes immune proteins involved in
the response to pathogen infection (Jones and Dangl, 2006). Most of the
identified R genes contain a nucleotide-binding site-leucine-rich
repeat (NBS-LRR) domain with a coiled-coil (CC) or a Toll/Interleukin-1
receptor (TIR) domain at the amino terminus (Dangl and Jones, 2001).
Emerging evidence has shown that cold and pathogen attack induced the
expression of some common genes, such as PR(PATHOGENESIS-RELATED) genes, which played roles in the response to
pathogen infection and low temperature stress (Snider et al., 2000; Seo
et al., 2008). Some R genes negatively regulated plants tolerance to low
temperature. In A. thaliana , the rpp4-1d mutant plant with
a gain-of-function mutation in RPP4 (TIR-NB-LRR protein
PERONOSPORA PARASITICA 4) was chilling-sensitive (Huang et al., 2010).
Mutations in HSP90 suppressed the chilling-sensitive phenotype ofrpp4-1d mutant plant, and HSP90 interacted with the RPP4 protein
in planta (Bao et al., 2014). Interestingly, a significant increase inHSP90 mRNA was detected in B. napus exposed to low
temperature (Krishna et al., 1995). In addition, a gain-of-function
mutant of CHS3 , encoding a TIR-NB-LRR-LIM protein, resulted in
enhanced defense responses and chilling-sensitivity phenotype (Yang et
al., 2010). Other studies found that some R genes played positive
roles in plants tolerance to low temperature. A point missense mutation
of CHS1 , encoding a TIR-NB protein, resulted in a
chilling-sensitive phenotype (Wang et al., 2013; Zbierzak et al., 2013).R genes were also regulated by genes that contributed to cold
tolerance. In tobacco, overexpression of SgRVE6 (REVEILLE )
improved plant tolerance to low temperature and up-regulated the
expression of genes encoding NB-LRR proteins (Chen et al., 2020).
Although many studies have investigated the molecular mechanisms
underlying low-temperature resistance, the roles of miRNAs in
low-temperature resistance in Brassica crops are largely unknown.
In this study, we constructed and sequenced small RNA libraries fromB. rapa under cold stress and performed a genome-wide association
study (GWAS) related to the phenotype of cold resistance using aB. napus population. Combing the results from both small RNA
sequencing and the GWAS, we found that Brassica -specific miR1885
was responsive to cold stress in both B rapa and B napus .
Previous studies have revealed that heat, as well as Turnip mosaic
virus (TuMV ) and Plasmodiophora brassicae infection,
induced miR1885 accumulation (He et al., 2008; Yu et al., 2011, Paul et
al., 2021). Here, we further demonstrated that miR1885 was also involved
in the response to low-temperature stress through its target genes and
functioned as a negative regulator of B. napus in low temperature
tolerance, providing potential genetic candidates for cold-resistant
rapeseed breeding.