The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis

Abstract

Long-chain acyl-CoA synthetase (LACS) activities are encoded by a family of at least nine genes in Arabidopsis (Arabidopsis thaliana). These enzymes have roles in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. Here, we show that the LACS2 gene (At1g49430) is expressed in young, rapidly expanding tissues, and in leaves expression is limited to cells of the adaxial and abaxial epidermal layers, suggesting that the LACS2 enzyme may act in the synthesis of cutin or cuticular waxes. A lacs2 null mutant was isolated by reverse genetics. Leaves of mutant plants supported pollen germination and released chlorophyll faster than wild-type leaves when immersed in 80% ethanol, indicating a defect in the cuticular barrier. The composition of surface waxes extracted from lacs2 leaves was similar to the wild type, and the total wax load was higher than the wild type (111.4 microg/dm(2) versus 76.4 microg/dm(2), respectively). However, the thickness of the cutin layer on the abaxial surface of lacs2 leaves was only 22.3 +/- 1.7 nm compared with 33.0 +/- 2.0 nm for the wild type. In vitro assays showed that 16-hydroxypalmitate was an excellent substrate for recombinant LACS2 enzyme. We conclude that the LACS2 isozyme catalyzes the synthesis of omega-hydroxy fatty acyl-CoA intermediates in the pathway to cutin synthesis. The lacs2 phenotype, like the phenotypes of some other cutin mutants, is very pleiotropic, causing reduced leaf size and plant growth, reduced seed production, and lower rates of seedling germination and establishment. The LACS2 gene and the corresponding lacs2 mutant will help in future studies of the cutin synthesis pathway and in understanding the consequences of reduced cutin production on many aspects of plant biology.

Figure 1.

A Simplified Outline of the Pathways of Fatty Acid Modification That Result in the Synthesis of Cutin and Cuticular Waxes in Epidermal Cells. FA, fatty acid.

Figure 2.

Expression Pattern of LACS2 in Arabidopsis Wild Type. Total RNA (15 μg) from wild-type tissues was probed with a full-length LACS2 cDNA. Lane 1, leaves from 14-d-old rosettes; lane 2, mature leaves (2 to 4 cm long) from 42-d-old rosettes; lane 3, roots; lane 4, buds and flowers; lane 5, siliques 1 to 5 d after flowering (DAF); lane 6, siliques 6 to 11 DAF; and lane 7, siliques 12 to 20 DAF. An ethidium bromide stain of the gel is shown to confirm equal RNA loading.

Figure 3.

The lacs2-1 T-DNA Knockout Mutant. (A) Diagram of the genomic sequence showing the location of the T-DNA insertion. The mutant was identified in a PCR screen of pooled genomic DNA using the 5′ gene-specific primer (2F) in combination with the T-DNA right border primer (XR2). (B) Photograph of 37-d-old wild-type (left) and lacs2-1 mutant plants. The lacs2-1 mutant has smaller, wrinkled leaves and displays overall reduced vigor.

Figure 4.

The lacs2-1 Phenotype Is Complemented by Expression of the LACS2 cDNA. (A) Photograph of the wild type (WT), lacs2-1 mutant, and lacs2-1 plant expressing a LACS2 cDNA under the control of the CaMV 35S promoter. Plants shown here are 24 d old. (B) Gel blot analysis of RNA from the wild type (lane 1), lacs2-1 heterozygote (lane 2), lacs2-1 homozygote (lane 3), and a complemented line (lane 4). Total RNA (15 μg) was probed with the LACS2 cDNA sequence. Ethidium bromide is shown as a loading control. (C) PCR analysis of genomic DNA from the wild type (1), lacs2-1 (2), and a complemented line (3). The complemented line did not contain a wild-type LACS2 genomic fragment, although the presence of the LACS2 transgene is indicated. Primer locations within the genomic sequence are shown in Figure 3.

Figure 5.

Leaf Phenotype of the lacs2-1 Mutant. (A) Leaves were removed from 22-d-old rosettes and measured. Scale bar = 1 cm. (B) Summary of leaf length versus leaf number at 22 d (n = 15). (C) Summary of leaf length versus leaf number at 35 d (n = 12).

Figure 6.

Scanning Electron Microscopy of Wild-Type and lacs2-1 Leaf Surfaces. (A) Wild-type leaf, adaxial side. (B) lacs2-1 leaf, adaxial side. (C) Wild-type leaf, abaxial side. (D) lacs2-1 leaf, abaxial side. Scale bar represents 75 μm.

Figure 7.

Histochemical Staining of Transgenic Lines Containing the LACS2 Promoter Fused to the GUS Reporter Gene. (A) Flower cluster showing GUS staining in the stigma, style, filament, sepals, and pedicel tissues. Staining can also be seen in developing seeds of a dissected developing silique (inset). (B) Overview of a leaf from a 15-d-old plant. (C) Cross section of a leaf from a 15-d-old plant showing expression only in epidermal cells.

Figure 8.

Electron Micrographs of the Cuticle Layer on Wild-Type and lacs2-1 Leaves. (A) Wild type, adaxial surface. (B) lacs2-1, adaxial surface. (C) Wild type, abaxial surface. (D) lacs2-1, abaxial surface. Scale bar = 100 nm.

Figure 9.

Chlorophyll Leaching from Wild-Type and lacs2-1 Leaves. Rosettes were placed in 80% EtOH, and aliquots were removed at the indicated time points. Results shown here are representative of three independent experiments.

Figure 10.

Pollen Germination on lacs2-1 Leaf Surfaces. Wild-type pollen was applied to the surfaces of wild-type or lacs2-1 leaves on 42-d-old plants. After 24 h, leaves were fixed for scanning electron microscopy analysis. (A) Wild-type adaxial surface. (B) Wild-type abaxial surface. (C) lacs2-1 adaxial surface. (D) lacs2-1 abaxial surface. The scale bar represents 75 μm.