The Regulatory Mechanism Of The Transcription Factor INO In Loading Iron Into Arabidopsis Seeds And Its Biological Fuction

Iron(Fe)is the essential micronutrients for living organisms,but excess iron causes cellular toxicity.In previous studies,due to an improved understanding of the Fe homeostasis regulatory mechanism in plants,major advances towards engineering biofortified crops have been made.However,there is still little known about the iron loading process and its regulatory mechanisms during seed development which is crucial for exploring new biofortification pathways of seed iron.After screening a large number of transcription factor mutants in Arabidopsis,we found that a transcription factor INO(INNER NO OUTER)mutant exhibited a phenotype of resistant to Fe-deficiency,as a member of the YABBY transcription factor family,which regulated the polar growth of the outer ovule integuments in early developing seeds.The three ino mutants were observed in seed shape and microstructures of outer ovule integuments,and then we selected two low-expression lines,ino-9 and ino-10,which did not affect the outer ovule integuments development.The ino-9 and ino-10 mutants showed more resistant after germination and growth under iron deficiency condition,but there was no significant difference among wildtype and two ino mutants which grown on Fe-sufficient(+Fe)medium 4 days previously and then transferred onto new medium lacking Fe for a further 3 days.In addition,the expression of FIT and IRT1,marker genes for iron deficiency in ino mutants,were no difference with wild type.On the contrary,INO overexpressed seedlings showed more sensitive under Fe-deficiency treatment,indicating that INO gene negatively regulated the seed iron content in Arabidopsis.By screening the downstream factors,we found the iron transporter NRAMP1 controlled by the INO.In the early stage of seed development,INO could significantly inhibit NRAMP1 expression and decrease the accumulation of NRAMP1 protein.We also show that the ino-9nramp1 double mutant restored the phenotype of increased iron content in ino mutant seeds,which also indicated that NRAMP1 could affect the iron loading as a downstream regulated by INO in seed development.In addition,the results of dualluciferase reporter gene,MST,EMSA and Ch IP experiments proved that INO could directly bind to the promoter region of NRAMP1 gene to regulate the NRAMP1 expression.In this paper,we treated the breeding ino mutants,wild type and ino-9nramp1 double mutant with high iron concentration.Our results showed that more iron was accumulated in ino siliques under high Fe concentration treatment.In addition,the seed abortion rate of ino mutants were dramatically increased,but there was no significant difference with wild type.A large of ROS was accumulated in these aborted seeds,indicating that early control of iron loading could prevent the production of ROS,due to excessive accumulation of iron,thus safeguarded successful reproduction in Arabidopsis.This new regulatory mechanism of iron loading into seeds not only reveals the important biological functions of INO,but also has important theoretical significance for exploring the new biofortification pathways of iron nutrition in seeds.