Validation of cold responsiveness of mature miRNAs in B. rapa
To validate the cold-responsive miRNAs detected by small RNA sequencing, we further analyzed the cold responsiveness of mature bra-miR1885, bra-miR398, bra-miR408, bra-miR161, bra-miR157, bra-miR393, and bra-miR168 using qRT-PCR. We found that the abundance of bra-miR1885, bra-miR398 and bra-miR168 were significantly up-regulated by cold stress, whereas bra-miR161, bra-miR157 and bra-miR393 were significantly down-regulated, which were consistent with the small RNA sequencing data (Figure 2A). Among these miRNAs, bra-miR398 and bra-miR168 were conserved miRNAs that were induced more than three-fold by cold treatment (Figure 2A). We then performed northern blotting analyses to further confirm the cold responsiveness of bra-miR398, bra-miR168, bra-miR1140 and bra-miR1885. Consistently, the accumulation of bra-miR398 and bra-miR168 was much higher in B_V7 than in B_V0, andBrassica -specific miR1140 was repressed after cold treatment (Figure 2B). In addition, the results of northern blot analyses further confirmed the abundance of mature bra-miR1885 was increased in both leaf and the shoot apical meristem (SAM) under cold stress (Figure 2C).
Next, to identify the potential cold-regulated cis -elements in the bra-MIR1885 promoter, we extracted the promoter sequence ofbra-MIR1885 using Promoter 2.0 Prediction Server (https://services.healthtech.dtu.dk/service.php?Promoter-2.0), and analyzed the cis- elements in the putative bra-MIR1885promoter sequence using PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). These analyses revealed that the promoter contained light-responsive, defense-responsive, stress-responsive, and low temperature-responsive elements (LTR: CCGAAA) (Figure S2). A previous study has revealed that LTR motif (CCGAAA) within the promoter region of CsINV5 was the core cis element in response to low temperature (Qian et al., 2018). This result suggested that the bra-MIR1885 promoter contained a low temperature-responsive element that potentially regulated the expression of bra-MIR1885 under cold stress.