SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds

Abstract

Triacylglycerol hydrolysis (lipolysis) plays a pivotal role in the life cycle of many plants by providing the carbon skeletons and energy that drive postgerminative growth. Despite the physiological importance of this process, the molecular mechanism is unknown. Here, a genetic screen has been used to identify Arabidopsis thaliana mutants that exhibit a postgerminative growth arrest phenotype, which can be rescued by providing sugar. Seventeen sugar-dependent (sdp) mutants were isolated, and six represent new loci. Triacylglycerol hydrolase assays showed that sdp1, sdp2, and sdp3 seedlings are deficient specifically in the lipase activity that is associated with purified oil bodies. Map-based cloning of SDP1 revealed that it encodes a protein with a patatin-like acyl-hydrolase domain. SDP1 shares this domain with yeast triacylglycerol lipase 3 and human adipose triglyceride lipase. In vitro assays confirmed that recombinant SDP1 hydrolyzes triacylglycerols and diacylglycerols but not monoacylglycerols, phospholipids, galactolipids, or cholesterol esters. SDP1 is expressed predominantly in developing seeds, and a SDP1-green fluorescent protein fusion was shown to associate with the oil body surface in vivo. These data shed light on the mechanism of lipolysis in plants and establish that a central component is evolutionarily conserved among eukaryotes.

Figure 1.

TAG Lipase Activity in Germinating sdp Seeds. Crude cell extracts (A) and purified oil body membranes (B) were prepared from imbibed seeds and 2-d-old seedlings of sdp mutants and the wild type, and their capacity to hydrolyze [¹⁴C]triolein was measured. Seedlings were grown on agar plates containing half-strength Murashige and Skoog (MS) salts plus 1% (w/v) sucrose. The values are the mean ± se of measurements on four separate preparations.

Figure 2.

Postgerminative Growth and Cell Ultrastructure in sdp1. (A) Five-day-old sdp1 and wild-type seedlings grown in the light on agar plates containing half-strength MS salts plus or minus 1% (w/v) sucrose. Bar = 1 cm. (B) Light microscopy of hypocotyl cells from whole 5-d-old sdp1-1 and wild-type seedlings that were grown on agar plates containing half-strength MS plus 1% (w/v) sucrose. Bar = 10 μm. (C) Electron microscopy images of cotyledon cells from 5-d-old sdp1-1 and wild-type seedlings that were grown on agar plates containing half-strength MS plus 1% (w/v) sucrose. P, peroxisome; OB oil body; M, mitochondrion; C, chloroplast. Bar = 1 μm.

Figure 3.

TAG Breakdown and Acyl-CoA Levels in sdp1. (A) Total TAG content of sdp1-1 and wild-type seeds germinated and grown in the light on agar plates containing half-strength MS salts plus 1% (w/v) sucrose. The values are the mean ± se of measurements from eight separate batches of 20 seeds or seedlings. (B) Acyl-CoA levels in 2-d-old sdp1-1 and wild-type seedlings germinated and grown in the light on agar plates containing half-strength MS salts plus 1% (w/v) sucrose. The values are the mean ± se of measurements from five separate samples, each containing ∼10 mg fresh weight (FW) of seedlings.

Figure 4.

Positional Cloning of SDP1. (A) A schematic diagram to illustrate how PCR-based simple sequence length polymorphism and cleaved-amplified polymorphic sequence markers were used to map SDP1 to a 12-kb region on chromosome 5 that contains four genes. The positions of markers a to h (listed in Supplemental Table 3 online) are denoted by bars, and the number of recombination events/total number of chromosomes (1536) is listed below each. (B) A representation of the SDP1 locus (At5g04040) showing the positions of mutations that were identified in five independent sdp1 alleles. Insertion/substitution positions are numbered relative to the initiation codon. Black bars are exons, and the white bars and arrow indicate the 5′ and 3′ untranslated regions.

Figure 5.

Sequence Similarity between SDP1 and Patatin-Like Proteins. (A) An amino acid sequence alignment of the sequence surrounding the lipase (GXSXG) motif found in the patatin-like domains of Arabidopsis SDP1, yeast TGL3, human ATGL, and potato patatin. The alignment was performed using ClustalX (version 1.83). (B) A schematic diagram showing the positions of the patatin-like (Pfam01734) domains in Arabidopsis SDP1, yeast TGL3, human ATGL, and potato patatin. The domains (marked in black) were identified by querying the NCBI Conserved Domain Database (Marchler-Bauer et al., 2005). aa, amino acids. (C) A phylogenetic tree illustrating the relationship between patatin-like domains found in TAG lipases from yeast (TGL3, 4, and 5), Arabidopsis (SDP1, SDP1-like, and ATGL-like), Drosophila (Brummer), and humans (ATGL). The prototype patatin domain from potato patatin and the most closely related of the 12 patatin-like proteins in Arabidopsis (Patatin-like1) are also included. The phylogenetic analysis was performed on the alignment shown in Supplemental Figure 1 online using MEGA (version 2.1). Bootstrap values were determined from 1000 trials and are shown at each node. The length of the branch line indicates the extent of divergence according to the scale (relative units) at the bottom.

Figure 6.

Expression of SDP1 in S. cerevisiae and Determination of TAG Lipase Activity. (A) A Coomassie-stained SDS-PAGE gel of His₆-tagged SDP1. Lane 1, total protein from uninduced cells; lane 2, total protein from induced cells; lane 3, affinity-purified rSDP1. (B) to (D) The effect of time (B), substrate concentration (C), and pH (D) on [¹⁴C]triolein hydrolysis catalyzed by purified rSDP1. The values are the mean ± se of measurements on four separate incubations. The standard amount of emulsified substrate used was 9 mg mL⁻¹ (∼10 mM), except in (C), where the amount of substrate was varied. FFA, free fatty acids.

Figure 7.

Expression and Localization of SDP1. (A) Real-time PCR analysis of the expression of SDP1 during seed development, germination, and early seedling growth. Values are the mean ± se of measurements on four separate RNA extractions. Equal quantities of total RNA were used from each sample. DAF, days after fertilization; DAI, days after imbibition. (B) A confocal image of a cell from a 5-d-old sdp1-1 seedling transiently expressing the SDP1-GFP fusion protein. Top left, GFP; top right, bright field; bottom left, superimposed images. Bar = 10 μm. (C) GFP fluorescence measured in different fractions from crude extracts of 5-d-old sdp1-1 seedlings expressing SDP1-GFP that were centrifuged at 10,000g for 15 min. Values are the mean ± se of measurements on three separate samples. O, oil pad; S, supernatant; P, pellet. (D) TAG lipase activity in oil body membranes purified from 5-d-old sdp1-1 seedlings (C) and 5-d-old sdp1-1 seedlings expressing SDP1-GFP (E). Values are the mean ± se of measurements on three separate samples.