The Peroxisome Deficient Arabidopsis Mutant sse1 Exhibits Impaired Fatty Acid Synthesis

GFP-SSE1 overexpression causes partially fused peroxisomal aggregates. A, Typical peroxisomes in the cotyledon of a wild-type seedling. Bar is 500 nm. B, A cotyledon cell in a seedling from GFP-SSE1 transgenic line 3. A peroxisomal aggregate is present. Bar is 2 μm. C, Enlargement of the peroxisomal aggregate in B. Peroxisomes are fused to each other at some areas. Bar is 500 nm. D, A peroxisomal aggregate in a mesophyll cell from a true leaf of GFP-SSE1 transgenic line 3. Peroxisomes are fused to each other in some areas. Bar is 1 μm. P, peroxisome; PA, peroxisomal aggregate; OB, oil body; C, chloroplast; M, mitochondrion.

RFP-labeled peroxisomes in wild-type (or sse1/+ heterozygous) and sse1 embryos revealed by fluorescence microscopy. A, Heterozygous (sse1/+) and sse1 mutant (sse1/sse1) embryos from an RFP-PTS1 transgenic line. Bright field images of the embryos are also shown. Bar is 150 μm. B, Heterozygous (sse1/+) and sse1 mutant (sse1/sse1) embryos from a PTS2-RFP transgenic line. Bar is 40 μm. C, RFP-PTS1 labeled peroxisomes in mature wild-type embryos. Bar is 40 μm.

Semiquantitative RT-PCR analyses of gene expression. RNA was isolated from flower buds (F), 7- to 21-DAF seeds (SEED), 2- and 4-d-old seedlings (SDL), and emerging leaves (EL). The same cDNA mixture was used to amplify different fragments through 30 to 40 cycle PCR reactions. PCR without cDNA templates was conducted simultaneously as a negative control (−). The PEX16 gene is SSE1. LEC1, Leafy Cotyledon 1; ACT2, Actin 2.

sse1 embryos exhibit reduced rates of fatty acid synthesis. A, Time course of starch and oil accumulation in wild-type (black circles) and developmentally advanced sse1 (white circles) embryos dissected from seed coats. The time course is determined from late torpedo and early cotyledon stage (9 DAF) until the mature embryo stage (22 DAF). Developmentally delayed sse1 embryos were excluded from the experiment. For each time point, 15 to 40 and 10 to 30 embryos were pooled for starch and oil determination, respectively. Each starch extract was measured twice and the error is less than 10% for values greater than or equal to 0.1 μg embryo⁻¹ (except sse1 at 9 DAF where it was 0.25 ± 0.11 μg embryo⁻¹). Each FAME extract was measured once. B, Starch and oil contents in the sse1adg1 and sse1pgm1 double mutant seeds. The original sse1 single mutant is in the C24 background (sse1-C24). The adg1 and pgm1 single mutants are in the Col background (adg1-Col and pgm1-Col). The sse1adg1 and sse1pgm1 double mutants are in the C24 and Col hybrid background (sse1adg1-H and sse1pgm1-H). Results from wild-type C24 (wt-C24) and Col (wt-Col) seeds, as well as sse1 single mutant seeds in the C24 and Col hybrid background (sse1-H), are shown as controls. Bars are ses. C, Suc contents in mature wild-type C24 (wt-C24) and sse1 seeds. Bars are ses. D, Incorporation of ¹⁴C-labeled precursors into triacylglycerol in developing sse1 and wild-type C24 seeds. Single representative experiments of two to three replicates are shown. Among different sets of experiments, seeds of the same developmental stage may differ in actual age of up to 1 DAF, due to seasonal or other variations. E, Oil synthesis precursors taken up and metabolized by the sse1 mutant embryos. Embryos of 11 to 12 DAF were fed [U-¹⁴C]Glc, [2-¹⁴C]pyruvate, or [1-¹⁴C]acetate as in D and washed with water before measurement of the total amount of label remaining in the embryo (total; black bars) and the amount incorporated into the 80% ethanol insoluble fraction (80% ethanol; white bars).

Tween 80 rescue of sse1 seedling growth. Arrows indicate shoot-like tissues in sse1 seedlings in the absence of Tween 80. In the presence of Tween 80, about 10% of the sse1 seedlings produce small and green translucent leaves. The sse1 seedling on Tween 80 is 4 weeks old, and all other seedlings are 3 weeks old. Bars are 4.0 mm for wild-type and 0.46 mm for sse1 seedlings.