Research ArticleResearch Article

Altered Lipid Composition and Enhanced Nutritional Value of Arabidopsis Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2

Sanjaya, Rachel Miller, Timothy P. Durrett, Dylan K. Kosma, Todd A. Lydic, Bagyalakshmi Muthan, Abraham J.K. Koo, Yury V. Bukhman, Gavin E. Reid, Gregg A. Howe, John Ohlrogge, Christoph Benning

Published February 2013. DOI: https://doi.org/10.1105/tpc.112.104752

Abstract

Enhancement of acyl-CoA–dependent triacylglycerol (TAG) synthesis in vegetative tissues is widely discussed as a potential avenue to increase the energy density of crops. Here, we report the identification and characterization of Chlamydomonas reinhardtii diacylglycerol acyltransferase type two (DGTT) enzymes and use DGTT2 to alter acyl carbon partitioning in plant vegetative tissues. This enzyme can accept a broad range of acyl-CoA substrates, allowing us to interrogate different acyl pools in transgenic plants. Expression of DGTT2 in Arabidopsis thaliana increased leaf TAG content, with some molecular species containing very-long-chain fatty acids. The acyl compositions of sphingolipids and surface waxes were altered, and cutin was decreased. The increased carbon partitioning into TAGs in the leaves of DGTT2-expressing lines had little effect on transcripts of the sphingolipid/wax/cutin pathway, suggesting that the supply of acyl groups for the assembly of these lipids is not transcriptionally adjusted. Caterpillars of the generalist herbivore Spodoptera exigua reared on transgenic plants gained more weight. Thus, the nutritional value and/or energy density of the transgenic lines was increased by ectopic expression of DGTT2 and acyl groups were diverted from different pools into TAGs, demonstrating the interconnectivity of acyl metabolism in leaves.

INTRODUCTION

In angiosperms, deposition of triacylglycerols (TAGs) in lipid droplets is most commonly associated with seed tissues. These TAGs represent a reduced form of carbon with high energy content essential for seed germination and seedling establishment. In addition, many microalgae accumulate considerable amounts of TAGs under stress conditions such as nitrogen deficiency, high light, or high salt. The utility of this storage lipid has been intensely explored in both organism groups as a source of food and nutraceuticals and as a feedstock for the petrochemical industry or biodiesel fuel production (Durrett et al., 2008; Halim et al., 2012). The mechanisms of TAG synthesis and its regulation have been mostly characterized in developing seed tissues. However, the molecular regulation and synthesis of TAGs in nonseed or vegetative cells of plants and microalgae is still largely uncharacterized but is thought to involve different regulatory mechanisms than those observed for developing seeds (Fan et al., 2011; Chapman and Ohlrogge, 2012).

In plants and microalgae, the plastid and the endoplasmic reticulum (ER) interact in the de novo formation of TAG, as the synthesis of fatty acids (FAs) occurs in the plastid, followed by the export of FAs and their assembly into TAGs at the ER. In both organism groups, the basic enzymatic mechanisms involved are similar for membrane glycerolipid and TAG synthesis. The carbon flux from the FA synthase in the plastid through the cytosolic acyl-CoA pool and the substrate specificities of the enzymes involved, especially that of the acyltransferases, likely affect the acyl composition of the DAG intermediates and ultimately the TAGs formed at the ER. Thus, the manipulation of carbon fluxes between acyl-CoA, TAG, and other acyl lipid pools are expected to provide insight into the lipid metabolic network and its regulation in vegetative tissues.

