pex5 Mutants that differentially disrupt PTS1 and PTS2 peroxisomal matrix protein import in Arabidopsis

Abstract

PEX5 and PEX7 are receptors required for the import of peroxisome-bound proteins containing one of two peroxisomal targeting signals (PTS1 or PTS2). To better understand the role of PEX5 in plant peroxisomal import, we characterized the Arabidopsis (Arabidopsis thaliana) pex5-10 mutant, which has a T-DNA insertion in exon 5 of the PEX5 gene. Sequencing results revealed that exon 5, along with the T-DNA, is removed in this mutant, resulting in a truncated pex5 protein. The pex5-10 mutant has germination defects and is completely dependent on exogenous Suc for early seedling establishment, based on poor utilization of seed-storage fatty acids. This mutant also has delayed development and reduced fertility, although adult pex5-10 plants appear normal. Peroxisomal metabolism of indole-3-butyric acid, propionate, and isobutyrate also is disrupted. The pex5-10 mutant has reduced import of both PTS1 and PTS2 proteins, and enzymatic processes that occur in peroxisomes are disrupted. To specifically study the import and importance of PTS1 proteins, we made a truncated PEX5 construct lacking the PTS1-binding region (PEX5(454)). Transformation of this construct into pex5-10 resulted in the rescue of PTS2 import, thereby creating a line with PTS1-specific import defects. The pex5-10 (PEX5(454)) plants still had developmental defects, although restoring PTS2 import resulted in a less severe mutant phenotype. Comparison of pex5-10 and pex5-10 (PEX5(454)) phenotypes can separate the import mechanisms for enzymes acting in different peroxisomal processes, including indole-3-butyric acid/2,4-dichlorophenoxybutyric acid oxidation, isobutyrate and propionate metabolism, and photorespiration.

Figure 1.

PEX5 gene expression and protein concentration. A, Cartoon representation of the PEX5 protein, the pex5 mutant proteins, and the PEX5₄₅₄ truncation. The pex5-1 Ser-to-Leu point mutation (Zolman et al., 2000) and the pex5-10 insertion site and resulting deletion are indicated by a star and a triangle, respectively. Gray circles indicate the PEX7-binding domain (PEX7), black ovals indicate the TPR domains, and rectangles represent the PPR domains. The goalpost under the protein indicates the recognition site of the PEX5 antibody (Zolman and Bartel, 2004). B, Total protein was extracted from wild-type Col-0 seedlings and adult plants and analyzed by western blot for PEX5 levels. C, Total protein from 5-d-old wild-type seedlings grown in the dark or light in the presence or absence of Suc was extracted and analyzed by western blot for PEX5 levels. For both B and C, equal amounts of protein were loaded, as confirmed by immunoblotting with an HSC70 antibody (bottom). D, Levels of PEX5 mRNA expression (absolute values) in dry and imbibed seeds, cotyledons, hypocotyls, cauline leaves, rosette leaves, mature pollen, seeds/siliques, carpels, petals, and roots. The graph was constructed using Arabidopsis eFP Browser data from http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi (Winter et al., 2007). Data were retrieved on March 6, 2010.

Figure 2.

T-DNA insertion effects in the pex5-10 mutant. A, Total protein extracted from leaves and roots of 12-d-old wild-type (Wt) and pex5-10 seedlings was analyzed by western blot with a PEX5 antibody. B, Total protein from 3-week-old wild-type and pex5-10 plants was analyzed by western blot for PEX5 levels. Seedlings were taken from the heterozygous progeny of a pex5-10 backcross to Col-0 (F₁c′′) and pex5-10 homozygous mutant plants over four generations (F2 through F6) and three backcrosses (F₄c for the first backcross; F₅c′ and F₆c′ for the second backcross; F₂c′′ and F₃c′′ for the third backcross). For all blots, equal amounts of protein were loaded, as confirmed by immunoblotting with an HSC70 antibody (bottom). C, To examine the effects of the insertion in exon 5, cDNAs from 3-week-old wild-type (W) and pex-10 (M) plants were amplified with PEX5 primers that directly surround (exons 5 + 6; expected cDNA size 340 bp/expected genomic size 774 bp) or span (exons 3 + 7; 780 bp/2,234 bp) the T-DNA. Genomic DNA (G) from wild-type plants was amplified to show the purity of cDNA synthesis. D, Cartoon representation of the PEX5 and pex5-10 genomic structures. Exon numbers are labeled. The pex5-10 insertion is shown by the triangle above the sequence, and the splice transition from the end of exon 4 to the beginning of exon 6 is indicated.

Figure 3.

