Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology

Abstract

An Arabidopsis mosaic death1 (mod1) mutant, which has premature cell death in multiple organs, was isolated. mod1 plants display multiple morphological phenotypes, including chlorotic and curly leaves, distorted siliques, premature senescence of primary inflorescences, reduced fertility, and semidwarfism. The phenotype of the mod1 mutant results from a single nuclear recessive mutation, and the MOD1 gene was isolated by using a map-based cloning approach. The MOD1 gene encodes an enoyl-acyl carrier protein (ACP) reductase, which is a subunit of the fatty acid synthase complex that catalyzes de novo synthesis of fatty acids. An amino acid substitution in the enoyl-ACP reductase of the mod1 mutant causes a marked decrease in its enzymatic activity, impairing fatty acid biosynthesis and decreasing the amount of total lipids in mod1 plants. These results demonstrate that a deficiency in fatty acid biosynthesis has pleiotropic effects on plant growth and development and causes premature cell death.

Figure 1.

The Phenotype of the mod1 Mutant. Wild-type and mod1 plants were grown under continuous light at 23°C and photographed at the indicated number of days after imbibition. (A) and (B) mod1 and wild-type plants at day 10. (C) and (D) mod1 and wild-type plants at day 20. (E) and (F) mod1 and wild-type plants at day 30. (G) and (H) mod1 and wild-type plants at day 40. (I) and (J) mod1 and wild-type siliques. (K) to (M) mod1 leaves. (N) Wild-type leaf. (A) (N) .

Figure 2.

Light and Electron Microscopic Analysis. (A) and (B) Scanning electron microscopy of mod1 and wild-type epidermal cells. (C) and (D) Differential interference contrast microscopy of mod1 and wild-type mesophyll cells. (E) and (F) A chlorotic sector (arrowhead) of a mod1 leaf (E) shows much weaker red fluorescence than a wild-type leaf (F) under UV light. (G) and (H) mod1 mesophyll cells (G) contain fewer chloroplasts than wild-type cells (H). (I) and (J) Transmission electron microscopy of mod1 and wild-type chloroplasts. (K) and (L) Transmission electron microscopy of mod1 and wild-type thylakoid membranes. ; ; .

Figure 3.

Histochemical Staining with Trypan Blue. (A) to (D) Trypan blue–stained mod1 and wild-type leaves and siliques, demonstrating a mosaic staining pattern in mod1 plants ([A] and [C]). (E) to (H) High magnification of stained single and clustered mod1 leaf cells. ; b; .

Figure 4.

DNA Fragmentation in mod1 Plants. The DNA ladder of mod1 plants was detected by DNA gel blot analysis with a ³²P-labeled total DNA digested with Sau3AI. Equal amounts of total DNA isolated from mod1 and wild-type plants at the same developmental stage were loaded in each lane. Lane 1, DNA isolated from mod1 plants; lane 2, DNA isolated from wild-type plants. Positions of DNA standards (identified in kilobases) are indicated at left.

Figure 5.

Effect of Temperature on mod1 Plants. (A) and (B) mod1 and wild-type plants grown under continuous light at 26°C for 40 days. (C) and (D) mod1 and wild-type plants grown under continuous light at 20°C for 40 days.

Figure 6.

Molecular Identification of the mod1 Gene. (A) Physical mapping of the mod1 locus. (B) The two DNA fragments used for complementation of the mod1 mutant. A, AccI; B, BamHI; E, EcoRI; X, XbaI. (C) Structure of the ENR gene and its mutation in the mod1 plant. The start codon (ATG) and stop codon (TAG) are indicated. The GenBank accession number for ENR cDNA (Col-0) is AF207593. Filled boxes denote the coding sequence; open boxes, 5′ and 3′ untranslated regions; lines between boxes, introns. (D) DNA polymorphism between mod1 and wild-type plants. The mutation in the mod1 genomic DNA introduces a new EcoRV site. The DNA fragments flanking the EcoRV site were amplified from the wild-type (Col-0) and mod1 plants, digested with EcoRV, and separated on an agarose gel.

Figure 7.

DNA Gel Blot Analysis. Arabidopsis Col-0 DNA was digested separately with BamHI and EcoRV and transferred onto a Hybond N⁺ membrane. The filter was probed with an ENR gene fragment. Lengths of DNA fragments in kilobases are indicated at right.

Figure 8.

ENR Protein Content and Its Enzymatic Activity in mod1 and Wild-Type Plants. (A) Protein gel blot analysis. Protein samples prepared from mod1 and wild-type (WT) plants 30 days after imbibition and from siliques 15 days after flowering were analyzed by immunoblotting. The arrow indicates the nonspecific protein, which served as a loading control. (B) ENR activity in Arabidopsis leaves and siliques. Samples were prepared from the leaves and siliques of mod1 and wild-type (WT) plants. ENR activity is shown as mean ±se ().

Figure 9.

Comparison of Total Lipid Contents between mod1 and Wild-Type Plants. Total lipid content was determined in 10-g samples of fresh rosette leaves from 4-week-old plants. The total lipid contents are shown as mean ±se ().