| Iron is definitely essential for fruit trees due to its various roles in life processes, fruit trees are one of the most affected crops by iron deficiency, which significantly decreases fruit yield and quality. Iron uptaking is regulated by various factors such as pH, mineral elements and hormones and so on. The pH is an important factor affecting iron uptaking. In order to maintain the balance in the tree-soil system during iron uptake, the root of plant excretes amount of protons mediated by PM H+-ATPase to lead to the acidification of rhizosphere, which in turn makes iron soluble and available in soil for uptake. Besides proton extrusion and the associated electrochemical gradient, PM H+-ATPase also supplies energy for iron uptake and transportation. Therefore, PM H+-ATPase plays a crucial role in iron acquisition and homeostasis in plant. The molecular mechanisms of iron deficiency response have been increasingly studied in model plants, the underlying molecular and genetic bases of this process remain unclear in apple trees.In Arabidopsis, Ib and IVc subgroups bHLH TFs, play a central role in modulating the expressions of the major Fe acquisition genes。In this study, we described an IVc subgroup bHLH TF gene MdbHLH104, which is up-regulated under iron deficiency conditions. 35S::MdbHLH104 transgenic apple plantlets exhibited an improved tolerance to Fe deficiency. The transgenic apple plantlets and calli showed an enhanced proton excretion, ferric oxidoreductase activities and Fe contents. Furthermore, we demonstrate that MdbHLH104 directly activates the expression of the MdAHA8 and Ib subgroup bHLH TFs by binding to the E-box element in its promoter.In order to identify the interaction genes MdbHLH104, yeast two-hybrid library screening was used with a full-length MdbHLH104 as bait. In our work, yeast two-hybrid and pull-down showed that MdbHLH104 interacts with other IVc subgroup members including MdbHLH105 (MdILR3), MdbHLH115, MdPYE, MdbHLH11 and MdbHLH121. The heterodimers also activated the expression of MdAHA8. Co-overexpression MdbHLH104 and MdbHLH105 (MdILR3), MdbHLH115, MdPYE, MdbHLH11 and MdbHLH 121, respectively, enhanced proton excretion, H+-ATPase activity and Fe contents in the transgenic apple calli.In addition, two BTB-TAZ proteins were found named MdBT1 and MdBT2 from apple. MdBT subfamily genes were identified from the apple genome database following a BLASTN homology search using each of the AtBT protein sequences, and the other three MdBT genes were found, named MdBT3.1, MdBT3.2 and MdBT4. Conserved domain analysis showed that All of them share similar structures, i.e. a BTB domain in the N-terminal part, a TAZ domain in the center and CaM domain in C-terminal. Yeast two-hybrid, pull-down and Co-IP showed that MdBTland MdBT2 interacted with MdbHLH104, and the TAZ domains of MdBT1 and MdBT2 interact with the C terminus of MdbHLH104. Western Blot analysis showed that more MdbHLH104 protein in 35S::MdBT1/2-Anti transgenic calli and less MdbHLH104 protein in 35S::MdBT2 transgenic calli, indicating that MdBTl and MdBT2 negative the MdbHLH104 protein accumulation. Furthermore, the BTB domain of MdBT2 protein is responsible for the interaction with MdCUL3 and the associated partners such as RBX1 to form CRL3BT2 complex. The cell-free degradation assays and ubiquitination modification in vivo and vitro were used, and the results showed that the BTB domain of MdBT2 recruits MdCUL3-containing CRL3 complex to regulate MdbHLH104 stability by ubiquitination and degradation via the 26S proteasome. Besides, the accumulation of MdBTl and MdBT2 protein are induced by Fe sufficiency, and reduced under Fe deficiency conditions. Taken together, our study provides evidence for that Iorn-MdBT2-MdbHLH104-MdAHA is an ubiquitin-dependent degradation pathway for development and growth for responding iron dificient in apple. |
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