Peroxisomal import defects in pex5 mutant lines. A, Localization of peroxisomally targeted GFP reporters with either PTS1 (Zolman and Bartel, 2004) or PTS2 (Woodward and Bartel, 2005) sequence. Wild-type (Wt) lines (Col-0 background) containing these constructs were crossed into the pex5-10 or pex5-10 (PEX5₄₅₄) background. Root hair cells for each line are shown, with all images taken at the same magnification. B, Total protein from wild-type and three independent transgenic Col-0 (PEX5₄₅₄) plants were analyzed by western blot for PEX5₄₅₄ accumulation using a myc antibody. C, Western blot of total protein from wild-type, pex5-10 (PEX5), pex5-1, pex5-1 (PEX5₄₅₄), pex5-10, and pex5-10 (PEX5₄₅₄) with PEX5, myc, and thiolase antibodies. Thiolase contains a PTS2 that is cleaved following import; protein size indicates if the protein is localized in the peroxisome (mature short form; two asterisks) or if it remains in the cytoplasm (unprocessed long form; asterisk). Four independent pex5-10 (PEX5₄₅₄) lines (p126, p122, p109, and p111) are shown to indicate the range of responses. For all blots, equal amounts of protein were loaded, as confirmed by immunoblotting with an HSC70 antibody. D, Extracts from 4-d-old Col-0 (wild-type), pex5-1, pex5-1 (PEX5₄₅₄), pex5-10, and pex5-10 (PEX5₄₅₄) seedlings were tested for acyl-CoA oxidase activity on n-hexanoyl-CoA (C6:0) and lauroyl-CoA (C12:0). Data are presented as pmol H₂O₂ produced per min. Error bars represent se of the rates from three independent experiments. The control samples used water in place of plant extracts to account for background readings.

Figure 4.

Fatty acid metabolism in pex5 mutant seedlings. A, Hypocotyl length of seedlings grown for 24 h in light and 6 d in dark on medium supplemented with increasing concentrations of Suc. Error bars represent se (n ≥ 12). B, Hypocotyl length of seedlings grown for 24 h in light and 6 d in dark on medium without Suc or with 0.5% Suc. Error bars represent se (n ≥ 12). C, Hypocotyl length of seedlings grown for 24 h in light and 6 d in dark on medium without Suc or with 0.5% Suc. pex5-10 seed coats were nicked immediately after plating. Error bars represent se (n ≥ 12). D, Photographs depicting pex5-10 plant lines. Plants were grown on complete medium for 7 d. pex5-10 seed coats were nicked after plating. E, Percentage of C20:1 fatty acid (versus total fatty acids) in 6-d-old seedlings grown in light on 0.5% Suc medium. Error bars represent se of three biological replicates. Wt, Wild type.

Figure 5.

pex5 mutant responses to IBA or the IBA analog 2,4-DB. A to C, Root length of 10-d-old seedlings on medium supplemented with IBA. Error bars represent se (n ≥ 12). D, Photographs of 11-d-old wild-type (Wt), pex5-10, and pex5-10 (PEX5) seedlings grown without hormone or on 10 μm IBA. E, Root length of 10-d-old seedlings grown under continuous light on medium supplemented with 2,4-DB. Error bars represent se (n ≥ 12).

Figure 6.

pex5 lateral root formation. A, Lateral root density of seedlings grown without hormone for 5 d and then transferred to medium with or without hormone for another 5 d. Data are shown as the number of lateral roots per mm root length. Error bars represent se (n ≥ 12). B, DR5-GUS expression in 8-d-old Col-0 and pex5-10 transgenic seedlings grown on complete medium with 0.5% Suc. Wt, Wild type.

Figure 7.

pex5 mutant responses to IAA, 2,4-D, and NAA. Root length is shown for 10-d-old seedlings grown on medium with IAA (A and C), 2,4-D (B), or NAA (C). The auxin transport mutant aux1 (Pickett et al., 1990) was used as a control to demonstrate insensitivity to IAA. Error bars represent se (n ≥ 12). Wt, Wild type.

Figure 8.

pex5-10 fertility, photorespiration, and H₂O₂ inactivation defects. A, Length of the fifth silique on the main stem. Error bars represent se (n ≥ 18). B, Average seed weight in mg per 100 seeds. Error bars represent se (n ≥ 4). C, Root length of 10-d-old seedlings grown on medium containing propionate or isobutyrate. Error bars represent se (n ≥ 12). D, Total chlorophyll content in mg per g fresh weight for 8-d-old seedlings and 4-week-old leaves. Error bars represent se (n ≥ 6). E, DAB staining showing the accumulation of H₂O₂ in 2-week-old leaves. Wt, Wild type.

Figure 9.

pex5-10 growth and development. A and B, Wild-type (Wt), pex5-10 (PEX5), and pex5-10 plants were grown under continuous white light at 22°C with normal watering two times per week. Plants were measured for rosette diameter (A) and plant height (B) on the indicated days. Error bars represent se (n ≥ 18). C, Photographs of 3-week-old soil-grown wild-type and pex5-10 plants.