Identification And Analysis The Function Of EPR1-A Phosphorylation-mediated Tomato Sl-EIL1 Transcriptional Activation Domain

Ethylene is an important plant hormone that regulates numerous growth and developmental processes in plant, including seedling germination and growth, lateral root development, leaf and petal abscission, fruit ripening, organ senescence, and stress and pathogen responses. Dozens of studies in Arabidopsis and tomato mutants have shown that ethylene responses have obvious temporal and spatial specificity. In plants, the same concentration of ethylene in different organizations or different developmental stages may lead to different ethylene responses.ETHYLENE INSENSITIVE3 (EIN3)/EIN3-like (EIL) are positive regulators in the ethylene signal pathway. EIN3/EIL locates in nucleolus that acts as a trans-activation factor during a downstream stage of the ethylene signal transduction pathway. Previous studies have shown that ERF (ETHYLENE response factor) genes were regulated by EIN3/EIL. Scientists speculated that EIN3/EIL protein cause temporal and spatial specific ethylene responses by regulating transcription of different ERF genes. However, EIN3/EILs genes belong to a small gene family that only four EIN3/EIL genes were isolated whether in Arabidopsis or in tomato. Furthermore, the EIN3/EIL genes are functionally redundant, and the transcription of EIN3/EILs was not regulated by ethylene. There are few reports about how EIN3/EIL controlling the ERF super gene family and leading to variety ethylene responses. Further studying to the mechanism of transcriptional regulation of EIN3/EIL is helpful to clarify the regulatory process of ethylene responses. So, our findings have important significance.Protein phosphorylation is an important and widely conserved mechanism underlying the regulation of a variety of biological processes including ethylene signaling pathway. In previous studies, we found that phosphorylation affects transcriptional activity of tomato EIN3-like 1(SL-EIL1). The present study identifies a new phosphorylation region named EPR1 (EIN3/EIL Phosphorylation Region 1) in tomato EIL1 proteins. Then, we investigated the function of EPR1 by using single cell system, BiFC(Bimolecular fluorescence complementation), transgenic technique and so on. The main results and conclusions are summarized below.(1) Identification of a new conserved phosphorylation site associated with the transcriptional activation domain of Sl-EIL1. The search for putative phosphorylation sites within Sl-EIL1 protein performed with Group-based Prediction system (GPS 2.1) program and ExPASy online prediction tools identified eight highly conserved phosphorylation sites. Amino acid sequence alignments of EIN3/EIL proteins from seven plant species revealed a highly conserved region located close to the N-terminal DNA-binding domain (DBD) of the EIN3/EIL protein (Figure 1A). This glutamine-rich (Q-rich) region contains a putative Protein Kinase A (PKA) phosphorylation site located at amino acids 92-95 and named here EIN3/EIL-phosphorylation-region-1 (EPR1).(2) The EPR1 domain is essential for the transcriptional activity of Sl-EIL1 protein. To gain insight on the function of the EPR1 region, two individual mutant versions of the Sl-EIL1 gene were generated. Sl-EIL1-Mutant1 (Sl-EM1) and Sl-EIL1-Mutant2 (Sl-EM2) were constructed by site-directed mutagenesis to introduce nucleotide substitutions within the EPR1 coding sequence. The results of single-cell transient expression analysis proved that EPR1 was a functional domain which was closely affected to the transcriptional function of Sl-EIL1 function.(3)The EPR1 region is located within the dimerization domain identified in Arabidopsis EIN3/EIL proteins. To investigate the putative role of the EPR1 domain in controlling the transcriptional function of Sl-EIL1 we explored its potential involvement in protein dimerization. Bimolecular Fluorescence Complementation (BiFC) approach was implemented to test how mutations in the EPR1 domain affect Sl-EIL1 dimerization.(4) Based our findings, a new model is proposed that takes into account the role of the EPR1 domain in promoting the ethylene-dependent transcriptional regulation. In this model, EIN3/EIL proteins with non-phosphorylated EPR1 domain are in inactive state and phosphorylation via PKA, triggers dimerization of EIN3/EIL proteins which activates the transcriptional function of EIN3/EIL.In addition, we have found that Sl-EIL1 plays an important role during the tomato flower development and fruit set by identificating and analyzing the transgenic tomatoes.