Fatty acid export from the chloroplast. Molecular characterization of a major plastidial acyl-coenzyme A synthetase from Arabidopsis

Abstract

Acyl-coenzyme A (CoA) synthetases (ACSs, EC 6.2.1.3) catalyze the formation of fatty acyl-CoAs from free fatty acid, ATP, and CoA. Essentially all de novo fatty acid synthesis occurs in the plastid. Fatty acids destined for membrane glycerolipid and triacylglycerol synthesis in the endoplasmic reticulum must be first activated to acyl-CoAs via an ACS. Within a family of nine ACS genes from Arabidopsis, we identified a chloroplast isoform, LACS9. LACS9 is highly expressed in developing seeds and young rosette leaves. Both in vitro chloroplast import assays and transient expression of a green fluorescent protein fusion indicated that the LACS9 protein is localized in the plastid envelope. A T-DNA knockout mutant (lacs9-1) was identified by reverse genetics and these mutant plants were indistinguishable from wild type in growth and appearance. Analysis of leaf lipids provided no evidence for compromised export of acyl groups from chloroplasts. However, direct assays demonstrated that lacs9-1 plants contained only 10% of the chloroplast long-chain ACS activity found for wild type. The residual long-chain ACS activity in mutant chloroplasts was comparable with calculated rates of fatty acid synthesis. Although another isozyme contributes to the activation of fatty acids during their export from the chloroplast, LACS9 is a major chloroplast ACS.

Figure 1

Accumulation of LACS9 mRNA in Arabidopsis tissues. Total RNA (15 μg) was probed with a fragment of the LACS9 transcript. Ethidium bromide staining of the major rRNA bands was used to confirm equal loading of total RNA. Lane 1, Flowers and buds; lane 2, 1- to 5-d after flowering (DAF) siliques; lane 3, 6- to 11-DAF siliques; lane 4, 12- to 20-DAF siliques; lane 5, primary leaves from 14-d old plants; lane 6, primary leaves from 16-d old plants; lane 7, leaves from 50-d old plants; lane 8, roots. LACS9 transcript was not present in appreciable amounts in mature seed (data not shown).

Figure 2

In vitro chloroplast import assays. ³H-labeled Rubisco small subunit (prSS), LeHPL, and LACS9 were incubated with intact chloroplasts. The chloroplasts were repurified, lysed, and the membranes pelleted by centrifugation. After resuspension in lysis buffer, samples of the membrane fraction were washed either with the same lysis buffer, or NaCl or Na₂CO₃. Samples were analyzed by SDS-PAGE and fluorography. TP, Translated product; P, membrane fraction; S, soluble fraction; p, precursor; m, mature protein. Results shown are from one of three separate experiments that showed comparable results.

Figure 3

Transient expression of LACS9-GFP and ACP-DsRED in a bombarded onion epidermal cell. A, LACS9-GFP is localized to the periphery of organelles tentatively identified as plastids. B, ACP-DsRED fluorescence confirms the identification of plastids. C, A and B merged, showing that LACS9 expression is localized in the plastidial envelope and associated stromules. The bar represents 10 μm.

Figure 4

A, The structure and organization of the LACS9 genomic sequence containing a T-DNA insertion. The lacs9-1 knockout mutant contains a T-DNA insertion in the third exon, 1,020 bp from the start codon in the genomic DNA. The LACS9 gene is located on chromosome I and is 3,324 bp in length (MIPS code At1g77590). B, Northern analysis of RNA isolated from the lacs9-1 homozygous and heterozygous mutant plants compared with wild type. Total RNA (15 μg) from tissues of wild-type (lane 1), heterozygous (lane 2), and homozygous (lane 3) mutants was separated by electrophoresis and probed with a fragment of the LACS9 cDNA. Ethidium bromide staining shows equal RNA loading.

Figure 5

Growth curves for wild type and the lacs9-1 plants grown at 22°C under 14:10 (light:dark) photoperiod. Growth measurements were made by taking the fresh weight of the aboveground portions of plants at the indicated intervals. The relative growth rate (ω⁻¹) for the wild type was 0.301 ± 0.007; for the mutant, it was 0.312 ± 0.013. Values shown are the means ± se (n = 10).

Figure 6

In vitro LACS assay on isolated chloroplasts from wild-type and lacs9-1 plants. Intact chloroplasts were isolated and then assayed with 1-[¹⁴C]18:1 or 1-[¹⁴C]16:0 substrates in hypotonic media (see “Materials and Methods” for details).