Different classes of diacylglycerol acyltransferases (DGATs) play distinct roles in determining the quality and quantity of acyl-CoA flux into TAG synthesis. In eukaryotes, several classes of DGATs have been identified based on structure and activity. The two most common are type 1 (DGAT1) and type 2 (DGAT2), which have both been shown to be involved in the synthesis of TAG (Yen et al., 2008). DGAT1 proteins are predicted to possess six or more transmembrane domains and belong to the membrane-bound O-acyltransferase family (Cases et al., 1998). Forward and reverse genetic approaches have confirmed that the Arabidopsis thaliana DGAT1 gene affects the TAG levels in seeds (Routaboul et al., 1999; Zou et al., 1999; Jako et al., 2001), and DGAT1 genes have been studied in detail in several species (Lung and Weselake, 2006; Shockey et al., 2006; Banilas et al., 2011). Structurally, DGAT2 proteins differ from DGAT1 and possess only two to three predicted transmembrane domains (Yen et al., 2008). Overexpression of fungal Umbelopsis ramannian DGAT2 increased seed oil content in soybean (Glycine max) and maize (Zea mays; Lardizabal et al., 2008; Oakes et al., 2011). A third type of DGAT includes the bifunctional enzyme ADP1 from Acinetobacter calcoaceticus, exhibiting both wax ester synthase (WS) and DGAT activity (Kalscheuer and Steinbüchel, 2003). ADP1 homologs have been identified in Arabidopsis and petunia (Petunia hybrida) and have been shown to have WS and DGAT activity at different levels (King et al., 2007; Li et al., 2008). A fourth type, a soluble cytosolic DGAT enzyme (DGAT3), was identified in peanut (Arachis hypogaea), with homologs in Arabidopsis (Saha et al., 2006; Hernández et al., 2012).

Like land plants, microalgae also possess DGAT-mediated TAG synthesis. For instance, in the diatom Phaeodactylum tricornutum, a cDNA encoding a DGAT1-like protein, was isolated and shown to incorporate saturated FAs into TAGs (Guihéneuf et al., 2011). In Ostreococcus tauri, a gene encoding DGAT2 has been identified and the enzyme has been shown to possess broad substrate specificity (Wagner et al., 2010). Previously, we identified five different genes encoding DGAT2 in Chlamydomonas reinhardtii and named them DGTT1 through DGTT5 (Miller et al., 2010). Recent studies have shown that of these five enzymes, DGTT1 and DGTT3 are active in TAG synthesis following nitrogen deprivation, but DGTT2, DGTT4, and DGTT5 are not (La Russa et al., 2012). Artificial microRNA silencing and overexpression of DGTT2 in C. reinhardtii did not influence TAG levels despite altered transcript abundance (Deng et al., 2012). Recently, a gene for a DGAT1-type enzyme was identified in C. reinhardtii after transcript-based correction of gene models (Boyle et al., 2012). Although the different types of DGATs differ structurally, whether they have redundant or specific functions in TAG synthesis in C. reinhardtii is not clear. The distinct substrate specificities of the various DGAT enzymes may determine the FA composition of TAG in microalgae and plants.

Here, we characterize DGTT2 of C. reinhardtii and use its ectopic production as a tool to affect carbon flux through the acyl-CoA pool in Arabidopsis, leading to the formation of TAGs in vegetative tissues. We focused on DGTT2 because it was the most active isoform of C. reinhardtii in our hands. We also present in vitro data on the acyl-CoA substrate specificity of DGTT2. Most notably, in DGTT2 transgenic plants, we observed a redirection of very-long-chain fatty acids (VLCFAs) from sphingolipids into TAGs. The implications of these results for plant lipid metabolism in vegetative tissues and for biotechnological applications are discussed.

RESULTS

Multiple Putative C. reinhardtii DGAT2 Isoforms

Amino acid sequence similarity to Arabidopsis DGAT2 was used to identify potential DGAT orthologs, using BLAST against version 3 of the C. reinhardtii genome. The BLAST search results returned five gene models with high sequence similarity to the Arabidopsis type 2 DGAT. These five candidates were named Diacylglycerol Acyltransferase Type Two (DGTT1 to DGTT5) and compared with type 2 DGATs from Arabidopsis, O. tauri, yeast (Saccharomyces cerevisiae), castor bean (Ricinus communis), and tung tree (Vernicia fordii). When analyzed with MEGA5 (Tamura et al., 2011), DGTT1 was the most closely related to Sc-DGA1. DGTT2, DGTT3, and DGTT5 formed their own clade, as did the three land plant DGATs. Ot-DGAT2B and DGTT4 were both more similar to the land plant DGATs than to the other C. reinhardtii DGTTs (Figure 1A; see Supplemental Data Set 1 online).

