OsEIL1 Regulates Auxin Biosynthesis And Root Growth In Rice

Rice is an important monocotyledonous crop worldwide,as a semi-aquatic plant,it differs from the dicotyledonous plant Arabidopsis in many aspects.In Arabidopsis,ethylene and auxin act synergistically to regulate root growth and development.However,their interaction in rice is still unclear.In this study,we found that ethylene-inhibited primary root elongation was abolished by yucasin,an inhibitor of YUC activity,indicating that ethylene-inhibited primary root elongation is dependent on YUC.To identify the function of YUC in ethylene-inhibited primary root elongation,we conducted a screening for ethylene-insensitive mutants from our mutagenesis libraries following ethylene treatment.A series rice ethylene-insensitive(rein)mutants were selected based on the phenotypes of the primary root.Compared to the wild type,all the mutants showed insensitivity or reduced sensitivity to ethylene in primary root elongation.One of mutants,rein7,exhibiting insensitive to ethylene,was selected to study.Through map-based cloning,we found that REIN7 encodes a member of YUC family,YUC8.The field-grown rein7 mutant exhibited narrow,curled leaves,and enhanced salt tolerance compared with wild-type plants.Moreover,overexpression of REIN7/YUC8 enhanced the primary root response to ethylene,indicating that REIN7/YUC8 plays an important role in ethylene-inhibited primary root elongation.The primary root of REIN7/YUC8 overexpression lines was shorter than that of the wild type under normal conditions,kynurenine(a potent inhibitor of in vivo TAA1/TAR activity)treatment suppressed the short-root phenotype of REIN7/YUC8 overexpression lines,indicating that REIN7/YUC8 and TAA1/TARs function in a linear pathway,and REIN7/YUC8 acts downstream of TAA1/TARs.Analysis with enzymatic assay evidenced that REIN7/YUC8 regulates rice auxin biosynthesis via the conversion of indole-3-pyruvic acid(IPA)to indole-3-acetic acid(IAA),and the C-terminus is important for REIN7/YUC8 enzymatic activity,loss of 47 amino acid residues from the C-terminal end of REIN7/YUC8 protein resulted in significant decrease of enzymatic activity,thus affecting auxin accumulation in primary root and insensitivity to ethylene.This finding was further supported by overexpression of REIN7/YUC8 in Arabidopsis yuc1-1 mutant background.Exogenous addition of low concentrations of IAA rescued the ethylene response in rein7 primary root,and this concentration of IAA had no obvious inhibitory effects on primary root growth in wild-type seedlings.Additional,ethylene promoted auxin accumulation in primary root,and this was dependent on induced REIN7/YUC8 expression,this induction was completely abolished in oseil1,indicating that ethylene-induced REIN7/YUC8 expression was dependent on OsEIL1.Furthermore,OsEIL1 directly binds to REIN7/YUC8 promoter and activate its expression.Overexpression REIN7/YUC8 in oseil1 background partially suppressed ethylene insensitivity of primary root in oseil1,and the inhibition of primary root growth of EIL1-OX seedlings was partially alleviated in the rein7-1 EIL1-OX seedlings,and addition of low concentrations of IAA rescued the ethylene response in oseil1 primary root,demonstrating that YUC8/REIN7-mediated auxin biosynthesis functions downstream of ethylene signaling to regulate the primary root elongation of rice,and there may have an OsEIL1-independent ethylene responsive pathway that regulates primary root elongation together with auxin.Thus,our findings indicate that YUC8/REIN7,as a downstream target of OsEIL1,is involved in ethylene-inhibited primary root elongation by regulating auxin biosynthesis in rice.This study reveals a model of interaction between ethylene and auxin in rice seedling primary root elongation,enhancing our understanding of ethylene signaling in rice.