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. 2017 Feb 3;8:107.
doi: 10.3389/fpls.2017.00107. eCollection 2017.

Proteasome Activity Profiling Uncovers Alteration of Catalytic β2 and β5 Subunits of the Stress-Induced Proteasome During Salinity Stress in Tomato Roots

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Free PMC article

Proteasome Activity Profiling Uncovers Alteration of Catalytic β2 and β5 Subunits of the Stress-Induced Proteasome During Salinity Stress in Tomato Roots

Judit Kovács et al. Front Plant Sci. .
Free PMC article

Abstract

The stress proteasome in the animal kingdom facilitates faster conversion of oxidized proteins during stress conditions by incorporating different catalytic β subunits. Plants deal with similar kind of stresses and also carry multiple paralogous genes encoding for each of the three catalytic β subunits. Here, we investigated the existence of stress proteasomes upon abiotic stress (salt stress) in tomato roots. In contrast to Arabidopsis thaliana, tomato has a simplified proteasome gene set with single genes encoding each β subunit except for two genes encoding β2. Using proteasome activity profiling on tomato roots during salt stress, we discovered a transient modification of the catalytic subunits of the proteasome coinciding with a loss of cell viability. This stress-induced active proteasome disappears at later time points and coincides with the need to degrade oxidized proteins during salt stress. Subunit-selective proteasome probes and MS analysis of fluorescent 2D gels demonstrated that the detected stress-induced proteasome is not caused by an altered composition of subunits in active proteasomes, but involves an increased molecular weight of both labeled β2 and β5 subunits, and an additional acidic pI shift for labeled β5, whilst labeled β1 remains mostly unchanged. Treatment with phosphatase or glycosidases did not affect the migration pattern. This stress-induced proteasome may play an important role in PCD during abiotic stress.

Keywords: 20S proteasome; activity-based protein profiling; catalytic subunit; immune proteasome; programmed cell death; salt stress; tomato root.

Figures

FIGURE 1
FIGURE 1
Phylogeny and variation of beta proteasome subunits of tomato. (A) Phylogenetic tree of beta subunit genes of tomato and Arabidopsis. Neighbor-joining tree of protein sequences was build using ClustalW2. (B) Summary of the variant amino acid residues that differ between β2a and β2b. e, putative solvent-exposed. Significant variation is printed in bold. (C) Location of variant residues in β2, modeled on the yeast proteasome. The tomato β2a protein was modeled using the β2 of yeast (2zcy) as a template. Residues that differ between β2a and β2b are highlighted in red in the topview (top) and sideview (bottom) of the β-ring of the proteasome and summarized in the table. The proteolytic chamber is highlighted with a dashed orange line and catalytic sites are indicated with orange arrows. (D) Transcript levels of β subunit-encoding genes in various tomato organs. These data were extracted from The Tomato Genome Consortium (2012). Reads per kilobase of transcripts per million mapped reads (RPKM) values were extracted from the database for each gene.
FIGURE 2
FIGURE 2
Salt treatment induces loss of viability in tomato roots. (A) Experimental assay. Tomato (Solanum lycopersicum) plants were grown in a hydroponic system and 5-weeks old plants were treated with 0-, 100- and 250 mM NaCl in the nutrient solution. Root tips were collected at 1, 6, and 24 h. (B) Loss of viability upon salt stress. Root tips were stained with fluorescein diacetate (FDA) to detect the viable cells. Top: representative images are shown. Scale bar, 0.5 mm. Bottom: fluorescent intensities of FDA fluorescence levels, when compared to the control. Error bars represent SEM of n = 3 biological replicates.
FIGURE 3
FIGURE 3
MV151 activity profile changes upon salt stress in roots. (A) Differential activity profiles with MV151. Tomato roots were treated with 0-, 100-, 250 mM NaCl. Root extracts were generated after 1-, 6- and 24 h and labeled with 2 μM MV151 at pH 6.0. A mix of all nine samples was pre-incubated with or without 50 μM E-64 and labeled with 2 μM MV151. Shown is a representative gel at long and short fluorescence exposure and upon coomassie staining. The other two experimental replicates are shown as Supplementary Figure S2. (B) Quantification of the upper differential MV151 signal (arrowhead) taken from three experimental replicates (A) (Supplementary Figure S2). Error bars represent SEM of n = 3 experimental replicates. (C) Differential signal is suppressed by proteasome inhibitor. The 6 h 0- and 250 mM NaCl treated samples were labeled with 2 μM MV151. A mix of the two samples was pre-incubated with or without 50 μM E-64 or epoxomicin and labeled with or without 2 μM MV151.
FIGURE 4
FIGURE 4
Proteasome activity profile changes upon salt treatment. (A) Tomato roots were treated with 0- and 250 mM NaCl and root extracts were generated after 6 h and pre-incubated with or without 100 μM epoxomicin and labeled with or without 0.2 μM MVB072. (B) Quantification of bands I–III indicated in (A). Error bars represent SEM of n = 3 experimental replicates.
FIGURE 5
FIGURE 5
Two-dimensional gels show molecular weight (MW) and pI shifts for labeled catalytic proteasome subunits. (A) Tomato roots were treated with 0- and 250 mM NaCl and root extracts were generated after 6 h and labeled with 0.2 μM MVB072. Samples were separated on IEF 2D gel. Spots are highlighted with different colors: β1 (green); β2 (blue); and β5 (red). Framed sections focus on β2 (blue) and β5 (red) catalytic subunits. (B) Quantification of the fluorescence signals of (A). Error bars represent SEM of n = 3 experimental replicates. (C) Schematic figures of 1D and 2D gel illustrating the effect of high salinity on the intensity of signals of β2 and β5 catalytic subunits. Closed and open spots indicate up- and down-regulated signals, respectively. (D) Ranking of detected catalytic subunits based on Mascot protein scores. (E) Assignment of catalytic subunits to some of the fluorescent spots, based on the detected proteins and their scores and ranking.
FIGURE 6
FIGURE 6
Subunit-specific labeling confirms modification of β5. (A) Tomato roots were treated with 0- and 250 mM NaCl and root extracts were generated after 6 h and pre-incubated with or without subunit-specific inhibitors, N3β1 and N3β5 and co- labeled with or without subunit-selective probes, LW124 (β1) and MVB127 (β5). Samples were separated on 1D gel. Differential signals are indicated by black arrowheads. (B) Tomato roots were treated with 0- and 250 mM NaCl and root extracts were generated after 6 h and co-labeled with LW124 (β1) and MVB127 (β5) and separated on IEF 2D gel. Differential signals are indicated by black arrowheads.
FIGURE 7
FIGURE 7
Phosphatase and glycosidase treatments do not affect altered proteasome activity profile. (A) Root extracts of 250 mM treated samples, labeled by MVB072 were treated with alkaline phosphatase at different conditions. (B) Dephosphorylation of MAP kinase, used as a positive control, detected by an anti-phosphoMAPK antibody. (C) Root extracts of 250 mM treated-samples, labeled by MVB072 were treated with and without PNGase F or deglycosylation mix. (D) Enzymatic deglycosylation of Bovine Fetuin was used as a positive control.

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