Molecular and Biochemical Characterization of VR-EILs Encoding Mung Bean ETHYLENE INSENSITIVE3-LIKE Proteins 1

ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the ethylene signal transduction pathway in Arabidopsis. Two full-length cDNA clones, pVR-EIL1 and pVR-EIL2, encoding EIN3-LIKE proteins were isolated by reverse transcriptase-polymerase chain reaction and by screening the cDNA library of mung bean ( Vigna radiata ) hypocotyls. VR-EIL1 and VR-EIL2 share 70% identity and display varying degrees of sequence conservation (39%–65%) with previously isolated EIN3 homologs from Arabidopsis, tobacco ( Nicotiana tabacum ) and tomato ( Lycopersicon esculentum ) plants. Gel retardation assay revealed that both VR-EILs were able to interact specifically with optimal binding sequence-1, the recently identified optimal binding sequence for tobacco TEIL, with the binding of VR-EIL2 being more efficient than that of VR-EIL1. Transient expression analysis using a VR-EIL::smGFP fusion gene in onion ( Allium cepa ) epidermal cells indicated that the VR-EIL proteins were effectively targeted to the nucleus. The fusion protein of VR-EIL2 with GAL4 DNA-binding domain strongly activated transcription of a reporter gene in yeast cells, and an essential domain for transcription-stimulating activity was localized to the amino-terminal acidic region that consists of 50 amino acid residues. In contrast with what has been previously found in EIN3 - and TEIL -overexpressing Arabidopsis plants, transgenic tobacco seedlings expressing the VR-EIL genes under the control of cauliflower mosaic virus 35S promoter did not exhibit a constitutive triple response. Instead, they displayed a markedly enhanced proliferation of root hairs, one of the typical ethylene response phenotypes, and increased sensitivity to exogenous ethylene. In addition, the pathogenesis-related (PR) genes encoding (cid:1) -1,3-glucanase, osmotin, and PR1 were constitutively expressed in 35S::VR-EIL lines without added ethylene, and were hyperinduced in response to ethylene treatment. These results indicate that VR-EILs are functional in tobacco cells, thereby effectively transactivating the GCC-box-containing PR genes and enhancing sensitivity to ethylene. The possible physiological role of VR-EILs is discussed in the light of the suggestion that they are active components of the ethylene-signaling pathway and their heterologous expressions constitutively turn on a subset of ethylene responses in tobacco plants. conditions. To examine the effect of ethylene on VR-EIL s mRNA expression, intact etiolated seedlings and light-grown plants were enclosed in 3-liter jars containing air or air plus 20 (cid:2) L L (cid:3) 1 ethylene. Afte r 6 h oftreatment, total RNAs were isolated from different parts of plants and were analyzed as described above. The blots were visualized by autoradiography. The equivalence of RNA loading among lanes of the agarose gel was demonstrated by ethidium bromide staining of rRNA on the gel. was blotted onto a membrane filter and the blot was hybridized to the 32 P-labeled cDNA probes for VR-EIL1 , VR-EIL2 , (cid:1) -1,3-glucanase (Ohme and Shinshi, 1990), osmotin (Sato et al., 1996), PR1 (Kim et al., 1998), and NG-ACO1 (Kim et al., 1998), respectively, under high stringent conditions. To investigate the transactivation activities of VR-EILs, total RNAs (20 (cid:2) g per lane) were isolated from 10-d-old green T 2 generation plants of various independent 35S::VR-EIL lines (VR-EIL1-2, VR-EIL1-3, VR-EIL1-4, VR-EIL1-8, VR-EIL2-1, and VR-EIL2-2), which had been grown without exogenous ethylene, and were similarly analyzed. The blots were visualized by autoradiography. The equivalence of RNA loading among lanes of the agarose gel was demonstrated by ethidium bromide staining of rRNA on the gel.