Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis

Abstract

Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a (15)N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.

Quantitative and functional posttranslational modification proteomics reveals that TREPH1 plays a role in plant touch-delayed bolting

“…Both the wild-type and Ser625Ala (S625A) recombinant TREPH1 genes were generated as described 73 . A 2.0 kb DNA fragment encoding the full-length TREPH1 was amplified from Arabidopsis genomic DNA by PCR using the following primers (Table S1) Point-mutated TREPH1 S625A was generated by PCR using mutagenic primers (mutation sites are indicated in bold): PPF, 5'-CCGAATCTAGCTGGAATCTTC-3' (mutation sites bolded); PPR, 5'-GTTGAAGATTCCAGCTAGATTCG-3' (mutation sites bolded).…”

“…The resulting LC-MS/MS raw data were converted to both mzXML and mgf formats following an established procedure 39 . All MS2 spectra contained in the mgf files were searched against TAIR10 (35,386 proteins) using the Mascot search engine (Version 2.3, Matrix Science) as described 73 . The false discovery rate (FDR) defined to select the PSM of phosphopeptides was set to 1% 80 .…”

Three-Dimensional Superresolution Imaging of the FtsZ Ring during Cell Division of the Cyanobacterium Prochlorococcus

“…After photobleaching, we were able to use STORM to visualize the localization of the plant plasma membrane intrinsic protein PIP2a ( Fig. S2 ), which is a water channel ( 29 ).…”

“…After section preparation and photobleaching, A. thaliana samples were incubated with an anti-PIP2a polyclonal antibody ( 29 ) for 12 h at 4°C. After washing with MTSB three times (10 min for each washing), samples were incubated for 5 h at 4°C with an anti-rabbit secondary antibody conjugated with Alexa Fluor 647 (Life Technologies).…”

Phosphatase PP2C6 negatively regulates phosphorylation status of Pti1b kinase, a regulator of flagellin-triggered immunity in tomato

“…Pti1b has S-acylation sites in its N terminus which play a role in its localization to the cell periphery (26). As a control, therefore, we applied the same purification method using another membraneassociated protein from Arabidopsis, PIP2A (AT3G53420) (35). The samples obtained were submitted for mass spectrometry analysis to identify potential Pti1b-interacting proteins.…”