The peroxisome deficient Arabidopsis mutant sse1 exhibits impaired fatty acid synthesis

Abstract

The Arabidopsis Shrunken Seed 1 (SSE1) gene encodes a homolog of the peroxisome biogenesis factor Pex16p, and a loss-of-function mutation in this gene alters seed storage composition. Two lines of evidence support a function for SSE1 in peroxisome biogenesis: the peroxisomal localization of a green fluorescent protein-SSE1 fusion protein and the lack of normal peroxisomes in sse1 mutant embryos. The green fluorescent protein-SSE1 colocalizes with the red fluorescent protein (RFP)-labeled peroxisomal markers RFP-peroxisome targeting signal 1 and peroxisome targeting signal 2-RFP in transgenic Arabidopsis. Each peroxisomal marker exhibits a normal punctate peroxisomal distribution in the wild type but not the sse1 mutant embryos. Further studies reported here were designed toward understanding carbon metabolism in the sse1 mutant. A time course study of dissected embryos revealed a dramatic rate decrease in oil accumulation and an increase in starch accumulation. Introduction of starch synthesis mutations into the sse1 background did not restore oil biosynthesis. This finding demonstrated that reduction in oil content in sse1 is not caused by increased carbon flow to starch. To identify the blocked steps in the sse1 oil deposition pathway, developing sse1 seeds were supplied radiolabeled oil synthesis precursors. The ability of sse1 to incorporate oleic acid, but not pyruvate or acetate, into triacylglycerol indicated a defect in the fatty acid biosynthetic pathway in this mutant. Taken together, the results point to a possible role for peroxisomes in the net synthesis of fatty acids in addition to their established function in lipid catabolism. Other possible interpretations of the results are discussed.

Figure 1.

GFP-SSE1 localizes to peroxisomes, and overexpression of GFP-SSE1 changes peroxisome distribution. Cotyledon and embryonic cells were examined by confocal microscopy and root cells were examined by fluorescent microscopy. A, The GFP-SSE1 fusion protein complements the lethal and shrunken seed phenotypes of the sse1 mutant. The plant and the seeds are from a homozygous sse1 line expressing the GFP-SSE1 transgene. Segregation of the shrunken seed phenotype is due to segregation of the GFP-SSE1 transgene, i.e. shrunken seeds do not carry the GFP-SSE1 transgene. Complementation was observed in all seven transgenic lines in the sse1 background. B, GFP-SSE1 containing organelles in young seedlings and developing embryos from GFP-SSE1 transgenic lines. Arrows indicate DAPI stained nuclei. The red color is generated by the autofluorescence of chloroplasts. Estimated borders of several embryonic cells are indicated to show the average cell size. cot, cotyledon cells from a seedling; emb, embryonic cells from a cotyledon stage embryo; root, root cells from a postgerminative seedling. Bar is 80 μm for cotyledon, 30 μm for embryo, and 25 μm for root. C, RFP-PTS1 and PTS2-RFP markers reveal normal peroxisome distributions in wild-type (+/+) or sse1/+ plants. The root hair cells are of +/+ and the embryonic cells sse1/+ genotypes. Estimated borders of several embryonic cells are indicated to show the average cell size. Bar is 100 μm for root and 50 μm for embryo. D, GFP-SSE1 colocalizes with RFP-PTS1 and PTS2-RFP markers, and overexpression of GFP-SSE1 changes peroxisome distribution. Each peroxisomal marker was introduced into the GFP-SSE1 transgenic line 3 through stable plant transformation. S, The GFP and RFP images are superimposed. Bar is 100 μm for root and 50 μm for embryo.

Figure 2.

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.

Figure 3.

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.

Figure 4

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.

Figure 5.

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).

Figure 6.

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.

Figure 7.

The developmental defects of the sse1 mutant. A, Embryos from an 8-DAF silique of a wild-type C24 and a heterozygous sse1/+ plant. Arrow indicates a green heterozygote sse1/+ embryo in the segregating population. B, The 11-DAF wild-type C24 and sse1 embryos as seen in the light microscope. C, Mature embryos from a dry silique from a heterozygous sse1/+ plant. Seeds were soaked in water for a few hours to reduce the shrunken appearance before embryos were dissected from the seed coats. Two heterozygote embryos in the population are indicated with arrows.