Ethylene-insensitive5 encodes a 5'->3' exoribonuclease required for regulation of EIN3-targeting F-box proteins EBF1/2

thylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F box proteins, EBF1 and EBF2 (1-3). Here, we report the identification of ETHYLENE INSENSITIVE5 as the 5'->3' exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response. Keywords: Arabidopsis growth regulation signal transduction For the oligonucleotide tiling array experiments 10 days-old light-grown Arabidopsis ein5-1 seedlings were transferred to a Petri-plate containing incubation buffer (1mM Pipes, 1mM sodium citrate, 1mM KCl, 15mM sucrose), and maintained in agitation at 75 rpm for 30 min covered with foil. At this point vacuum was applied for exactly 15 seconds and cordycepin (Sigma Chemical Co., St. Louis) was added to a final concentration of 150 ug/ml. The seedlings were then incubated for 2 more hours in the dark at room temperature and kept in agitation at 75 rpm in Petri-plates. The seedlings were then briefly blotted to on filter paper to dry and kept in liquid nitrogen. Total RNA was extracted from the samples using the RNAeasy plant kit (Qiagen, Valencia, CA). Biotinylated target RNA was prepared from 120 ug of total RNA from each sample using the GeneChip® Expression Analysis system (Affymetrix, Santa Clara, CA). Each sample of the purified and fragmented labeled target cRNA was hybridized to one set of Arabidopsis genome tiling arrays consisting of twelve custom made arrays (Yamada, K et al., 2003). Hybridization, washes and the staining were carried out as described above for the Affymetrix array ATH1. Cell files were obtained using the GCOS software (Affymetrix, Santa Clara, CA) and the ChipViewer software was used to visualize and analyze the tiling chip data (Yamada et al. 2003. Empirical Analysis of Transcriptional Activity in the Arabidopsis Genome. Science 302, 842-846). Data submitted by Huaming Chen.