Preliminary Study Of Arabidopsis E3 Ubiquitin Ligase RING1 In Response To Iron Deficiency

Iron is an essential microelement involved in various physiological processes in plants.Despite iron is abundant in the earth's crust,its bioactivity is extremely low in the alkaline or calcareous soils,leading to plant iron deficiency on these soils.And then restrict agricultural production.Although great progresses have been made in the molecular mechanism of transcriptional regulation in iron deficiency response,the regulation at post-translational level is largely unknown.In order to identify novel regulatory genes in response to rapid iron deficiency,we performed RNA-seq for Arabidopsis wild-type(WT)seedlings under short-term iron deficiency treatment,and found that 144 and 92 genes were up-and down-regulated by iron deficiency,respectively.Among the differentially expressed genes,RING1 was found to be significantly induced by iron deficiency.RING1 encodes a RING type E3 ubiquitin ligase.Its gain-of-function T-DNA insertion mutant ring1-1 was more sensitive to iron deficiency than WT,which was similar to RING1 overexpression lines.Further studies showed that the root iron content was reduced in ring1-1 mutant versus WT under iron deficiency,which was probably attributed to the lower activity of root ferric-chelate reductase(FCR)in ring1-1(versus WT).In accordance with this,the expression of Ferric Reductase-Oxidase 2was significantly decreased in the ring1-1 mutant and RING1 overexpression lines compared with WT under iron deficiency for 1 day.Further GUS staining showed that RING1 was mainly expressed in shoots and root tips of seedlings,and in rosette leaves and axillary leaves of mature plants under iron supply;while under iron deficiency condition,RING1 expression was strongly induced in root mature zone,and was enhanced in shoots.In addition,RING1 was found to be expressed in both cytoplasm and nucleus.Finally,we identified several potential interactants of RING1 including the transcription factor PYE,through screening of yeast-two hybrid nuclear library,suggesting that RING1 may be involved in iron deficiency response by regulating the stability of these proteins.In conclusion,we propose that the E3 ubiquitin ligase RING1 may be a novel important factor that negatively regulates the response of iron deficiency response,and that it may be involved in the regulation of iron homeostasis in Arabidopsis by regulating the stability of key regulatory proteins such as PYE.This study hence expanded the regulatory network of plant in response to iron deficiency,and provided new gene resource and novel insight for iron biofortification in crops.