CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis

Abstract

The gaseous phytohormone ethylene C(2)H(4) mediates numerous aspects of growth and development. Genetic analysis has identified a number of critical elements in ethylene signaling, but how these elements interact biochemically to transduce the signal from the ethylene receptor complex at the endoplasmic reticulum (ER) membrane to transcription factors in the nucleus is unknown. To close this gap in our understanding of the ethylene signaling pathway, the challenge has been to identify the target of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) Raf-like protein kinase, as well as the molecular events surrounding ETHYLENE-INSENSITIVE2 (EIN2), an ER membrane-localized Nramp homolog that positively regulates ethylene responses. Here we demonstrate that CTR1 interacts with and directly phosphorylates the cytosolic C-terminal domain of EIN2. Mutations that block the EIN2 phosphorylation sites result in constitutive nuclear localization of the EIN2 C terminus, concomitant with constitutive activation of ethylene responses in Arabidopsis. Our results suggest that phosphorylation of EIN2 by CTR1 prevents EIN2 from signaling in the absence of ethylene, whereas inhibition of CTR1 upon ethylene perception is a signal for cleavage and nuclear localization of the EIN2 C terminus, allowing the ethylene signal to reach the downstream transcription factors. These findings significantly advance our understanding of the mechanisms underlying ethylene signal transduction.

Fig. 1.

CTR1 phosphorylates specific serine/threonine residues in the EIN2 C-terminal domain in vitro. (A) Cartoon of CTR1 and EIN2 protein domain structure. Position of the predicted NLS in EIN2 is shown (19). (B) Yeast two-hybrid assay showing that the CTR1 kinase domain (residues 551–821) interacts with the EIN2 soluble domain (residues 516–1294). Bait vector (pLEXA), prey vector (pACTII), and lamin were negative controls. (C) BiFC interaction of full-length EIN2 and CTR1 in tobacco leaf epidermal cells. Merged image shows BiFC, DIC, and chlorophyll. (Scale bar: 20 µM.) (D) Coomassie-stained SDS/PAGE gel of purified WT and mutant versions of CTR1 kinase domain (KD) and soluble C-terminal domain of EIN2 (EIN2-C). Molecular weight markers are shown on Left. (E) In vitro kinase assay of purified CTR1 kinase domain (residues 531–821) with the EIN2 C-terminal domain (residues 479–1294). The indicated proteins (WT or mutant) were incubated together in kinase reaction buffer, separated by SDS/PAGE, and the incorporated radiolabel detected with a phosphorimager. (F) The relative phosphorylation of various peptides by CTR1-KD^WT. Peptides used: 1: KAAPTSNFTVGSDGPPS⁶⁴⁵FRSLSGK; 1m: KAAPTSNFTVGSDGPPA⁶⁴⁵FRSLSGK; 2. KAAVANEKKYSS⁹²⁴MPDISGLSMSAR; 2m: KAAVANEKKYSA⁹²⁴MPDISGLSMSAR; 3: KPVGMNQDGPGS¹²⁸³RKNVTAYG; 3m: KPVGMNQDGPGA¹²⁸³RKNVTAYG; 4:KQQRTPGS⁷⁵⁷IDSLYGLQR; 5: KKGMDS⁷³⁹QMTSSLYDSLKQQRT. (G) Kinetic analysis of CTR1 phosphorylation of peptide 1. A total of 20 ng of His₆-CTR-KD^WT protein was incubated with increasing concentrations of EIN2 peptide1 in kinase buffer with γ-labeled [³²P]ATP, and the amount of radioactivity incorporated determined.

Fig. 2.

Ser⁶⁴⁵Ala and Ser⁹²⁴Ala substitutions in EIN2 confer constitutive ethylene responses in Arabidopsis. (A) The ethylene-response phenotype in 4-d-old dark-grown seedlings in the absence of ethylene treatment is shown for ein2-5 transformed with WT and mutant versions of genomic transgene EIN2p-EIN2. Representative seedlings are shown in comparison with the WT (Col-0), ein2-5, and ctr1-1. Mean hypocotyl length ± SD is averaged for two independent lines, 20–30 seedlings per line. (B) Inhibition of leaf cell expansion in representative rosettes of 5-wk-old soil-grown plants of the same transgenic lines as in A. Mean rosette diameter ± SD at 4 wk old is averaged for two independent lines, 20–35 rosettes per line. (C) Real-time quantitative PCR expression analysis for EIN2 and the ethylene-responsive transcription factor gene ERF1. Two independent lines (1 and 2) are shown for each transgene. (D) Four-d-old dark-grown Col-0 seedlings stably transformed with WT and mutant versions of 35S-EIN2 treated with or without 20 μM ACC. Representative seedlings are shown in comparison with Col-0 and ctr1-1.

Fig. 3.

EIN2^AA results in constitutive nuclear localization of the EIN2 C-terminal domain. (A) Western blot of EIN2-GFP in the microsomal fraction. Four-d-old dark-grown ein2-5 seedlings stably transformed with EINp-EIN2-GFP were treated without or with ethylene gas for 3 h. The microsomal fraction was analyzed by Western blotting using an anti-GFP antibody. EIN2 (without GFP) is 141 kDa. ECA1 (41) was a loading control. (B) Differential localization of the N and C termini of EIN2 in onion epidermal cells. 35S-YFP-EIN2 and 35S-EIN2-GFP were delivered into onion cells by particle bombardment and fluorescence was visualized by confocal microscopy. The YFP tag and the ER marker (ER-RFP) colocalized both without and with 3–6 h ethylene treatment. The GFP tag colocalized with the ER marker without ethylene treatment, but predominantly colocalized with the nuclear DAPI stain after ethylene treatment. (C) Ethylene-responsive nuclear localization of EIN2^WT-YFP and constitutive nuclear localization of EIN2^AA-YFP in Arabidopsis hypocotyl cells. Four-d-old dark-grown Col-0 seedlings stably transformed with 35S-EIN2^WT-YFP were treated for 3 h with H₂O (0 µM ACC) or 100 µM ACC, and then examined by confocal microscopy. Col-0 seedlings transformed with 35S-EIN2^AA-YFP were treated for 3 h with only H₂O (0 µM ACC). (D) Constitutive nuclear localization of EIN2^WT-GFP and EIN2^AA-GFP in hypocotyl cells of ctr1-1 and ein2-5, respectively. Four-d-old dark-grown seedlings stably transformed with EIN2p-EIN2^WT-GFP or EIN2p-EIN2^AA-GFP without ethylene treatment were examined by confocal microscopy. (Scale bar: B–D, 20 μm.)

Fig. 4.

Model of ethylene signaling. In the absence of ethylene (Left), the ethylene receptors (e.g., ETR1) at the ER membrane activate the CTR1 protein kinase, a dimer (42), which phosphorylates the C-terminal domain of EIN2, preventing its nuclear localization. Without ethylene, EIN2 is targeted for 26S proteasomal degradation by F-box proteins ETP1/2 (16). Transcription factors EIN3/EIL1 are also targeted for degradation by F-box proteins EBF1/2 (17). In the presence of ethylene (Right), the receptors are inactivated and therefore the CTR1 kinase is no longer active. The absence of phosphorylation on EIN2 results in EIN2 C terminus being cleaved and localizing to the nucleus where it can activate the downstream transcriptional cascade.