Introduction
Low temperature stress is the major abiotic stress that is not conducive to plant growth and yield. Basing the different effects of cold stress in plants, it can be classified chilling stress and freezing stress (Jan, Mahboob ul, & Andrabi, 2009). Chilling stress disturbs membrane processes including opening of ion channels, membrane associated electron transfer reactions (Ruelland, Vaultier, Zachowski, & Hurry, 2009). The freezing temperature has more negative effects in plants. The extracellular freezing which means cellular water migrates to this extracellular ice causing cell dehydration and shrinkage could occur in freezing stress (Dowgert & Steponkus, 1984). Ultimately, ice can penetrate the symplast (Gusta & L., 2004), causing a deterioration of the intracellular structures and death of tissues. In addition, ROS is accumulated continuously, no matter under chilling stress or freezing stress, which leads to the degradation of various proteins and protein complexes in plants and destroys the process of plant biogenesis (Ruelland et al., 2009). Unraveling the mechanisms of how cold resistant plant varieties adapt to extremely cold environments could provide valuable information for enhancing the performance of conventional crops under freezing stress, via genetic engineering. Plants have evolved sophisticated mechanisms that limit cold-induced damage. A series of comprehensive physiological and biochemical events take place during plants withstanding cold stress (Y. Ding, Shi, & Yang, 2019). At the physiological level, many substances or protective proteins are synthesized in plants, such as soluble sugars, proline, and cold-resistance proteins, of which are involved in osmotic potential, ice crystal formation, membrane stability and ROS scavenging in plants. These physiological and biochemical changes in plants are regulated by different signaling and metabolic pathways (Asif et al., 2014), such as CBF signaling pathway, sugar metabolism and ROS scavenge system. Exploring upstream regulation mechanism of these pathways is of great significance for revealing the molecular basis of plant freezing tolerance.
CBF signal pathway endows plants cold tolerance by regulating the expression of downstream cold-resistance proteins (Chinnusamy et al., 2003a). CBF/DREB1 (C-REPEAT BINDING FACTOR/DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN1) genes are involved in cold acclimation of plants and rapidly induced under cold stress (Q. Liu et al., 1998; Stockinger, Gilmour, & Thomashow, 1997). Subsequently, COR genes, a class genes encoding osmolyte and cryprotective proteins, are induced by CBFs to protect plant from freezing injury (Shi, Ding, & Yang, 2018; Yamaguchi-Shinozaki & Shinozaki, 1994). Inducer of CBF expression transcription factor (ICE) is a MYC type bHLH transcription factor, which is the most characteristic transcription activator of CBF gene (Chinnusamy et al., 2003b). The ICE1 has been proved to play an important role in response to low temperature stress in varied plants, such as rice (Dianjun, Yu, & Kuide, 2007), Arabidopsis (Chinnusamy et al., 2003b) and tomato (Juan et al., 2015). Not only CBFs gene, ICE1 can also bind CORs promoter to activate CORs gene expression (Tang et al., 2020). Due to the importance of ICE1 in CBF signaling pathway, its upstream regulation mechanism has also been widely studied. Ding et al. report that ubiquitination, sumo acylation and phosphorylation are important for ICE1 in Arabidopsis cold tolerance (Yanglin Ding et al., 2019). ICE1 is ubiquitinated and degraded by E3 ubiquitin ligase HOS1 (high expression of osmotically responsive gene 1), while sumo E3 ligase SIZ1 (SAP and Miz) mediates sumo acylation of ICE1 and stabilizes ICE1 under low temperature stress (Miura et al., 2007). In addition, protein kinase OST1 (open stomata 1) mediates ICE1 phosphorylation and cold tolerance in Arabidopsis. OST1 is cold activated and is negatively regulated by protein phosphatase EGR2 (clade e growth regulating 2) and ABI1 (abscisic acid insensitive 1), independent of ABA (Y. Ding et al., 2019; Y. L. Ding et al., 2015). Cold activated OST1 phosphorylates ICE1 to enhance its stability by destroying its interaction with HOS1 (Y. L. Ding et al., 2015). Other study also finds that the protein stability and transcriptional activity of OsICE1 are positively regulated by OsMPK3 (MAP kinase 3) in rice (Z. Zhang et al., 2017). However, the upstream regulation mechanism of ICE1 in plant freezing tolerance is still elusive.