Figure 1.
Figure 1.

Identification of C. reinhardtii Type 2 DGATs and Expression of DGTT Constructs in Yeast.

(A) Phylogenetic tree of C. reinhardtii DGTT1-5 compared with DGAT2s from other species: Arabidopsis (NP_566952.1), O. tauri (XP_003083539.1), S. cerevisiae (NP_014888.1), castor bean (DQ923084.1), and tung tree (DQ356682.1).

(B) Predicted structure of DGTT1-5. Dark-gray boxes indicate predicted transmembrane domains (TM), and light-gray boxes indicate the conserved enzymatic domain (DAGAT).

(C) ESI-MS quantification of TAG levels extracted from transformed yeast. The total amount of TAG was normalized based on the DW of the yeast. Tritridecanoin (tri13:0) and tripentadecanoin (tri15:0) TAGs were added as internal standards (n = 4; average ± sd). H1266 indicates the empty vector control.

Structural analysis of the gene model–translated protein sequences was performed in silico using the TMHMM server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) to predict transmembrane sequences. All five candidates had one to three transmembrane domains in the N-terminal half of the protein, consistent with other type 2 DGATs. SignalP V3.0 (http://www.cbs.dtu.dk/services/SignalP/) and TargetP V1.1 (http://www.cbs.dtu.dk/services/TargetP/) failed to identify potential cellular localization signals. Searching against the National Center for Biotechnology Information Conserved Domain Database revealed a DAGAT domain in the C-terminal half of all five candidates, although DGTT5 has an apparent disruption in its domain (Figure 1B).

Expression of DGTT Constructs in Yeast

To test the function of the predicted DGTT genes, the coding sequences of DGTT2-5 were isolated and expressed in yeast strain H1266, a triple knockout mutant for DiacylGlycerol Acyltransferase1 (DGA1), Lecithin cholesterol acyl transferase Related Open reading frame1 (LRO1), and Acyl-coenzyme A: cholesterol acyl transferase-Related Enzyme2 (ARE2) (Sandager et al., 2002). This strain has very little native DGAT activity, providing a suitable background for testing the C. reinhardtii DGAT2 candidates. We were unable to isolate a DGTT1 cDNA based on the available gene model. We confirmed the presence of the DGTT2-5 proteins in yeast by immunoblotting (see Supplemental Figure 1 online). To quantify the amount of TAG produced, electrospray ionization–mass spectrometry (ESI-MS) was performed on lipid extracts from the transgenic yeast (Figure 1C). Both DGTT2- and DGTT3-producing yeast accumulated considerable levels of TAG (13.4 and 13.9% per dry weight [DW], respectively) compared with the empty vector control (0.5% per DW). Both DGTT4- and DGTT5-producing strains showed TAG levels equivalent to the empty vector (0.6 and 0.5% per DW, respectively), indicating that these may not have DGAT activity or lack appropriate cofactors or substrates in yeast necessary for proper DGAT activity.

In Vitro DGAT Activity of Recombinant Proteins

The substrate preference of the DGTT proteins was estimated in an in vitro reaction with isolated microsomes. DGTT-containing microsomes were incubated with radiolabeled palmitoyl-CoA (16:0) and oleoyl-CoA (18:1), along with dioleoyl DAG. The level of DGAT activity was estimated from their incorporation of radiolabel into isolated TAGs. Comparison of the results using the two different substrate acyl-CoAs suggested an apparent difference in substrate specificity, with DGTT2 and DGTT3 preferring 16:0-CoA and DGTT4 18:1-CoA (Figures 2A and 2B). These assays also confirmed that DGTT5 has essentially no activity under these assay conditions. This result, in conjunction with the apparent lack of expression of its gene in C. reinhardtii and disruption in the DAGAT domain, suggests that DGTT5 is a nonactive pseudogene.

Figure 2.
Figure 2.

