tae-miR399-TaUBC24 module has a positive regulatory role
in Dn1 response to freezing stress
miR399 is sensitive to environmental temperature(Kim, Ahn, Chiou, &
Ahn, 2011). The expression change of miR399 is different in different
plants and different low temperature treatments. The expression of
miR399 in tobacco was significantly up-regulated at 10 for 1 d (R. S. Hu
et al., 2019), but in Arabidopsis, the expression of miR399 at 16℃ was
significantly lower than that at 23℃ (Kim et al., 2011). However, it has
not been reported the expression pattern of miR399 in wheat under low
temperature stress. In this study, we found that the expression of
miR399 in Dn1 tillering nodes of winter field was not always up or down,
it was down-regulated at 0℃, then gradually increased, and the
expression increased rapidly at -25℃ (Fig. 2A). According to the
standards of previous studies, the average temperature of winter wheat
cold acclimation period in the north of China is about 5℃ (Li, Fu, Wang,
Sun, & Tong, 2010), and 0℃ is the critical point of water freezing. At
0℃, the expression of a little winter wheat cold resistant genes is
unchanged or slightly decreased, such as TaMYB1 , TabZIP1and TaWABI5 (Lv et al., 2018). Previous studies have shown that
when plants are exposed to similar environment for a long time, the
expression of stress genes may decrease (J. Kilian et al., 2007).
Therefore, the expression of tae-miR399 decreased at 0℃ in this study.
We speculate that this may be due to the small temperature difference
between 5℃ and 0℃, Dn1 did not initiate cold response, and the
expression of stress genes returned to the level before cold acclimation
due to cold adaptation. When the temperature drops to -10℃, the
physiological and biochemical level and molecular level of winter wheat
begins to change significantly (Lu, Guo, Xu, & Cang, 2020; Lv et al.,
2018). Previous studies have shown that most cold resistance genes
begins to increase significantly at -10℃, such as TaWABI5 ,TaCSD1 and TaFBA-A10 (Lu, Guo, et al., 2020; Lv et al.,
2018; K. K. Peng et al., 2021). In this study, the expression of Dn1
tae-miR399 at -10℃ was lower than that at 5℃, but it was higher than
that at 0℃, which was similar to the previous results, indicating that
the expression of miR399 was started at -10℃. At the extremely low
temperature of -25℃, the expression of transcription factors, such asTaMYB1 , TabZIP1 and TaWABI5 , tends to decrease
slightly, while the expression of genes encoding key enzymes in
metabolism pathway still rises sharply, such as TaCSD1 ,TaG6PDH and Ta6PGDH (Lu, Guo, et al., 2020; Lv et al.,
2018; Tian et al., 2021). In this study, we found that at -25℃, the
expression of Dn1 tae-miR399 increased significantly, while the target
gene TaUBC24 decreased significantly. According to the function
of miR399 in maintaining Pi homeostasis in plants(Bari et al., 2006),
these results imply that the Dn1 tillering node still needs to absorb
and transport Pi at -25℃, which may be affected by the high expression
of genes encoding phosphatase in Dn1 metabolism pathway to provide
substrate phosphate.
At present, it has been shown that miR399 is involved in plant growth
and development. For example, silencing the expression of miR399 can
damage the floral meristem and anther development of Citrus (Pegler,
Oultram, Grof, & Eamens, 2020), and overexpressing miR399 can affect
the fruit quality of strawberry (Y. Wang et al., 2017). However, it has
not been reported whether miR399 is involved in the development of
tillering nodes in plant seedlings. Combined with previous studies, we
found that the date of sampling Dn1 tillering nodes in winter is
similar, and it took about three months for the temperature to drop from
5℃ to -25℃ (K. K. Peng et al., 2021). Therefore, there might be temporal
and spatial differences in gene expression in wheat. To further confirm
that the high expression of miR399 in Dn1 under low temperature was
indeed induced by low temperature, we treated Dn1 wheat seedlings
cultured for about 15 d in the laboratory with short-term low
temperature. The results showed that the expression of miR399 in Dn1
seedlings increased rapidly at -10℃ (Fig. 2C), which indicated that
freezing stress could induce the expression of miR399 in Dn1 seedlings
and decrease the expression of its target gene TaUBC24 in
response to low temperature stress. However, after cold acclimation at
4℃ for 30 d, the expression of miR399 decreased slightly, and the
expression of its target gene TaUBC24 increased significantly
(Fig. 2C and D). This may be due to the long-term cold acclimation,
which make Dn1 adapt to the current temperature.
UBC24 (PHO2) is a ubiquitin-conjugating enzyme, its encoding geneUBC24 is targeted by miR399. In this study, sequence analysis of
miR399 and TaUBC24 showed that both miR399 and TaUBC24belonged to highly conserved families. tae-miR399 precursor could be
folded into a perfect stem-loop structure with high negative minimum
free energy and minimum free energy index. In the 5’UTR ofTaUBC24 , we predicted six response elements/binding sites (MREs).
Similarly, there are five MREs of ath-miR399 in AtPHO2 ofArabidopsis thaliana (Fujii et al., 2005), and six MREs of
zm-miR399 in ZmPHO2 of maize(Du et al., 2018). Using tobacco
transient co-transfection and 5’RLM-RACE analysis, we demonstrated thatTaUBC24 , which encodes ubiquitin binding E2 enzyme, is the target
gene of tae-miR399 and is negatively regulated by tae-miR399 (Fig. 3).
These results indicate that miR399-UBC24 module is highly
conserved in wheat.