Plant Molecular Biology 44: 11–25, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
ERN1, a novel ethylene-regulated nuclear protein of Arabidopsis
Stefan M. Trentmann
Botanisches Institut der Universität zu Köln, Gyrhofstrasse 15, 50931 Köln, Germany; present address: Priontest
GmbH, Robert-Bosch-Str. 19, 85748 Garching bei München, Germany (e-mail: StefanTrentmann@Euroﬁns.com)
Received 10 February 2000; accepted in revised form 8 May 2000
Key words: Arabidopsis, differential display, ethylene signaling, GUS expression, nuclear localization
Employingdifferential displayof mRNA to investigatethe transcriptionallyregulated part of the ethylene response
pathway in etiolated seedlings of Arabidopsis thaliana, a novel ethylene-regulated nuclear-localized protein, des-
ignated ERN1, was identiﬁed. ERN1 is one of four genes whose differential expression was conﬁrmed by RNA
blot analysis. ERN1 is represented by a single-copy gene in the Arabidopsis genome. Its expression is suppressed
by ethylene in wild-type Arabidopsis but not in the ethylene-insensitive etr1-1 mutant. To gain ﬁrst insight into
the biological role of ERN1, a promoter-β-glucuronidase(GUS) gene fusion was constructed and the expression in
various organs from early to late developmental stages was examined. The analysis revealed spatial and temporal
expression patterns that correlate with developmental processes known to be affected by ethylene. Evidence is
given that the level of expression of ERN1 is regulated through the ethylene signal transductionpathway via CTR1
and EIN3, indicating that ERN1 acts downstream of EIN3.
The gaseous hormone ethylene affects many aspects
of plant growth and development, including seed ger-
mination, seedling growth, root initiation, epinastic
growth and fruit ripening (Abeles et al., 1992). Al-
thoughthe ethylene biosynthetic pathway is well char-
acterized (reviewed by Kende, 1993), the molecular
events underlying the ethylene-mediated transforma-
tion of external stimuli into a physiological response
have only begun to be understood (Ecker, 1995;
Bleecker and Schaller, 1996; Chang, 1996).
In the past few years genetic efforts have been un-
dertaken in Arabidopsis thaliana to elucidate the eth-
of a classic response of dark-grown dicotyledonous
seedlings to ethylene known as the ‘triple response’.
The morphological changes associated with this re-
sponse consist of an inhibition of hypocotyl and root
elongation, a radial swelling of the hypocotyl and the
The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession numbers Y15066 (cDNA) and Y17509 (promoter).
root, and a negative geotropic growth, leading to an
exaggerated curvature of the apical hook (Neljubow,
1901). By looking for morphological changes in the
triple response of seedlings to ethylene, a number
of mutants with altered responses to ethylene were
identiﬁed (reviewed by Ecker, 1995; Kieber, 1997).
The cloning of seven genes of the ethylene signal
transduction pathway in Arabidopsis, ETR1 (Chang
et al., 1993), ETR2 (Sakai et al., 1998), EIN4 (Hua
et al., 1998), RAN1 (Hirayama et al., 1999), CTR1
(Kieber et al., 1993), EIN2 (Alonso et al., 1999) and
EIN3 (Chao et al., 1997), which were identiﬁed by
mutational analysis, has been reported. The mutants
etr1, etr2, ein4, ein2 and ein3 belong to the class of
ethylene-insensitive mutants, whereas ctr1 displays a
constitutive triple response in the absence of ethylene.
A library screen under less stringent conditions re-
vealed two ETR-related genes, designated ERS1 (Hua
et al., 1995) and ERS2 (Hua et al., 1998). Muta-
tions in ETR1 and the four ETR1-related genes, ETR2,
EIN4, ERS1, ERS2, conferred dominant ethylene in-
sensitivity, and double-mutant analysis indicated that
they act upstream of CTR1 in the ethylene response