Global impacts of peroxisome and pexophagy dysfunction revealed through multi-omics analyses of lon2 and atg2 mutants

Abstract

Peroxisomes house diverse metabolic pathways that are essential for plant and animal survival, including enzymes that produce or inactivate toxic byproducts. Despite the importance of peroxisomes and their collaborations with other organelles, the mechanisms that trigger or prevent peroxisome turnover and the cellular impacts of impaired peroxisomes are incompletely understood. When Arabidopsis thaliana LON2, a peroxisomal protein with chaperone and protease capacity, is disrupted, metabolic dysfunction and protein instability in peroxisomes ensue. Paradoxically, preventing autophagy in lon2 mutants appears to normalize peroxisomal metabolism and stabilize peroxisomal proteins-hinting at a role for autophagy in causing the peroxisomal defects observed in lon2 seedlings. Using a combination of transcriptomics, proteomics, and in silico investigations, we compared wild type to lon2 and autophagy null mutants and double mutants. Through this analysis, we found that impeding autophagy via an atg2 null mutation alleviated several of the global defects observed when LON2 is absent. Moreover, we revealed processes influenced by LON2 that are independent of autophagy, including impacts on lipid droplet and chloroplast protein levels. Finally, we identified and classified potential LON2 substrates, which include proteins that might provide signal(s) for pexophagy.

Keywords: Arabidopsis thaliana; ATG2; LON proteases; LON2; autophagy; peroxisomal homeostasis; pexophagy.

Figure 1

lon2‐8 decreased peroxisome function is alleviated when autophagy is disrupted by the atg2‐4 mutation. (a) lon2‐8 seedlings are slightly smaller than wild‐type seedlings. Seedlings of the indicated genotypes were grown for 6 days in constant light before being moved to a new plate and photographed. The range of seedling sizes are represented by the selected seedlings, including the largest (left) and smallest (right) germinated seedlings observed. (b) LON2 is not detected in lon2‐8 or lon2‐8 atg2‐4, and the PTS2‐processing defect of lon2‐8 is alleviated in lon2‐8 atg2‐4. 6‐day‐old light‐grown seedlings were processed for immunoblotting with the indicated antibodies. NBR1 is a selective autophagy receptor that accumulates in autophagy mutants. Mature PMDH is synthesized as a precursor; its targeting sequence is removed after peroxisomal import. HSC70 is a cytosolic loading control. (c) The decreased IBA responsiveness of lon2‐8 is recovered in lon2‐8 atg2‐4. Lateral root density of 8‐day‐old wild type (Wt), atg2‐4, lon2‐8, and lon2‐8 atg2‐4 seedlings grown without (mock) or with 10 μM IBA is shown. Error bars show standard deviations (n = 3 biological replicates with 8–12 seedlings each). One‐way ANOVA with Tukey's post‐hoc test was separately performed on mock and IBA. Data not sharing a letter (uppercase for mock and lowercase for IBA) above the bar are significantly different. (d, e) lon2‐8 displays growth defects. Seedlings germinated on agar‐based medium were transferred to soil after 7 days and 21‐day‐old plants were photographed (d) and rosette diameters were measured (e). Data not sharing a letter above the bar significantly differ according to a one‐way ANOVA with Tukey's post‐hoc test.

Figure 2

Differentially expressed genes (DEGs) and differentially accumulated proteins (DAPs) in atg2‐4, lon2‐8, and lon2‐8 atg2‐4. (a–d) Volcano plots of detected transcripts using a q < 0.01 (a–c) or <0.05 (d) significance cutoff (horizontal dashed lines). (e–h) Volcano plots of detected proteins using a P < 0.1 significance cutoff. Additional DAPs that were detected in at least two replicates of one genotype and not detected in all three replicates of the comparison genotype are indicated, but do not have a P‐value and are not represented as points on the volcano plots. Purple dots indicate known peroxisomal (Araperox; Reumann et al., 2004) genes. Points above y‐axis breaks indicate P or q‐value approaching zero. DEGs and DAPs are listed in Data S1, S2.

Figure 3

Gene ontology (GO) analysis of transcripts and proteins altered in atg2‐4, lon2‐8, or lon2‐8 atg2‐4. (a) Selected significant GO biological processes identified from DEGs of atg2‐4, lon2‐8, and lon2‐8 atg2‐4 compared to Wt, and lon2‐8 atg2‐4 compared to atg2‐4. GO terms with >30% associated genes altered in at least one genotype comparison are separated into functional groups. The full list of significant GO terms for the transcriptome analysis is in Data S5A–H. (b) Significant GO biological processes identified from DAPs of atg2‐4, lon2‐8, and lon2‐8 atg2‐4 compared to Wt (Data S6A–F). No GO terms were significantly enriched in the lon2‐8 atg2‐4 versus atg2‐4 comparison. The size of the circle reflects the percentage of genes with the corresponding GO annotation that are impacted, and the intensity of shading reflects the significance of the enrichment. Blue and purple text highlight GO terms that are altered in both the transcriptome and proteome.

