Interplay Between Ethylene, ETP1/ETP2 F-Box Proteins, and Degradation of EIN2 Triggers

Supplemental Data

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

Hong Qiao, Katherine N. Chang, Junshi Yazaki and Joseph R. Ecker

Supplemental Method

Microarray Experiments and Analysis

All RNA extractions were performed using the RNeasy Kit (Qiagen) per manufacturers instructions. cRNA synthesis, labeling, and hybridization to Arabidopsis ATH1 gene expression arrays (Affymetrix Inc) were performed according to manufacturer’s recommendations except that the labeling reactions were scaled down by 50%. After hybridization, the arrays were scanned and the.CEL files were used for further analysis. All normalization and quality controls were performed using the packages from the remote analysis computation for gene expression data (RACE, http://race.unil.ch) (Psarros et al. 2005). After normalization, present, marginal, and absent flags, together with the intensity values converted from logarithmic to linear scales, were exported to GeneSpring GX (Agilent). Ethylene-regulated genes were selected using a linear model approach (Smyth 2004) implemented in the limma package from BioConductor (Smyth 2004) . This analysis was done using the Remote Analysis Computation for Gene Expression (Psarros et al. 2005). Genes that had a P value of <0.05 and a fold change between control and treatment or control and mutants experiments greater than 1.5 were selected. Finally, only genes that were present or marginal in both replicates in the treated (when selecting upregulated genes) or in the untreated (when selecting for down regulated genes) samples were further considered. The microaray data has been deposited in the Gene Expression Omnibus (GE0) database (Accession # XXXXX).

Supplemental References

Psarros, M. et al., RACE: Remote Analysis Computation for gene Expression data. Nucleic Acids Res 33 (Web Server issue), W638 (2005).

Smyth, G. K., Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3, Article3 (2004).

Supplemental Figures and Legends

Figure S1. The effect of ethylene on EIN2 protein accumulation. The abundance of EIN2 protein correlates with the triple response. Etiolated wild-type seedlings (Col-0) were grown on MS medium supplemented with 10um ACC or 10uM AVG for 3 days. Total membrane protein extracts were subjected to immunoblot with EIN2 antiserum. H+-ATPase was used as a lane loading control.

Figure S2. The most conserved domain of EIN2 is essential for the interaction of EIN2 and ETP1/ETP2. (A) Yeast strains which carried different combination of constructs (as indicated) were grown in SD-Leu/-Trp liquid medium overnight and subjected to SD–leu/-Trp/-His/+3AT (20 mM) selective medium by the indicated start titer (OD600). (B) Expression of truncated forms of EIN2 protein in yeast. Yeast strains which carried different truncations of EIN2 and ETP1 or ETP2 were cultured in SD-Leu/-Trp liquid medium overnight and total protein lysates were separated by PAGE and subjected to blotting using anti-GAL4 DNA-binding domain (DB) antibody. The EIN2:GAL4 fusion proteins tested are indicated, Coomassie blue staining was used as a lane loading control.

Figure S3. The alignment of EIN2 from different plant species. Alignment of EIN2 amino acid sequences from different species generated with the ClustalW program. The positions of amino acid residues are indicated with numbers; asterisks and dots indicate identical and conserved amino acids respectively.

Figure S4. Mutation of ETP1 Leads to Slightly Hypersensitive to Ethylene Phenotype. (A) Schematic representation of T-DNA insertions in the related F-box genes ETP1 and ETP2 respectively. Coding regions are indicated by boxes and non-codling regions are indicated by lines. F-box and FBA_1 domains are colored yellow and green respectively. A triangle represents a T-DNA insertion event and the positions are indicated. (B-C) Hypocotyl length measurement of etp1 and etp2 mutants. 3-day-old etiolated seedlings were grown on MS medium supplemented with or without 10µM ACC. Each measurement is the average length (mean± standard error) of >20 hypocotyls. (D) ACC dosage response of etp1 and etp2 mutants. Etiolated seedlings were grown on MS medium supplemented with the indicated amount of ACC. Each measurement is the average length (mean± standard error) of >20 hypocotyls.

Figure S5. EIN2 protein accumulates in amiR-ETP1/ETP2 mutant plants. Anti-EIN2 antibody specifically recognizes EIN2 protein in amiR-ETP1/ETP2 mutant plants. Total proteins from wild type (Col), ein2-5 or amiR-ETP1/ETP2 plants were subjected to PAGE, and immunoblotting with anti-EIN2 antibody. Coomassie blue staining was used as a lane loading control.

Figure S6. Venn diagrams showing the overlap of genes up or down regulated in wild-type Col-0 plants treated with ethylene or amiR-ETP1/ETP2 plants. Both wild-type Col-0 ethylene treated and amiR-ETP1/ETP2 plants were first compared to an air treated control.

Figure S7. The mRNA level of ETP1 and ETP2 were accumulated in 35S::MYC-ETP1/ETP2 transgenic plants. mRNA level of ETP1 (A) or ETP2 (B) were detected by RT-PCR using ETP1 or ETP2 specific primers (upper panel), and the relative density was quantified (lower panel). Total RNA was extracted from the leaves of 1-week-old dark-grown plants. The data were normalized to the corresponding tubulin (input) controls. The relative density quantification was done by software ImageGauge.

Figure S8. The level of ETP1 and ETP2 mRNA is not regulated by ethylene. qPCR analysis of ETP1(A) and ETP2 (B) transcript levels in wild type plants treated with ethylene for different time periods. Total RNA was extracted from the leaves of 1-week-old dark-grown plants. The data were normalized to the corresponding actin (input) controls. The data shown are the mean ± SD of three independent experiments.