Interaction Of AhFIT And AhbHLH100 And Functional Identification Of AhIRT1 In Peanut

Iron（Fe）is an important trace element in the growth and development of plants.Iron exists in the form of Fe（II）and Fe（III）in soil,but most of them are insoluble Fe（III）.Before being absorbed by plants,Fe（III）needs to be reduced to Fe（II）in rhizosphere.In calcareous soil,plants often subject to iron deficiency due to the low efficiency of iron absorption.In order to overcome iron deficiency,plants have evolved two mechanisms of Fe absorption.Peanut,as a non-gramineous plant,can take up iron by the reduction mechanism（mechanism type I）.Based on the previous transcriptome analysis,it is inferred that a bHLH transcript factor,AhFIT（or AhbHLH29）,might regulate the downstream gene AhIRT1 by interacting with AhbHLH100.In order to test the hypothesis,coding region sequences of AhFIT and AhbHLH100 were cloned from peanut,and the interaction between AhFIT and AhbHLH100 was verified by the yeast two hybrid（Y2H）and bimolecular fluorescence complementation（Bi FC）.Moreover,the coding region sequence and the promoter sequence of AhIRT1 were cloned,and the physiological function of IRT1 was analyzed by using yeast heterologous complementary experiment,subcellular localization,GUS activity analysis,RT-q PCR and heterologous overexpression.The findings would lay a foundation for understanding the regulation mechanisms of iron homeostasis in peanut.Phylogenetic analysis revealed that AhFIT and AhbHLH100 from peanut show closest phylogenetic relationship with At FIT and AtbHLH100 from Arabidopsis,respectively.Y2 H results showed that AhFIT interacted with AhbHLH100 in yeast cells.Bi FC experiment verified interaction between AhFIT and AhbHLH100 in onion epidermal cells.These results indicate that AhFIT and AhbHLH100 can form heterodimer,which might regulate the expression of downstream genes.The target gene prediction shows that AhFIT contains a G-box,which can combine with the G-box on the AhIRT1 promoter to regulate the expression of AhIRT1.Yeast complementation experiment showed that AhIRT1 could heterologously transport zinc and cadmium,but had no effect on manganese.Subcellular localization experiment indicated that fluorescence was located on the plasma membrane of onion,indicating that AhIRT1 was located on the cell membrane.GUS activity analysis showed that AhIRT1 was mainly located in the root tip and leaves of Arabidopsis,especially in the leaves,which may have the role of metal transport in the roots and leaves.RT-q PCR showed that the expression of AhIRT1 was up-regulated by iron deficiency,and the up-regulation was the most in leaves,followed by roots.In conclusion,our results demonstrated that AhFIT might regulate the downstream gene AhIRT1 by interacting with AhbHLH100.AhIRT1 is induced by iron deficiency,located on the plasma membrane and expressed in root tips and leaves.Therefore,it is speculated that AhIRT1 might be involved in iron transport in root tips and leaves.