Figure 4

Comparative analysis of DEGs and DAPs. (a–d) Scatter plots showing mRNA and protein log₂ fold‐change relationships for genes with coverage in both transcript and protein datasets. atg2‐4 (a), lon2‐8 (b), and lon2‐8 atg2‐4 (c), were compared to wild type, and lon2‐8 atg2‐4 was compared to atg2‐4 (d). Blue indicates significant mRNA changes (q < 0.01 in panels a–c; q < 0.05 in panel d) with unchanged protein levels. Orange indicates significant protein changes (P < 0.1) with unchanged mRNA levels. Green indicates significance in both mRNA and protein changes. Gray indicates protein/mRNA values without significant changes. The number of significant mRNA/protein pairs is shown in green in each sector, and significant RNA‐ (blue) or protein‐only (orange) changes are seen at the end of each dashed line. Points beyond broken x‐axes indicate DAPs that were detected in at least two replicates of one genotype and not detected in all three replicates of the comparison genotype (true zeros). The solid black line shows the Pearson correlation (Corr) and fit (R ²) within each comparison (excluding the true zero points). (e–h) Overlapping DEGs and DAPs (Data S7A–D) of atg2‐4 (e), lon2‐8 (f), lon2‐8 atg2‐4 (g) compared to Wt and lon2‐8 atg2‐4 compared to atg2‐4 (h) were categorized based on predicted subcellular localization using SUBA5 software (Data S8A–D). Additional localization data for peroxisomes (Araperox; Reumann et al., 2004) and lipid droplets (Gene Ontology lipid droplet query) were also documented. Shades of brown indicate proteins that accumulated with or without changes to the mRNA, and shades of blue indicate proteins that were depleted with or without changes to the mRNA. The number of mRNA/protein pairs in each category is indicated within the bar, and the total is indicated to the right.

Figure 5

Gene ontology (GO) analysis of overlapping DEGs and DAPs. (a–f) Significant GO biological processes identified from DEG‐DAP pairs of atg2‐4, lon2‐8, and lon2‐8 atg2‐4 compared to Wt, and lon2‐8 atg2‐4 compared to atg2‐4 (Data S9). The size of the circle reflects the percentage of genes with the corresponding GO annotation that are impacted, and the intensity of color reflects the significance of the enrichment.

Figure 6

Potential LON2 substrates include enzymes involved in β‐oxidation, the glyoxylate cycle, photorespiration, and ROS scavenging. (a) Volcano plots of significantly altered proteins in atg2‐4, lon2‐8, and lon2‐8 atg2‐4 with peroxisomal localization predicted by SUBA5 (https://suba.live/index.html). Blue and red numbers indicate the number of less or more abundant proteins, respectively, including proteins (not shown) that were absent in one genotype. Selected DAPs (dark purple spots) are named; asterisks indicate proteins analyzed by immunoblotting in panel (c). DAPs and predicted localizations are listed in Data S4A–D. (b) Scatter plot showing mRNA and protein log₂ fold‐change relationships of known and predicted peroxisomal proteins in lon2‐8 atg2‐4 compared to atg2‐4. Blue indicates significant mRNA changes (q < 0.05) and unchanged protein levels. Orange indicates significant protein changes (P < 0.1) and unchanged mRNA levels. Green indicates significance in both mRNA and protein changes. Gray indicates protein and mRNA values without significant changes. Points beyond broken x‐axes were detected in at least two replicates of one genotype and not detected in any replicates of the comparison genotype. Accumulated proteins (to the right of the vertical dashed line) include potential LON2 protease substrates, whereas depleted proteins (to the left of the vertical dashed line) may include potential LON2 chaperone substrates. (c) Immunoblots of 6‐day‐old light‐grown seedling extracts probed with antibodies recognizing peroxisomal proteins marked by asterisks in panels (a) and (b). Four replicate membranes were prepared and probed, and the corresponding HSC70 cytosolic loading control is shown for each membrane above the other proteins probed. MLS/CAT, ICL/HPR/PEX11E, and ACX4/CSY3 were serially probed on the same membrane. The CSY2 antibody was probed on the membrane that was used for LON2 in Figure 1b, and the HSC70 panel is duplicated here for clarity. The anti‐cottonseed catalase antibody may recognize any or all of the three CAT isozymes accumulating in panels (a) and (b), and the PEX11E antibody is expected to recognize PEX11C, PEX11D, and PEX11E. Numbers below bands indicate levels of protein of interest divided by the HSC70 signal relative to the level in Wt.