This journal is c The Royal Society of Chemistry 2011 Mol. BioSyst., 2011, 7, 2637–2650 2637 Cite this: Mol. BioSyst., 2011, 7, 2637–2650 Proteomic responses in Arabidopsis thaliana seedlings treated with ethylenew Ruiqiang Chen, a Brad M. Binder, b Wesley M. Garrett, c Mark L. Tucker, d Caren Changz* a and Bret Cooperz* d Received 26th April 2011, Accepted 2nd June 2011 DOI: 10.1039/c1mb05159h Ethylene (ET) is a volatile hormone that modulates fruit ripening, plant growth, development and stress responses. Key components of the ET-signaling pathway identified by genetic dissection in Arabidopsis thaliana include five ET receptors, the negative regulator CTR1 and the positive regulator EIN2, all of which localize to the endoplasmic reticulum. Mechanisms of signaling among these proteins are still unresolved and targets of ET responses are not fully known. So, we used mass spectrometry to identify proteins in microsomal membrane preparations from etiolated A. thaliana seedlings maintained in ambient air or treated with ET for 3 h. We compared 3814 proteins from ET-exposed seedlings and controls and identified 304 proteins with significant accumulation changes. The proteins with increased accumulation were involved in ET biosynthesis, cell morphogenesis, oxidative stress and vesicle secretion while those with decreased accumulation were ribosomal proteins and proteins positively regulated by brassinosteroid, another hormone involved in cell elongation. Several proteins, including EIN2, appeared to be differentially phosphorylated upon ET treatment, which suggests that the activity or stability of these proteins may be controlled by phosphorylation. TUA3, a component of microtubules that contributes to cellular morphological change, exhibited both increased accumulation and differential phosphorylation upon ET treatment. To verify the role of TUA3 in the ET response, tua3 mutants were evaluated. Mutant seedlings had altered ET-associated growth movements. The data indicate that ET perception leads to rapid proteomic change and that these changes are an important part of signaling and development. The data serve as a foundation for exploring ET signaling through systems biology. Introduction Ethylene (ET) is a gaseous molecule that has numerous effects on plant growth and displays complex interactions with other endogenous plant hormones, growth regulators and environmental factors to provide fine-tuning of development and adaptation. 1 Components of the ET-perception signal transduction pathway have been identified, mostly through forward-genetics in Arabidopsis thaliana. ET is perceived by a family of membrane-bound, homo- and heterodimeric receptors residing at the endoplasmic reticulum (ER) and possibly the Golgi apparatus. 2–7 One receptor displays His auto-kinase activity while the others exhibit Ser/Thr kinase activity, or both, in vitro. 8,9 The roles of their kinase activities and their in vivo substrates are unknown. The receptors repress ET responses in the absence of ET by activating CTR1 10 although there may also be an alternate ET-response pathway that bypasses CTR1. 11,12 CTR1 is a negative regulator of ET response, resides at the ER due to physical association with the receptors and has sequence similarity to Raf-like protein kinases. 13,14 The next known genetic downstream component, EIN2 (ETHYLENE INSENSITIVE 2), is a critical, positive regulator of ET-signal transduction, but its biochemical function is unknown. 15 EIN2 also localizes to the ER where it possibly interacts with the ETR1 ethylene receptor. 16 Genetically downstream of EIN2 are transcription factors (TF) EIN3 and EIL1, which activate the expression of another TF, ERF1. 17 a Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA. E-mail: carenc@umd.edu b Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA c Animal Biosciences and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA d Soybean Genomics and Improvement Laboratory, USDA-ARS, Beltsville, MD 20705, USA. E-mail: bret.cooper@ars.usda.gov w Electronic supplementary information (ESI) available: Tables S1 and S2. PANORAMICS 2 output of peptides and proteins. XLSX file. This includes peptide sequence matches, amino acid modifications, Mascot scores, peptide spectral counts and protein group probabilities for combined data from replicates of AIR control (S1) and ET-treated seedlings (S2). Table S3. Differentially accumulating proteins. XLSX file. Data include proteins with significantly different summed spectral counts, protein annotation, GOslim descriptions and presence of transmembrane domains. Dataset S4. Manually-annotated spectra of phosphopeptides. Zipped PDF files. See DOI: 10.1039/c1mb05159h z These authors made equal contributions to this project. Molecular BioSystems Dynamic Article Links www.rsc.org/molecularbiosystems PAPER