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Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis

ABSTRACT: A complex interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 is essential for triggering ethylene responses in plants. The gaseous plant hormone ethylene can trigger myriad physiological and morphological responses in plants. While many ethylene signaling pathway components have been identified and characterized, little is known about the function of the integral membrane protein EIN2, a central regulator of all ethylene responses. Here, we demonstrate that Arabidopsis thaliana EIN2 is a protein with a short half-life that undergoes rapid proteasome-mediated protein turnover. Moreover, EIN2 protein accumulation is positively regulated by ethylene. We identified two F-box proteins, EIN2 TARGETING PROTEIN and 2 (ETP1 and ETP2), that interact with the EIN2 carboxyl-terminal domain (CEND), which is highly conserved and sufficient to activate most ethylene responses. Overexpression of ETP1 or ETP2 disrupts EIN2 protein accumulation, and these plants manifest a strong ethylene insensitive phenotype. Furthermore, knocking down the levels of both ETP1 and ETP2 mRNAs using an artificial microRNA (amiRNA) leads to accumulation of EIN2 protein, resulting in plants that display constitutive ethylene response phenotypes. Finally, ethylene down-regulates ETP1 and ETP2 proteins, impairing their ability to interact with EIN2. Thus, these studies reveal that a complex interplay between ethylene, the regulation of ETP1/ETP2 F-box proteins, and subsequent targeting and degradation of EIN2 is essential for triggering ethylene responses in plants. Keywords: ethylene treatment, genetic modification

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE14247 | GEO | 2009/12/01

SECONDARY ACCESSION(S): PRJNA111175

REPOSITORIES: GEO

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Project description:Ethylene 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 5â?3â 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. Experiment Overall Design: Cold-treated seeds in MS plates were placed in chambers at 24oC in the dark with hydrocarbon-free airflow for three days, after which some of the chambers were connected to ethylene gas at 10 ppm while the others remained on air treatment. Four hours later the seedlings of each plate were quickly collected and frozen in liquid nitrogen. Total RNA was prepared from both air-treated and ethylene-treated etiolated seedlings of Arabidopsis Col-0, ein2-5 and ein5-1 using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Biotinylated target RNA was prepared from 16 ?g of total RNA using the procedure described by the manufacturer (Affymetrix, Santa Clara, CA). Briefly, a primer encoding a T7 RNA polymerase promoter fused to (dT)24 (Genset Oligos, La Jolla, CA) was used to prime double-stranded cDNA synthesis using the SuperScript Choice System (Life Technologies). The resulting cDNA was transcribed in vitro using the BioArray High-Yield RNA Tran-script Labeling Kit (Enzo Biochem, New York, NY) in the presence of biotinylated UTP and CTP to produce biotinylated target complementary RNA (cRNA). The labeled target cRNA was purified, fragmented, and hybridized to Arabidopsis microoarrays (Affymetrix whole genome tiling, and ATH1 gene expression arrays) according to protocols provided by the manufacturer in a hybridization oven model 640 (Affymetrix, Santa Clara, CA). The arrays were washed and stained with streptavidin-phycoerythrin using a GeneChip Fluidics Station model 400 and then scanned with a Gene Array Scanner (Hewlett-Packard, Palo Alto, CA). Scanned images were processed and quantified using GeneChip Suite 3.2. Genespring software (Silicon Genetics, Redwood City, CA) was used to manage and filter the array data. Each measurement was divided by the 50.0th percentile of all measurements in that sample. The percentile was calculated with all normalized measurements above 10. For samples where the bottom tenth percentile was less than the negative of the 50.0th percentile, it was used as a background, and subtracted from all the other genes first. Experiments were carried out in duplicate and hybridized to two sets of expression chips. Expression values were analyzed using GeneSpringâ¢ software version 4.2 (Silicon Genetics, Redwood, California).

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Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis

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