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. 2006 Nov;18(11):3047-57.
doi: 10.1105/tpc.106.046508. Epub 2006 Nov 3.

The exoribonuclease XRN4 is a component of the ethylene response pathway in Arabidopsis

Thomas Potuschak  1 , Amérin Vansiri, Brad M Binder, Esther Lechner, Richard D Vierstra, Pascal Genschik
Affiliations

The exoribonuclease XRN4 is a component of the ethylene response pathway in Arabidopsis

Thomas Potuschak et al. Plant Cell. 2006 Nov.

Abstract

EXORIBONUCLEASE4 (XRN4), the Arabidopsis thaliana homolog of yeast XRN1, is involved in the degradation of several unstable mRNAs. Although a role for XRN4 in RNA silencing of certain transgenes has been reported, xrn4 mutant plants were found to lack any apparent visible phenotype. Here, we show that XRN4 is allelic to the unidentified components of the ethylene response pathway ETHYLENE-INSENSITIVE5/ACC-INSENSITIVE1 (EIN5/AIN1) and EIN7. xrn4 mutant seedlings are ethylene-insensitive as a consequence of the upregulation of EIN3 BINDING F-BOX PROTEIN1 (EBF1) and EBF2 mRNA levels, which encode related F-box proteins involved in the turnover of EIN3 protein, a crucial transcriptional regulator of the ethylene response pathway. Epistasis analysis placed XRN4/EIN5/AIN1 downstream of CTR1 and upstream of EBF1/2. XRN4 does not appear to regulate ethylene signaling via an RNA-INDUCED SILENCING COMPLEX-based RNA silencing mechanism but acts by independent means. The identification of XRN4 as an integral new component in ethylene signaling adds RNA degradation as another posttranscriptional process that modulates the perception of this plant hormone.

