PMC

Model for PXA1 Activity in Plant Cells.
PXA1 is required for uptake of from (and other cellular sources) for β-oxidation, import of for conversion to the auxin hormone , and import of into peroxisomes for conversion to . GL-18:3, galactolipid containing 18:3. See text for additional details.

Evaluation of the Signaling Pathway in Wild-Type and cgi-58, pxa1, and cgi-58 pxa1 Mutant Arabidopsis Plants in Response to Wounding.
(A) Plants were mechanically wounded and then amounts of (left graph) and (right graph) were measured over time. FW, fresh weight; WT, the wild type.
(B) Changes in CGI-58 and AOS expression as measured by . The plant genotype is shown at the bottom, and specific transcripts quantified are shown above. a = P < 0.05 between the wild type and pxa1; b = P < 0.05 between cgi-58 and pxa1; c = P < 0.05 between the wild type and cgi-58 pxa1; d = P < 0.05 between cgi-58 and cgi-58 pxa1.
Values in (A) and (B) represent averages and sd (n = 4).

Analysis of and Composition in Leaves of Wild-Type and Mutant Arabidopsis Plants.
(A) Representative confocal fluorescence micrographs of mesophyll cells of mature (i.e., 35 d old) leaves from wild-type (WT) and cgi-58, pxa1 and cgi-58 pxa1 mutant plants. Chloroplast autofluorescence is red and (stained with BODIPY 493/503) are green. Quantification revealed that the average numbers of were approximately six- to sevenfold higher in all mutant cells compared with the wild-type, but the values were not statistically different between mutants. Bars = 10 μm.
(B) Compositional analysis of extracted from leaves of wild-type and cgi-58, pxa1, and cgi-58 pxa1 mutant 35-d-old plants. Values represent averages and sd of five biological replicates.

Measurement of Seedling Establishment and Seed Oil Breakdown in Wild-Type and Mutant Arabidopsis Plants.
(A) Seeds of the indicated plant lines were germinated on plant nutrient medium lacking or containing 0.5% Suc and then grown under constant light for 7 d. Note the inability of pxa1 to grow in the absence of Suc, while cgi-58 mutants are similar to the wild type (WT).
(B) Measurement of total content in germinating seedlings, illustrating that CGI-58 is not required for the breakdown of derived from storage oil. Values represent averages and sd (n = 4). FW, fresh weight.

Measurement of Auxin Responses in Wild-Type and cgi-58, pxa1, and cgi-58 pxa1 Mutant Arabidopsis Plants.
Seeds were germinated in media lacking hormones, transferred to plates lacking or containing the indicated amounts of or , and then grown under constant light for 8 d before root length was measured.
(A) Representative images of wild-type (WT) and cgi-58 plants showing long root growth for each plant type on media lacking auxins (Control) but significantly shorter growth of wild-type plants when grown in the presence of or . Note that cgi-58 plants are resistant to the effects of in comparison to the wild type.
(B) Dose–response curves showing resistance of various plant lines to (left graph) and similar sensitivities to (right graph). Values represent averages and sd (n > 15), and significant differences (indicated by asterisks; P < 0.05) are relative to the wild type.

Localization of CGI-58 to the Cytosol and Peroxisomes in Tobacco Leaf Cells.
Shown are representative confocal micrographs of tobacco epidermal cells transiently cotransformed with GFP-CGI-58 and the peroxisomal matrix marker protein Cherry-PTS1 (consisting of the Cherry fluorescent protein linked to a type 1 peroxisomal targeting signal) at either 2 or 5 d after Agrobacterium coinfiltration. Arrowheads illustrate the localization of GFP-CGI-58 to the periphery of Cherry-PTS1–labeled peroxisomes. The bottom row of images shows a portion of the cell (dashed boxes in middle row) 5 d after Agrobacterium coinfiltration, shown at higher magnification. Shown also in the top two rows of images are the differential interference contrast (DIC) images of each cell to help delineate cell borders. Note that the majority of cell volume is occupied by the vacuole(s), and as such, the cytoplasm is mostly appressed into to a narrow region near the cell surface. Bar = 15 μm.

Interaction of CGI-58 with Various Regions of PXA1.
(A) Cartoon of PXA1 structure and topology showing peroxisomal membrane-associated regions and two , each composed of Walker A and B motifs.
(B) Summary of yeast two-hybrid (Y2H; [C]) and nuclear relocalization (NRA; [E]) assays, where CGI-58 was tested for its ability to interact with specific regions (or modified versions thereof) of PXA1 (constructs 1 to 8, numbered on the left). n.s., results of the NRA were similar to those of yeast two-hybrid, but data are “not shown.”
(C) Yeast two-hybrid assay showing the relative growth of yeast strains on media for either plasmid selection only (left panel) or high stringency conditions that are dependent on two-hybrid protein interactions (right panel). Numbers on the left correspond to PXA1-based constructs described in (B). The dilution series was prepared by first adjusting the yeast cell culture density to 0.5 OD₆₀₀, then plating, from left to right, 5 μL of a 1:5 dilution series on the plates, with the 0.5 OD₆₀₀ culture being the left-most spot.
(D) Coimmunoprecipitation of CGI-58 and PXA1. Whole-cell lysates from Escherichia coli expressing S-tagged CGI-58 or an empty vector were incubated with in vitro–synthesized full-length HA-tagged PXA1 and complexes were immunoprecipitated using anti-S-tag antibodies. PXA1-HA was detected by immunoblotting using anti-HA antibodies. The position of a molecular mass marker (117 kD) is shown to the left. A Coomassie blue–stained gel of lysates used to program the reactions, including an immunoblot of S-tagged CGI-58, is shown in Supplemental Figure 3 online.
(E) Nuclear relocalization results, whereby each row of epifluorescence micrographs shows a representative plant cell that is transiently coexpressing NLS-RFP alone (top row) or NLS-RFP fused to a portion of PXA1 (numbers correspond to PXA1-based constructs described in [B]) and GFP-CGI-58. Protein–protein interactions were scored based on recruitment of a portion of GFP-CGI-58 to the nucleus (white arrowheads). Bar = 10 μm.