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.