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Figures

Figure 1.
Figure 1.
EBF1 and EBF2 Transcript Levels Are Upregulated in ein5 Mutant Plants, and Epistasis Analysis of ein5. (A) RNA gel analysis of EBF1 and EBF2 transcript accumulation in ecotype Columbia (Col-0), ein5-1, and xrn4-3. EtBr, ethidium bromide. (B) Relative EBF1 (hatched bars) and EBF2 (dotted bars) transcript accumulation in Col-0, ein5-1, and xrn4-3 by quantification using a phosphor imager. (C) Phenotypes of 3-d-old etiolated seedlings of the indicated genotypes grown without ACC. (D) Hypocotyl (light gray) and root (dark gray) length measurements of 3-d-old dark-grown Col-0, ebf1-1 ebf2-1, ein5-1, and ein5-1 ebf1-1 ebf2-1 seedlings germinated in the absence of ACC. Values shown are average lengths (means ± se) of >10 hypocotyls or roots. (E) Phenotypes of mature ebf1-1 ebf2-1, ein5-1 ebf1-1 ebf2-1, and wild-type (Col-0) plants grown on soil. ein5-1 plants are similar to wild-type plants on soil and therefore are not shown. (F) Phenotypes of 3-d-old etiolated seedlings of the indicated genotypes grown on Murashige and Skoog (MS) medium supplemented with 10 μM ACC. (G) Hypocotyl (light gray) and root (dark gray) length measurements of 3-d-old dark-grown Col-0, ein5-1, xrn4-3, ctr1-1, ctr1-1 ein5-1, and ctr1-1 xrn4-3 seedlings in the presence of 10 μM ACC. Values shown are average lengths (means ± se) of >10 hypocotyls or roots.
Figure 2.
Figure 2.
ein5 and ein7 Are Mutants in the Exoribonuclease XRN4. (A) Phenotypes of 4-d-old etiolated seedlings of the indicated genotypes grown on MS medium. (B) Hypocotyl (light gray) and root (dark gray) length measurements of 4-d-old dark-grown Col-0, xrn4-3, and ein5-1 seedlings germinated in the absence of ACC. Values shown are average lengths (means ± se) of >10 hypocotyls or roots. (C) Hypocotyl (light gray) and root (dark gray) length measurements of 4-d-old dark-grown Col-0, xrn4-3, ein5-1, and F1 seedlings of a xrn4-3 × ein5-1 cross in the presence of 10 μM ACC. Values shown are average lengths (means ± se) of >10 hypocotyls or roots. (D) Gene structure of XRN4. Introns are indicated by lines. Shaded and hatched boxes indicate coding regions and 5′ and 3′ untranslated regions, respectively. The positions of ein5-1 and ein7 mutations are indicated and correspond to cytosine deletions creating truncated proteins in both cases. The position of the T-DNA of the xrn4-3 allele is also indicated. (E) EBF1 transcript levels in Col-0 and ein7 at different time points during treatment with 50 μM ACC. RNA was extracted from 3-week-old Col-0 and ein7 seedlings at different time points of the ACC treatment, subjected to RNA gel blot analysis, and hybridized with the indicated probes. EtBr, ethidium bromide. (F) EIN3 protein accumulation in Col-0 and ein7 at different time points during 50 μM ACC treatment. Total protein extracts from the same sample as indicated in (E) were subjected to immunoblot assays. (G) RNA gel analysis of XRN4 transcript accumulation in different mutant backgrounds as indicated.
Figure 3.
Figure 3.
Growth Kinetics of Etiolated Arabidopsis Hypocotyls in Response to Ethylene. Growth rates were recorded for 1 h in air followed by a 2-h exposure to 10 μL/L ethylene. This was followed by 5 h in air. The responses of wild-type Col-0 hypocotyls are shown in both panels (open squares) for comparison with the homozygous mutants (closed symbols) xrn4-3 (A), ein5-1 (B), and ein7 (C). All data represent averages of at least five seedlings ± sd.
Figure 4.
Figure 4.
EBF1 and EBF2 mRNAs Remain Short-Lived in xrn4/ein5 Mutants, and Their Turnover Is Ethylene Independent. (A) RNA gel analysis of a time-course experiment for EBF1 and EBF2 transcript half-life determination in Col-0 and the ein5-1 mutant. The blot was hybridized with either the 3′ untranslated region (UTR; detecting endogenous transcripts) or ORF probes (also detecting ectopically expressed EBF1) as indicated, and EBF1/2 transcript half-lives were determined relative to EF1-α, which is a stable transcript. The asterisk indicates the possible EBF1 mRNA cleavage product as indicated by Souret et al. (2004), and the arrowhead indicates the shorter EBF1 transcript originating from the 35S:EBF1 transgene. EtBr, ethidium bromide. (B) Strong EBF1 overexpression exacerbates the ethylene-insensitive phenotype of ein5. An Arabidopsis T-DNA–transformed line that expresses a high level of EBF1 mRNA (see [A]) exhibits strong ethylene insensitivity when germinated in the dark in the presence of 10 μM ACC. This phenotype is maintained when the transgene is introduced into the ein5-1 mutant. (C) Hypocotyl (light gray) and root (dark gray) length measurements of 3-d-old dark grown Col-0, ein5-1, Col-0∷EBF1ox, and ein5-1∷EBF1ox seedlings in the presence of 10 μM ACC. Values shown are average lengths (means ± se) of >10 hypocotyls or roots. (D) RNA gel analysis of a time-course experiment for EBF1 and EBF2 endogenous transcript half-life determination in Col-0 and in ein5-1 and ein7 mutants pretreated with or without 50 μM ACC. EBF1/2 transcript half-lives were determined relative to EF1-α.
Figure 5.
Figure 5.
Representation of the Ethylene Signaling Cascade Including XRN4/EIN5. Ethylene is perceived by the ETHYLENE RESPONSE (ETR/ERS) receptors located in the endoplasmic reticulum membrane. Binding of ethylene to the receptors results in the inactivation of both the receptors and CTR1, thereby causing the derepression of positive regulatory factors, such as EIN2 (data not shown) and XRN4/EIN5. How CTR1 regulates EIN2 and XRN4/EIN5 and the relationship of EIN2 with XRN4/EIN5 in the ethylene signaling cascade are unknown. Strikingly, our model proposes the existence of two degradation pathways in ethylene signaling: one RNA decay pathway into the cytosol that indirectly controls the steady state levels of EBF1/2 mRNAs, which are components of a protein degradation pathway that controls EIN3 stability in the nucleus.
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NOTE ADDED IN PROOF

    1. While this manuscript was under review, a report by Olmeda et al. (2006) identified EIN5 as XRN4.
    1. Olmedo, G., Guo, H., Gregory, B.D., Nourizadeh, S.D., Aguilar-Henonin, L., Li, H., An, F., Guzman, P., and Ecker, J.R. (2006). ETHYLENE INSENSITIVE5 encodes a 5′ →3′ exoribonuclease required for regulation of the EIN3-targeting F-box proteins EBF1/2. Proc. Natl. Acad. Sci. USA 103 13286–13293. - PMC - PubMed

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