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 . Semi-winter type WT K407 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 S6A). This result indicated
miR1885 functioned as negative regulator in rapeseed response to low
temperature. We further performed phenotypic studies using WT K407 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 K407 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 K407
(Figure S6B-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 test that whether the cold-responsive miR1885 can be applied to
improve low temperature tolerance of Spring type Brassica , the
miR1885 knockdown plants (STTM1885 ) generated in Sping typeBrassica 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 STTM1885 plants. Compared with those in wildtype
Westar, the expression levels of Bn.TNL.A03 and Bn.TIR.A09were increased in STTM1885 plants (Figure 7B). To study
morphological responses to cold stress, we then moved the Westar andSTTM1885 plants into 2 °C environment. After 14 days cold
treatment, a cold-tolerant phenotype was observed in STTM1885plants (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 S7A). 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 S7A and
Figure S7C). 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 S7D). 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.