miR1885 negatively regulated low temperature tolerance inBrassica
A previous study reported that knockdown of CHS1 , encoding a TIR-NB protein, led to a chilling-sensitive phenotype in A. thaliana (Zbierzak et al., 2013). We found that two TIR-NB transcripts were the targets of miR1885 and both miR1885 and these two TIR-NB genes were cold-responsive. Therefore, we further tested the effects of miR1885 and its targets on low-temperature resistance in B. napus . WT and miR1885-OE plants were grown for 6 weeks at 22 °C, and then were transferred to 4 °C for 30 days or 2 °C for 7 days. We found that the miR1885-OE plants were more sensitive as compared to WT under 2 °C for 7 days (Figure 7A) but showed no apparent phenotypic difference under 4 °C (Figure S5A). This result indicated miR1885 functioned as negative regulator in rapeseed response to low temperature. We further performed phenotypic studies using WT and miR1885-OE seeds during post-germination under normal and cold environment. The seeds were harvested at the same season and the cold environment was 6 °C. The hypocotyl length (NHL) of WT and miR1885-OE showed no difference after growing for 2 days on the germination bed without cold treatment. However, after 7 days cold treatment, the miR1885-OE exhibited shorter NHL compared with WT (Figure S5B-D), indicating that miR1885 also played critical role in cold response in the early developmental stage, when no significant phenotypic difference between WT and miR1885-OE was observed under normal condition.
To confirm that whether miR1885 and their targets negatively regulated low temperature tolerance in Brassica , the miR1885 knockdown plants (STTM1885 ) generated in Brassica napus cv. ‘Westar’ were used for further study (Cui et al., 2020). The qRT-PCR results showed that the accumulation of miR1885 was reduced in STTM1885plants. Compared with those in wildtype Westar, the expression levels ofBn.TNL.A03 and Bn.TIR.A09 were increased inSTTM1885 plants (Figure 7B). To study morphological responses to cold stress, we then moved the Westar and STTM1885 plants into 2 °C environment. After 14 days cold treatment, a cold-tolerant phenotype was observed in STTM1885 plants (Figure 7C). This finding further suggested that attenuating miR1885 resulted in enhanced cold tolerance of rapeseed potentially via their targets.
We further tested whether overexpression of miR1885 affected plant growth at freezing temperature compared to its background winter ecotype K407, while the spring ecotype Westar was used as negative control (Figure S6A). The abundance of mature miR1885 in wild-type B. napus under freezing stress was examined by real-time PCR. Based on previous studies, we set the freezing temperature at −10 °C (Xu and Cai, 2019; Ljubej et al., 2021). We found that miR1885 was up-regulated under freezing stress, and its expression level was approximately 2.4-fold than in wild type at 2.5 h (Figure S6B). The wild type and miR1885-OE lines were subjected to a −10 °C treatment for 2.5 h, and then allowed to recover at 22 °C for 7 days. The survival rate of wild type K407 was 78.00%, and those of miR1885-OE were much lower, ranging from 22.33% to 55.33%, whereas 100% of spring ecotype Westar died (Figure S6A and Figure S6C). Accumulation of free proline (Pro) during cold or freezing stress is thought to protect plants (Xin and Browse, 1998). To test whether miR1885-OE had altered Pro content, we determined the Pro content in wild type and miR1885-OE with or without freezing treatment (Figure S6D). As we expected, the Pro content was higher in wild type and miR1885-OE grown at −10 °C than in those grown at 22 °C. In plants grown at −10 °C, more Pro accumulated in the wild type plants than in the miR1885-OE. This finding was consistent with the freezing-sensitive phenotype of transgenic lines subjected to a −10 °C treatment. These results demonstrated that overexpression of miR1885 increases plant sensitivity to freezing at the seedling stage.