Starch degradation is involved in cold‐induced sugar accumulation (Ruelland et al., 2009). When suffering cold stress, amylases activity in plants increases. Subsequently, starch is degraded to glucose and fructose, which is the substrate for the accumulation of soluble sugar (Kaplan et al., 2007; Ruelland et al., 2009). The AtBAM3(At4g17090) encoding β-amylase, is induced by low temperature stress (Kaplan & Guy, 2005; Lundmark, Cavaco, Trevanion, & Hurry, 2006). Thebam3 Arabidopsis mutant is sensitive to cold, and the accumulation of soluble sugar is significantly reduced(Kaplan & Guy, 2005; Yano & R., 2005). Overexpressing PbrBAM3 in tobacco (Nicotiana tabacum ) and pear (P.ussuriensis ) can increase β-amylase activity, promote starch degradation under low temperature stress, and enhance its cold tolerance (Zhao et al., 2019). ExceptBAM3 , another gene encoding β-amylase, BAM1 , also plays an important role in starch degradation under chilling and freezing stress (T. Peng, Zhu, Duan, & Liu, 2014). Moreover, α-glucan hydration dikinase (GWD) can phosphorylate C-3 and C-6 of α-glucan, this starch phosphorylation may promote starch degradation by increasing the water-insoluble glucan to become hydrophilic ensuring better access to starch degrading enzymes(Hejazi, Fettke, Haebel, Edner, & Ritte, 2010). And its coding genes SEX1 and SEX4 are induced by low temperature stress (Berrocal-Lobo et al., 2011; Hejazi et al., 2010). Based on the importance of starch metabolism in plant response to low temperature stress, its upstream regulatory mechanism is still to be explored.
miR399 is an essential regulator of UBC24 (PHO2 ) expression during plant growth and response to stress. The miR399-UBC24 (PHO2 ) regulation module is conserved in plants including Arabidopsis (Bari, Datt Pant, Stitt, & Scheible, 2006), rice (Oryza sativa ) (Hu et al., 2011), wheat (Triticum aestivum ) (J. Wang, Sun, et al., 2013) and maize (Zea mays ) (Du, Wang, Zou, Xu, & Li, 2018). The miR399-UBC24 model was first found to play a role in plant response to Pi deficiency stress (Fujii, Chiou, Lin, Aung, & Zhu, 2005). When Pi is sufficient, UBC24 can degrade the Pi transporter PHOSPHATE TRANSPORTER 1 (PHT1) by ubiquitination (Huang et al., 2013). Under Pi-deficient condition, miR399 is strongly induced to downregulate the expression of UBC24 to increase the level of PHT1 (Aung et al., 2006; Hu et al., 2011). miR399 has been shown to regulate plant reproductive development. The miR399-UBC24 module regulates SEPALLATA MADS box transcription factor genes (SEPs) and ICE1 protein level to effect floral organ development in Citrus (R. Wang et al., 2020). The miR399-UBC24 module is also involved in plant other process such as sugar metabolism (Y. Wang et al., 2017), nutrienrt starvation responses (Hu et al., 2015), salt, drought and ABA signaling (Baek et al., 2016). In addition, the results of multi plant species miRNA sequencing showed that miR399 was induced by low temperature (R. Hu et al., 2019; Koc, Filiz, & Tombuloglu, 2015). Overexpressing miR399 can improve plant growth in low temperature (Gao, Qiang, Zhai, Min, & Shi, 2015). However, the mechanism of miR399 in response to freezing stress is unknown.
Wheat (Triticum aestivum ) is one of the main food crops for the global population (Rizwan et al., 2019). Freezing stress is one of the main factors damaging the yield and quality of economic crops such as wheat. To avoid winterkill it is very important to obtain the wheat freezing resistance gene (Babben et al., 2018). Dongnongdongmai1 (Dn1) is the first winter wheat cultivar that can overwinter safely in alpine region of Heilongjiang province (The minimum temperature in this area can reach -30℃) and the rate of returning green is greater than 85% (K. K. Peng et al., 2021; Tian et al., 2021). Winter wheat’s resistance to cold includes microRNAs (miRNAs)-short, single-stranded, non-coding RNAs that regulate the posttranscriptional gene expression by targeting mRNAs for cleavage or repressing translation (Lu, Xu, et al., 2020). Therefore, Dn1 is valuable germplasm resources for mining freezing resistance genes for crop variety improvement.
In this work, we systematically studied the interaction of tae-miR399-TaUBC24 and the expression of tae-miR399-TaUBC24 in winter wheat using bioinformatics and research methods in molecular biology, physiology and biochemistry. Then we determined the physical and genetic interaction of TaUBC24 and a inducer of CBF expression transcription factor protein, TaICE1, and reported that in addition to its role in Pi homeostasis and starch degradation, the mechanism of how tae-miR399-UBC24 module and ICE1 synergistically regulated plant tolerance to freezing stress, which providing a broader view on the novel upstream regulating mechanism of both CBF singnaling pathway and starch degradation response to freezing stress.