Analysis of 26S Proteasome Activity across Arabidopsis Tissues

Abstract

Plants utilize the ubiquitin proteasome system (UPS) to orchestrate numerous essential cellular processes, including the rapid responses required to cope with abiotic and biotic stresses. The 26S proteasome serves as the central catalytic component of the UPS that allows for the proteolytic degradation of ubiquitin-conjugated proteins in a highly specific manner. Despite the increasing number of studies employing cell-free degradation assays to dissect the pathways and target substrates of the UPS, the precise extraction methods of highly potent tissues remain unexplored. Here, we utilize a fluorogenic reporting assay using two extraction methods to survey proteasomal activity in different Arabidopsis thaliana tissues. This study provides new insights into the enrichment of activity and varied presence of proteasomes in specific plant tissues.

Keywords: 26S proteasome; cell-free system; fluorogenic probes; methods; plant proteolysis; protein degradation; proteolytic activity; ubiquitin system.

Figure 1

Schematic overview the 26S proteasome activity analysis across Arabidopsis tissues performed in this study. (a) Schematic representation of the proteasome activity assay using LLVY-AMC and the tissue sample preparation from Arabidopsis thaliana mature plant and seedlings using two different lysis strategies that were performed in this study. Image was created with BioRender.com. (b) Upper panel, heatmap comparison of several subunit genes from the 19S regulatory particle and 20S core particle in the seedlings, flower, stem, leaf, and root tissues of Arabidopsis thaliana. Data was obtained using the Klepikova Atlas from the Bio-Analytic Resource for Plant Biology and analyzed in R-Studio. Seedling expression combined and averaged the data from the seedling meristem, cotyledons, hypocotyl, and root. The highest levels of expression in the roots and flowers are shown in a darker red color. Lower panel, architecture of plant proteasome from Spinacia oleracea (PDB: 7QVG and 7QVE) [29]. Graphical representation of the proteasome (shown as surface representation, 19S RP colored raspberry, 20S CP colored in light blue) was illustrated and analyzed via PyMOL-2.5.4. TAIR accession for the 19S RP presented are: RPN7 (At4g24820), RPN10 (At4g38630), RPN12a (At1g64520), RPT1a (At1g53750), RPT3 (At5g58290), and RPT5a (At3g05530). TAIR accession for the 20S CP presented are: PAC1 (At3g22110), PAD1 (At3g51260), PAF1 (At5g42790), PBC1 (At1g21720), PBE1 (At1g13060), and PBF1 (At3g60820).

Figure 2

Proteasomal activity in different Arabidopsis tissues. (a) The dissected tissues used in the proteasomal activity assays were extracted and resolved via SDS-PAGE followed by Coomassie brilliant blue staining. Arabidopsis thaliana plant was drawn using Notability. (b,c) Proteasomal activity was measured in the presence and absence of proteasome inhibitor, MG132, for the different lysates 1 (L1, (b)) and 2 (L2, (c)) using the different tissues. (d) Comparative representation (L1 vs. L2) of the proteasomal activity-based assays shown in (b,c). Proteasome activity was plotted after subtracting the RFU value observed in the reactions with MG132 from the reactions without MG132. The fluorescence signal emitted by AMC cleavage was visualized and measured via a microplate reader. The relative fluorescence unit (RFU) value was measured via Gen5 3.08 software, averaged using Microsoft excel, and plotted using Graphpad Prism 10.0.1. All experiments were repeated at least three times, and the plotted data show the mean of three technical replicates. Error bars represent standard deviation (SD).