Phosphate and phosphite differentially impact the proteome and phosphoproteome of Arabidopsis suspension cell cultures

Abstract

22Phosphorus absorbed in the form of phosphate (H2PO4 -) is an essential but limiting macronutrient 23for plant growth and agricultural productivity. A comprehensive understanding of how plants 24respond to phosphate starvation is essential to develop more phosphate-efficient crops. Here we 25 employed label-free proteomics and phosphoproteomics to quantify protein-level responses to 48 26 h of phosphate versus phosphite (H2PO3 -) resupply to phosphate-deprived Arabidopsis thaliana 27 suspension cells. Phosphite is similarly sensed, taken up, and transported by plant cells as 28phosphate, but cannot be metabolized or used as a nutrient. Phosphite is thus a useful tool to 29 delineate between non-specific processes related to phosphate sensing and transport, and specific 30 responses to phosphorus nutrition. We found that responses to phosphate versus phosphite 31 resupply occurred mainly at the level of protein phosphorylation, complemented by limited 32 changes in protein abundance, primarily in protein translation, phosphate transport and 33 scavenging, and central metabolism proteins. Altered phosphorylation of proteins involved in core 34 processes such as translation, RNA splicing, and kinase signalling were especially important. We 35 also found differential phosphorylation in response to phosphate and phosphite in 69 proteins, 36including splicing factors, translation factors, the PHT1;4 phosphate transporter and the HAT1 37 histone acetyltransferase-potential phospho-switches signalling changes in phosphorus nutrition. 38Our study illuminates several new aspects of the phosphate-starvation response and identifies 39 important targets for further investigation and potential crop improvement. 40

Library-free BoxCarDIA solves the missing value problem in label-free quantitative proteomics

“…Ler) suspension cells grown for five days in either constant light or dark. Trypsin digestion of the extracted proteome was performed BoxCar, data-independent and -dependent acquisition analysis of Arabidopsis proteomes Mehta et al, 2020 4 using an automated sample preparation protocol, with 1ug of digested peptide subsequently analysed using an Orbitrap Fusion Lumos mass spectrometer operated in either DDA, DIA, or BoxCarDIA acquisition modes over 120-minute gradients. Two separate experiments were performed using independent digests of the extracted Arabidopsis proteins.…”

“…BoxCar, data-independent and -dependent acquisition analysis of Arabidopsis proteomes Mehta et al, 2020 16 https://github.com/UhrigLab/BoxCarMaker under a GNU Affero General Public License 3.0.…”

“…BoxCar, data-independent and -dependent acquisition analysis of Arabidopsis proteomesMehta et al, 2020 …”

“… Abstract The last decade has seen significant advances in the application of quantitative mass spectrometry-based proteomics technologies to tackle important questions in plant biology. This has included the use of both labelled and label-free quantitative liquid-chromatography mass spectrometry (LC-MS) strategies in model 1,2 and non-model plants 3 . While chemical labelling-based workflows (e.g.…”