Root Membrane Ubiquitinome under Short-Term Osmotic Stress

Abstract

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress.

Keywords: aquaporin; mass spectrometry; osmotic stress; ubiquitination.

Figure 1

Workflow for quantitative profiling of the proteome and the ubiquitinome in Arabidopsis root membrane proteins upon mannitol treatment. LC-MS/MS: liquid chromatography–tandem mass spectrometry. IP: immunopurification.

Figure 2

Functional enrichment analysis of ubiquitinated root microsomal proteins. The percentage is calculated with regard to the number of identified ubiquitinated proteins (black) and the total number of Arabidopsis proteins (white). Numbers indicate the fold enrichment by comparison with the Arabidopsis genome. Underlined biological processes concern proteins involved in intracellular trafficking and include 15 genes (Table S3).

Figure 3

Motif analysis of identified K-Ub residues in root microsomes. (A) Ubiquitination motifs and the conservation of K-Ub residues. The height of each letter corresponds to the frequency of the amino acid residue in that position. The central K refers to the K-Ub residue. (B) The number of identified peptides containing a K-Ub residue in each motif. (C) The number of K-Ub sites per protein.

Figure 4

Relative abundance of PIP2 isoforms in Arabidopsis suspension cells overexpressing PIP2;1 WT and carrying a point mutation of K3 in alanine (K3A) and in arginine (K3R). ELISA assays were performed with total protein extracts and anti-PIP2 antibody [19]. The number of independent stable cell lines is indicated. Data were from three individual ELISA assays per cell line. Standard error is shown.

Figure 5

The interaction network of ubiquitinated proteins. Interactants from a Y2H approach [15] and Split-Ub approach [16,20] were considered, and the network was visualized by Cytoscape (version 3.7.2). (A) The network includes ubiquitinated proteins (blue) together with their reported interactants (orange) (details in Table S7a). (B) The UBC32 subnetwork (details in Table S7b). (C) The UBC34 subnetwork (details in Table S7c).

Figure 6

Inhibition of primary root length by mannitol in Arabidopsis thaliana WT plants (Col) and ubc32, ubc33, ubc34, and ubc32xub33xubc34 mutants. Fifteen plants per condition were grown for 5 days in MS medium and then transferred to MS medium and MS medium supplemented with 200 mM mannitol. Primary root length was monitored up to 5 days after transfer (Figure S9). Asterisks (*) mean that the WT is significantly different from at least one mutant in a one-way ANOVA test combined with a Tukey test (p-value between 0.01 and 0.001 (statistics in Table S8)).