MdbHLH4 negatively regulates apple cold tolerance by inhibiting MdCBF1/3 expression and promoting MdCAX3L-2 expression

Abstract

Low temperature affects the yield and quality of crops. Inducer of CBF expression 1 (ICE1) plays a positive role in plant cold tolerance by promoting the expression of CRT binding factor (CBF) and cold-responsive (COR) genes. Several ICE1-interacting transcription factors (TFs) that regulate plant cold tolerance have been identified. However, how these TFs affect the function of ICE1 and CBF expression under cold conditions remains unclear. Here, we identified the MYC-type TF MdbHLH4, a negative regulator of cold tolerance in Arabidopsis (Arabidopsis thaliana) and apple (Malus domestica) plants. Under cold conditions, MdbHLH4 inhibits the expression of MdCBF1 and MdCBF3 by directly binding to their promoters. It also interacts with MdICE1L, a homolog of AtICE1 in apple, and inhibits the binding of MdICE1L to the promoters of MdCBF1/3 and thus their expression. We showed that MdCAX3L-2, a CAX (Ca2+/H+ exchanger) family gene that negatively regulates plant cold tolerance, is also a direct target of MdbHLH4. MdbHLH4 reduced apple cold tolerance by promoting MdCAX3L-2 expression. Moreover, overexpression of either MdCAX3L-2 or MdbHLH4 promoted the cold-induced ubiquitination and degradation of MdICE1L. Overall, our results reveal that MdbHLH4 negatively regulates plant cold tolerance by inhibiting MdCBF1/3 expression and MdICE1L promoter-binding activity, as well as by promoting MdCAX3L-2 expression and cold-induced MdICE1L degradation. These findings provide insights into the mechanisms by which ICE1-interacting TFs regulate CBF expression and ICE1 function and thus plant cold tolerance.

MdNAC104 positively regulates apple cold tolerance via CBF‐dependent and CBF‐independent pathways

“…Some NAC TFs bind to CBF promoters and regulate their expression; thus, participating in the regulation of the cold stress response (An et al ., 2018a; Hao et al ., 2011; Shan et al ., 2014). As overexpressing MdNAC104 significantly increased the transcription levels of MdCBF1/3 and their downstream cold‐responsive genes (Figure 3) (Yang et al ., 2023), MdCBF1/3 may be the target gene of MdNAC104. We cloned the MdCBF1 / MdCBF3 promoter sequences and identified the transcriptional activation ability of MdNAC104 on their promoters using the LUC/REN dual‐luciferase system.…”

“…In Arabidopsis , the CBF‐COR pathway includes several cold‐responsive genes, such as CBF1 and CBF3 , as well as their downstream target genes RD29A , COR15A , COR47 and KIN1 (Aslam et al ., 2022; Ding and Yang, 2022; Kidokoro et al ., 2022). The orthologs of these genes in apples were also shown to significantly respond to cold stress (An et al ., 2018a; Feng et al ., 2012; Wang et al ., 2021b; Xie et al ., 2018; Yang et al ., 2023). The expression changes in these cold‐responsive genes were analysed in MdNAC104 transgenic and GL‐3 plants to clarify the MdNAC104 mechanism of the apple cold stress response.…”

“…Each group contained three biological replicates, with 20 plants per line (OE#1, OE#2, OE#3 and GL-3) in each replicate. The freezing treatments were carried out referring to the methods of Xie et al (2018) and Yang et al (2023) with some modifications.…”

“…These CBF TFs activate the transcription of downstream cold‐responsive ( COR ) genes to transmit and amplify the cold signal; thus, triggering a series of physiological responses (Kidokoro et al ., 2022; Liu et al ., 2019). Studies in other plant species indicate that this CBF‐COR pathway is well‐conserved, such as Arabidopsis (Liu et al ., 2019), rice (Li et al ., 2022b; Nakamura et al ., 2011), tomato (Wang et al ., 2018a) and apple (An et al ., 2018a; Feng et al ., 2012; Xie et al ., 2018; Yang et al ., 2023). In apples, five DREB1/CBF family genes were identified (Feng et al ., 2012).…”

EjFAD8 Enhances the Low-Temperature Tolerance of Loquat by Desaturation of Sulfoquinovosyl Diacylglycerol (SQDG)

“…ICE induces transcriptional activation of CBF gene family expression at low temperatures. The induced CBF protein binds to the cis-element present in the COR genes promoter and activates the expression of the COR genes, thus increasing the cold tolerance of the plant [ 25 ]. Chloroplasts also participate in the ICE-CBF-COR regulatory pathway.…”