BTB Protein MdBT2 Interacts With MdNAC1 To Negatively Regulates Iron Homeostasis In Apple

Iron is a very important trace element in the growth and development of fruit trees,which affects the fruit yield and quality.However,iron deficiency is still the key to limit the yield and quality of crops,especially fruit trees,because most of the iron in soil exists in the form of oxides,which cannot be directly absorbed and utilized by plants.To cope with iron deficiency,plants have developed two survival strategies:The first strategy is that all dicotyledons,such as apples,as well as non-gramineous monocotyledons,can reduce soil p H by H~+-ATPase-mediated proton exhalation from the plasma membrane,converting insoluble iron to soluble iron,which is then transported to the roots via IRT1(iron-regulated transporter1).The second strategy is that gramineous plants secrete siderophores to chelate Fe,and the chelated Fe complex travels through a specific transport system to the root system,where it is absorbed.NAC transcription factors play an important role in plant abiotic stress response,but the molecular mechanism of their regulation of iron uptake and utilization in apple remains unclear.In this study,apple plants,callus and Arabidopsis thaliana were used as materials,and molecular biology and biochemistry techniques were employed to reveal the molecular mechanism of MdBT2-mediated MdNAC1 posttranslational modification regulating iron homeostatic.The specific results are as follows:1.MdNAC1 protein contained a conserved NAC domain at the N-terminal,and phylogenetic analysis showed that MdNAC1 protein had the highest similarity to Pb NAC1.MdNAC1 showed a constitutive expression pattern in different apple tissues,suggesting that MdNAC1 may be involved in multiple biological process during apple growth and development.q RT-PCR was used to detect the expression of MdNAC1 after iron deficiency treatment,and it was found that the transcription level of MdNAC1 in roots increased when apples were transferred from iron-sufficient liquid medium to iron-deficient liquid medium,indicating that MdNAC1 was sensitive to iron deficiency.2.MdNAC1 was located in the nucleus by subcellular localization.Yeast two-hybrid experiments showed that MdNAC1 had transcriptional activity,and the C-terminal domain of MdNAC1 had the autonomic activating activity.3.Proton efflux staining showed that the proton efflux of MdNAC1 gene transformed apple callus was significantly enhanced under iron deficiency condition,and the plasma membrane H~+-ATPase activity was significantly increased compared with the wild type.The results showed that overexpression of MdNAC1 could promote iron accumulation in apple callus under iron deficiency condition.By q RT-PCR analysis,it was found that the plasma membrane H~+-ATPase genes Md AHA3 and Md AHA12 were significantly induced in apple overexpressing MdNAC1.In addition,Md FIT,a major transcriptional regulator of plant iron deficiency response,and FRO2,a downstream gene,were also significantly induced in transgenic callus.MdNAC1 was expressed ectopically in Arabidopsis thaliana and was found to be similar to that in apple.These results suggest that overexpression of MdNAC1 enhances plant tolerance to iron starvation.4.MdBT2,a negative regulator of iron absorption,was screened by yeast two-hybrid(Y2H)assay.Further yeast two-hybrid assay showed that the BTB-back domain of MdBT2and the NAC domain of MdNAC1 were essential for the interaction between the two proteins.The interaction between MdBT2 and MdNAC1 was verified by pull-down assay and Bi FC assay.In vitro protein degradation experiments were carried out using transgenic callus.The results showed that the protein extract from MdBT2-OX transgenic apple callus could accelerate the degradation of MdNAC1 protein,while the protein extract from MdBT2-anti apple callus improved the stability of MdNAC1 protein.Ubiquitin analysis showed that there was a strong ubiquitin band in MdNAC1-GFP/MdBT2-OX callus.In addition,overexpression and inhibition of MdBT2 in MdNAC1-GFP apple callus showed that the abundance of MdNAC1 protein in MdBT2-TRV/MdNAC1-GFP was significantly increased compared with that in MdBT2-p IR/MdNAC1-GFP.In conclusion,MdBT2 promotes the ubiquitination and degradation of MdNAC1.5.Phenotypic analysis showed that there was no significant difference in phenotype between transgenic plants and wild type when iron ion was sufficient.However,under the condition of iron deficiency,the MdBT2 transgenic plants showed an obvious chlorotic phenotype in the new leaves,and the contents of iron and chlorophyll were significantly lower than those of the wild-type control.This suggests that MdBT2 negatively regulates iron ion absorption.The proton outflow trend of MdNAC1-GFP/MdBT2-OX apple callus under iron starvation was weaker than MdNAC1-GFP,but stronger than wild-type callus.The activity of H~+-ATPase and iron content in plasma membrane were determined.It was found that the activity of H~+-ATPase and iron content in MdNAC1-GFP/MdBT2-OX apple callus and MdNAC1-GFP apple callus were higher than those in wild-type apple callus.The results were consistent with the phenotype.In conclusion,MdBT2 negatively regulated iron utilization in iron deficiency by degrading MdNAC1 protein.Overexpression of MdNAC1 increased iron uptake and utilization and chlorophyll content by activating H~+-ATPase in plasma membrane.Meanwhile,MdBT2 ubiquitinated MdNAC1 through the 26S proteasome pathway and negatively regulated the protein stability of MdNAC1.In addition,overexpression of MdBT2 in apple and Arabidopsis showed inhibition of iron uptake and utilization,and chlorosis in new leaves.In conclusion,this study provides molecular and genetic evidence that MdBT2 may negatively regulate iron ion uptake and utilization by mediating protein stability of MdNAC1.