Plant hormone ethylene plays important roles in plant growth, development, and stress responses. Decades of molecular genetic work has established a central dogma for the major ethylene signaling pathway, in which the ethylene signal is perceived by a family of ethylene receptors located on the endoplasmic reticulum endomembrane systems and its signal is transduced down to the C-terminal domain dephohsphoryaltion of a positive ethylene response regulator, Ethylene Insensitive 2 (EIN2). This dephosphorylated C-terminal fragment of EIN2 protein migrates into the nucleus to activate and stabilize both Ethylene Insensitive 3 (EIN3) and Ethylene Insensitive 3-Like 1 (EIL1) transcription factors and to further regulate the expression of a large set of downstream Ethylene Response Fact...[ Read more ]

Plant hormone ethylene plays important roles in plant growth, development, and stress responses. Decades of molecular genetic work has established a central dogma for the major ethylene signaling pathway, in which the ethylene signal is perceived by a family of ethylene receptors located on the endoplasmic reticulum endomembrane systems and its signal is transduced down to the C-terminal domain dephohsphoryaltion of a positive ethylene response regulator, Ethylene Insensitive 2 (EIN2). This dephosphorylated C-terminal fragment of EIN2 protein migrates into the nucleus to activate and stabilize both Ethylene Insensitive 3 (EIN3) and Ethylene Insensitive 3-Like 1 (EIL1) transcription factors and to further regulate the expression of a large set of downstream Ethylene Response Factors (ERFs), which leads to diverse ethylene responses. However, the emerging functional phosphoproteomic and molecular genetic studies have also demonstrated that there exist alternative ethylene signaling pathways independent to EIN3 and EIL1.

Protein acetylation is one of post-translational modifications (PTMs) and involved in many biological and cellular processes. To identify novel acetylation sites and monitor ethylene-induced dynamic acetylome changes, we have developed a dimethyl-labelling-based extracted ion chromatogram (XIC) workflow of quantitative PTM proteomics and applied it on Arabidopsis ein3/eil1 double mutant. The workflow comprised 4-component (4C) steps: c̲hemical labelling (i.e., dimethyl labelling), c̲hromatographic enrichment, c̲omputational analysis (i.e., identification and quantification), and c̲onfirmation of identified PTM sites (i.e., immunoblot analysis). Ultimately, 5,250 acetylation sites on 2,638 acetylproteins were repeatedly identified. Among them, 4,228 acetylation sites (i.e., 80.5%) were newly discovered. As a result, we have substantially expanded the database of Arabidopsis acetylation sites from 3,122 sites to 7,350 sites. Previous study showed that acetylation is mainly concentrated on histone proteins, which facilitate transcription. However, based on our results, the acetylproteins mainly derived from histone proteins, ribosomal proteins, heat shock proteins and proteins related to stress/stimulus responses and energy metabolism. That means acetylation may be involved in protein translation, protein folding, stress/stimulus response, photosynthesis and energy metabolism. In addition, as a global PTM, acetylation may be also involved in signalling transduction. Quantitative proteomics quantified 33 ethylene-enhanced and 31 ethylene-suppressed u̲nique P̲TM peptide a̲rrays (UPAs), which suggested that their ethylene-regulated acetylation was independent of EIN3 and EIL1. Among them, the acetylation site histone H4K5 has been validated by immunoblot using the acetylation site-specific antibody. Some deacetylases have been reported to be ethylene-related, including histone deacetylase HDA6, HDA19 (ATHD1), SRT1 and SRT2. Moreover, deacetylases HDA19 (ATHD1) and HDA6 and acetylases HAM1 and HAM2 have been demonstrated involving the acetylation on histone H4K5. Based on all these information, HDA19 (ATHD1) is predicted to be the deacetylase of histone H4K5. Since dimethyl labelling can applied for the peptides from any organism theoretically, this workflow of quantitative PTM proteomics has potential to be used in model eukaryotes, agricultural crops and even tissue samples from animals and humans.

We also have developed an in planta chemical cross-linking-based quantitative interactomics (IPQCX-MS) 4C workflow. A chemical cross-linker, azide-tag modified disuccinimidyl pimelate (AMDSP), was designed to investigate in vivo global protein-protein interactions in Arabidopsis ein3/eil1. In total, 354 non-redundant cross-linked peptides (i.e., 61 inter- and 293 intra-protein cross-linked peptides) were identified. This is the first time to in vivo identify hundreds of cross-linked peptides in a plant at a multicellular organismal level. The identified PHB3 and PHB6 protein-protein interaction was validated and confirmed using co-immunoprecipitation and super-resolution microscopy. The potential application of this IPQCX-MS workflow is to in vivo identify protein-protein interaction networks in agricultural crops and plant-microbe interactions