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.