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.