Disruption of the Arabidopsis CGI-58 homologue produces Chanarin-Dorfman-like lipid droplet accumulation in plants

Abstract

CGI-58 is the defective gene in the human neutral lipid storage disease called Chanarin-Dorfman syndrome. This disorder causes intracellular lipid droplets to accumulate in nonadipose tissues, such as skin and blood cells. Here, disruption of the homologous CGI-58 gene in Arabidopsis thaliana resulted in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common leaf-specific fatty acids. Leaves of mature cgi-58 plants exhibited a marked increase in absolute triacylglycerol levels, more than 10-fold higher than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-of-function plants were unchanged, and unlike mutations in β-oxidation, the cgi-58 seeds germinated and grew normally, requiring no rescue with sucrose. We conclude that the participation of CGI-58 in neutral lipid homeostasis of nonfat-storing tissues is similar, although not identical, between plant and animal species. This unique insight may have implications for designing a new generation of technologies that enhance the neutral lipid content and composition of crop plants.

Fig. 1.

Description of the Arabidopsis CGI-58 homolog. (A) Partial amino acid sequence alignments of the Arabidopsis CGI-58 splice variants (At4g24160.1 and At4g24160.2) and homologs from grape, rice, human, mouse, Caenorhabditis elegans, zebrafish, moss (Physcomitrella), and yeast. Motif analysis by MEME/MAST (http://meme.sdsc.edu/meme4_4_0/intro.html) revealed three distinct domains common to all proteins (red, green, and blue). A fourth conserved region (yellow) was evident upon visual inspection. Two positions in the Arabidopsis protein marked 176 and 315 correspond to amino acid residues that, when mutated in the human sequence, interfere with lipid droplet-binding (and cause disease) (47). (B) The At4g24160 locus gives rise to two transcripts and the relative gene exon/intron/UTR structures are shown (redrawn based on information from www.arabidopsis.org). Two T-DNA insertional mutant lines were annotated in the SALK collection.

Fig. 2.

Accumulation of lipid droplets in Arabidopsis plants that harbor a disruption in the CGI-58 gene. Representative confocal fluorescence micrographs of (A) wild-type (Col 0) and two homozygous T-DNA mutant lines [SALK_136871 (B), and SALK_127083 (C)] stained with Nile red to reveal lipid droplets in 21-d-old seedlings (petiole region of true leaves). (Scale bar, 20 μm.) (D) TLC separation of the neutral lipid fraction isolated from liquid cultured 21-d-old seedlings (450 mg FW each) of wild-type (lane 1) and T-DNA mutants (lanes 2 and 3). Standards are steryl esters (StE), TAG (arrow), free fatty acids (FFA), diacylglycerols (DAG), and monoacylglycerols (MAG). (E–G) Representative epifluorescence micrographs of purified lipid droplets that were obtained from equivalent amounts of wild-type and mutant seedlings (stained with BODIPY 493/503). (Scale bar, 20 μm.)

Fig. 3.

MS analysis of TAGs in tissues and in isolated lipid droplets from leaves of wild-type (red) and cgi-58 disrupted mutants (black). (A) Positive-ion MS analysis of neutral lipids extracted from leaves (overlay of representative spectra). TAG species are labeled according to the total acyl chain length followed by the number of double bonds present in each TAG molecular species. (B) Representative MS scans of direct organelle MS analysis of lipids in isolated droplets from wild-type and mutant leaves.

Fig. 4.

Lipid droplets are abundant in leaves of cgi-58 mutants. (A) Representative confocal fluorescence micrograph of mesophyll tissues of mature wild-type leaves, showing chloroplasts (red) and a few lipid droplets (arrows) stained with BODIPY 493/503 (green). (B) Confocal fluorescence micrograph of mesophyll tissues of same-age leaves of cgi-58 T-DNA knockouts. (C) A z-stack of thirteen optical sections of the cgi-58 T-DNA knockout mutant (Salk_136871). (D) Averages and SDs of lipid droplet numbers are plotted for 10 digital images of 25,000 μm², each taken from several leaves (P < 0.005). (E) The frequency of images with different numbers of lipid droplet numbers. (Scale bars, 20 μm.)

Fig. 5.

Lipid composition analysis of cgi-58 T-DNA knockout plants. (A) Total fatty acid composition and content (Inset) of leaves of 35-d-old plants. Means and SDs for five samples (**P < 0.01; ***P < 0.000001). (B) TLC separation of neutral lipid fractions collected from five replicate samples. Samples were spiked with heptadecanoic acid hence the appearance of substantial amounts of FFAs in all samples. (C) Fold-differences of cgi-58-derived lipid amounts compared with wild-type are plotted for neutral lipids (NL; includes both TAGs and StE); glycolipids (GL; includes monogalactosyldiacylglcerols, digalactosyldiacylglycerols, and sulfoquinovosyldiacylglycerols), and phospholipids (PL; includes phosphatidyl-choline, -ethanolamine, -inositol, glycerol, and phosphatidic acid) quantified by direct infusion ESI-MS (35). Values are averages summed from all major molecular species of polar and nonpolar lipids identified and then plotted as higher or lower relative to wild-type (dotted line). Quantities of TAG are plotted in the inset (*P < 0.05, n = 5).