Global Quantitative Proteomics Studies Revealed Tissue-Preferential Expression and Phosphorylation of Regulatory Proteins in Arabidopsis

Abstract

Organogenesis in plants occurs across all stages of the life cycle. Although previous studies have identified many genes as important for either vegetative or reproductive development at the RNA level, global information on translational and post-translational levels remains limited. In this study, six Arabidopsis stages/organs were analyzed using quantitative proteomics and phosphoproteomics, identifying 2187 non-redundant proteins and evidence for 1194 phosphoproteins. Compared to the expression observed in cauline leaves, the expression of 1445, 1644, and 1377 proteins showed greater than 1.5-fold alterations in stage 1–9 flowers, stage 10–12 flowers, and open flowers, respectively. Among these, 294 phosphoproteins with 472 phosphorylation sites were newly uncovered, including 275 phosphoproteins showing differential expression patterns, providing molecular markers and possible candidates for functional studies. Proteins encoded by genes preferentially expressed in anther (15), meiocyte (4), or pollen (15) were enriched in reproductive organs, and mutants of two anther-preferentially expressed proteins, acos5 and mee48, showed obviously reduced male fertility with abnormally organized pollen exine. In addition, more phosphorylated proteins were identified in reproductive stages (1149) than in the vegetative organs (995). The floral organ-preferential phosphorylation of GRP17, CDC2/CDKA.1, and ATSK11 was confirmed with western blot analysis. Moreover, phosphorylation levels of CDPK6 and MAPK6 and their interacting proteins were elevated in reproductive tissues. Overall, our study yielded extensive data on protein expression and phosphorylation at six stages/organs and provides an important resource for future studies investigating the regulatory mechanisms governing plant development.

Global Quantitative Proteomics Analysis Reveals the Downstream Signaling Networks of Msx1 and Msx2 in Myoblast Differentiation

“…To provide insight into the possible cellular mechanisms, comprehensive protein-protein interactions (PPIs) were used to depict numerous biological aspects of complex regulatory networks and signaling pathways. Hence, we used a multi-omics data analysis tool, OmicsBean, to construct PPI networks (Huang et al 2020;Lu et al 2020b;Szklarczyk et al 2016) for DEPs. Before the induced differentiation stage, we found that the PPIs from Msx1-mediated differentiation might play important roles in amino sugar and nucleotide sugar metabolism, arginine and proline metabolism, glycolysis/gluconeogenesis (Fig.…”

“…Peptide Preparation was performed previously (Lu et al 2020a). Briefly, after sonicating the cells with lysis buffer.…”

“…200 μg proteins was taken from each sample and digested at 37 °C for 12 h with trypsin at a ratio 1-20 (trypsin to protein). 100 g peptides were labelled from each samples according to the manufacturer's protocol (Lu et al 2020a). 113-iTRAQ tag for C2C12 wild-type cells before induced differentiation (WT_B).…”

“…Mass Spectrometry Analysis was first conducted in a nanoflow EASY-nLC 1000 system (Thermo Fisher Scientific) as previously reported (Lu et al 2020a). Briefly, the peptides were separated using a prepMap100 C18 Column and then examined on an PrepMap RSLC C18 Column.…”

“…The proteomics data were processed in Mascot search engine (Matrix Science, London, UK) (Lu et al 2020b) based on the mouse database. Peptides scored by Mascot software greater than the 99% confidence interval are considered to be successfully identified.…”