tae-miR399-UBC24 module regulates Arabidopsis CBF signaling pathway in response to freezing stress
In this study, we analyzed the expression pattern and biological function of Dn1 tae-miR399 and its target gene TaUBC24 under low temperature stress, and provided evidence for tae-miR399 mediatedTaUBC24 expression as a new regulatory mechanism in winter wheat response to low temperature stress. Due to the limitation of experimental conditions, it is very difficult to carry out tissue transfection in wheat. Therefore, Arabidopsis thaliana was used for functional gain analysis. The results showed that in transgenic Arabidopsis plants, OEmiR399 lines had a cold tolerance phenotype, and OEmiR399 Arabidopsis plants had a higher survival rate, lower ion permeability and membrane lipid damage than WT (Fig. 4). These results indicates that tae-miR399 has a certain biological function of cold resistance.
Although a large number of cold responsive miRNAs have been identified, further functional analysis is needed to determine their roles in plant cold response, especially resistance to freezing stress. It has found that overexpressing miR394 transgenic Arabidopsis plants show stronger cold tolerance, and the expression of multiple genes in CBF signaling pathway are higher than those of WT, indicating that miR394 is a positive regulator of plant response to low temperature stress (Song, Li, Cao, & Qi, 2019). Overexpressing miR319 in rice has a better survival rate than that of wild-type, the expressions of CBFsincrease, and the content of ROS is also significantly lower than that of wild-type, which indicates that miR319 is involved in plant cold tolerance (S. T. Wang et al., 2014). At present, the mechanism of miRNA regulating wheat response to low temperature stress is still limited, and the specific mechanism of wheat miRNAs mediated CBF signaling pathway is still unclear. ICE1, as a MYC bHLH protein, also plays a role in CBF signaling pathway by regulating downstream target genes (Deng, Ye, Fan, Pu, & Yan, 2017). The previous study showed that CsUBC24 and CsICE1 interact in citrus, and CsUBC24 mediate degradation of CsICE1 but not regulate the transcription level of CsICE1 (R. Wang et al., 2020). Therefore, we tested the transcription level of AtICE1 in OEmiR399 Arabidopsis plants and found that only at 4°C, the expression level of AtICE1 in OEmiR399 Arabidopsis was higher than that of WT (Fig. 7C). However, the ICE1 protein level of OEmiR399 Arabidopsis plants was always higher than that of WT (Fig. 7A and B). These results suggests that overexpressing tae-miR399 can increase the protein content of AtICE1 by down-regulating the expression of AtUBC24 . In Arabidopsis, ICE1 plays a key role in the development of leaf stomata and the response to chilling and freezing stress, and can combine with the CBFs promoter sequence to promote its expression (Miura et al., 2007). Recent studies have shown that except binding to the promoter sequence of CBFs, ICE1 can also directly initiate the expression of most genes in the CBF signaling pathway, such asCOR47 , COR413IM , MPK and HOS (Tang et al., 2020). In this study, the expression of CORs detected in all Arabidopsis lines were up-regulated in different degrees under low temperature. It is worth noting that only the expression levels ofAtCOR47 and AtCOR413IM in OEmiR399 Arabidopsis plants were significantly higher than those in WT under cold acclimation and freezing stress (Fig. 6F and G). The expression of AtCBFs in OEmiR399 Arabidopsis plants were significantly higher than that in WT under freezing stress (Fig. 6A, B and C). The results indicate that AtICE1 with high protein level promotes the expression of its downstream target genes (AtCBFs, AtCOR47 and AtCOR413IM ) by binding to the promoter region under freezing stress. Some studies have shown that plants can activate the expression of CBFs in the early stage of cold acclimation, and when the content of CBFs protein reaches a certain level in the later stage, the expression of CBFs is inhibited to prevent plants from over chilling defense (Chinnusamy et al., 2003a; Novillo, Alonso, Ecker, & Salinas, 2004). For example, when Arabidopsis was treated at 4℃ for 24 h, its AtCBFs peaked at 3 h and then gradually decreased(Joachim Kilian et al., 2007). In this study, AtCBFs also showed similar expression changes. At 3 days of cold acclimation, the expression of AtCBFs in WT had returned to the level before untreated, while the expression of AtCBFs in OEmiR399 Arabidopsis plants was significantly lower than that at 24℃ (Fig. 6A, B and C). It implies that during cold acclimation, the AtCBFs protein content of OEmiR399 Arabidopsis plants is significantly higher than that of WT. In addition, we found that TaUBC24 interacts with TaICE1 through Y2H and BiFC experiments (Fig. 13). These results suggested that the interaction between UBC24 and ICE1 may be conserved in plants. Also in wheat, tae-miR399-TaUBC24 module may mediate the CBF signaling pathway in response to freezing stress by inhibiting the degradation of TaICE1 protein.
tae-miR399-UBC24 module regulates Pihomeostasis and phosphorus utilization efficiency in Arabidopsis in response to freezing stress
PHO2 down-regulates the expression of PSI-related genes (such as PHTs) by promoting the degradation of PHO1, and negatively regulates Pi uptake and translocation (Chiou & Lin, 2011; Liang et al., 2014). Under phosphorus deficiency, plants adapt complex strategies to enhance the acquisition of phosphorus, for example, the remodeling of the root structure and the reactivation of phosphorus in plants (Chiou & Lin, 2011; Liang et al., 2014). In this study, at 24℃, the Pi content of OEmiR399 Arabidopsis plants was significantly higher than that of WT, and the primary roots of OEmiR399 Arabidopsis plants were elongated, which was similar to the phenotype of overexpressing miR399 Arabidopsis (Chiou et al., 2006), rice (Hu et al., 2011) and maize(Du et al., 2018). These results imply that overexpressing tae-miR399 may promote the absorption and transport of Pi in Arabidopsis. During the cold acclimation, the total phosphorus content and the expression ofAtPHT1; 1 and AtPHT1; 4 in OEmiR399 Arabidopsis plants were significantly higher than that in WT (Fig. 8D and F). However, the Pi content of OEmiR399 Arabidopsis plants was significantly lower than that of WT (Fig. 8B). These results may imply that during cold acclimation, the phosphorous transporter in OEmiR399 Arabidopsis plants is active, and the transported Pi has been rapidly converted to organophosphorus. Previous reports have reported that at 5°C, the Pi and organophosphates of pho2 Arabidopsis mutants are significantly higher than those of WT, and the many phosphatases and other metabolic enzymes activities of pho2 increase (Hurry, Strand, Furbank, & Stitt, 2000), which is consistent with our research results. Therefore, we speculate that overexpressing tae-miR399 may increase Pi absorption and phosphorus utilization efficiency of Arabidopsis during cold acclimation. The Pi content and the expression of AtPHTs in OEmiR399 Arabidopsis plants under freezing stress were similar to cold acclimation, indicating that under freezing stress, the phosphorus utilization efficiency of OEmiR399 Arabidopsis plants is higher than that of WT. These results demonstrate that the function of tae-miR399 in response to Pi signal in Arabidopsis is conserved, and that Pi signal plays an important and positive role in response to low temperature stress in Arabidopsis. In addition, the expression of AtPHO1 of OEmiR399 Arabidopsis plants has been at a low level under low temperature in this study, which may be a feedback regulation of increasing AtPHO1 protein content caused by down-regulatingAtUBC24 expression (Fig. 8C).