DGTT Activity in Transgenic Yeast Microsomes.

(A) and (B) Autoradiograph of [1-14C]-16:0-acyl-CoA and [1-14C]-18:0-acyl-CoA radiolabeled lipids separated on a TLC plate. Signals representing TAG, free fatty acids (FFA), and diacylglycerol (DAG) are indicated. H1266 indicates the empty vector control.

(C) Competition assay. For each reaction, 1.725 nmol of [1-14C]-16:0-acyl-CoA was added. Competitors consisting of 1.725 nmol of unlabeled 16:0-acyl-CoA, 18:1-acyl-CoA, or 22:1-acyl-CoA was added to the respective reactions and compared with a no-competitor control (0). The newly synthesized 14C-labeled TAGs were separated and quantified by scintillation counting. The results were normalized by subtracting the background (in dpm) and then dividing all the results by the no-competitor control (n = 3; average ± sd).

Although DGTT2 and DGTT3 belong to the same clade, following nitrogen deprivation the level of DGTT2 transcripts remained unchanged, whereas DGTT3 transcript levels increased (Miller et al., 2010; Boyle et al., 2012). When expressed in yeast, DGTT2 activity in vitro was higher than that of the other isoforms. Based on the observed activity levels, we focused on the characterization of this enzyme as a potential tool for the manipulation of cytosolic acyl-CoAs in vegetative tissues of plants, the primary goal of this study. A microsome-based competition assay was used to further probe the substrate specificity of DGTT2. When incubated with equimolar amounts of 14C-labeled and unlabeled 16:0-CoA, the radioactivity in the TAG band was reduced to ∼50% compared with the reactions with only 14C-labeled 16:0-CoA (Figure 2C). When incubated with equal moles of unlabeled 18:1-CoA, the decrease was only ∼40%. However, when incubated with equal moles of unlabeled 22:1-CoA, the decrease was ∼75%. Together, these data suggested that DGTT2 is able to incorporate varying acyl-CoA species into TAG, perhaps preferring very-long-chain acyl groups, which will become relevant for the interpretation of lipid data for the transgenic plants described below.

Production of DGTT2 in Arabidopsis Affects Seedling Growth

To explore DGTT2 as a tool to manipulate acyl-CoA pools in plants and to engineer TAGs in vegetative tissues, we expressed the full-length DGTT2 coding sequence in Arabidopsis under the control of the constitutive 35S cauliflower mosaic virus promoter, 35S:DGTT2. Five homozygous lines, 14, 22, 46, 52, and 57, were carried forward to the T4 generation and used for detailed analysis.

The hypocotyls of transgenic 10- to 12-d-old seedlings grown on Murashige and Skoog agar plates supplemented with 1% Suc were strikingly more elongated than those of control plants and slightly pale in color. Consistent with the pale-green phenotype, the total chlorophyll content in the transgenic lines was 28 to 30% lower. On soil, all transgenic lines followed wild-type growth and development patterns. To determine the abundance of DGTT2 mRNA in the seedlings of overexpressors (15 d old), we used quantitative RT-PCR. The expression of DGTT2 relative to ACTIN2 ranged from 2 to 7 (ratio of DGTT2/ACTIN2) in the independent transgenic lines tested (see Supplemental Figures 2A to 2D online), while no transcripts were detected in the wild type.

DGTT2 Production Causes Accumulation of TAGs with VLCFAs

To determine the effect of DGTT2 production on the accumulation of TAG, we analyzed 15-d-old whole seedlings by ESI-MS. The TAG levels in 15-d-old seedlings of transgenic lines were increased up to 22- and 25-fold (Figure 3A). Lines 22 and 57 contained 0.88 and 1.00% TAG per DW, respectively, compared with 0.04% TAG per DW in the wild type. The increase in TAG levels correlated well with the relative abundance of DGTT2 transcript in each line (see Supplemental Figure 2D online). Comparison of ESI-MS spectra of neutral lipid extracts from the wild type and line 57 indicated an abundance of TAG molecular species containing VLCFAs in the latter (Figures 3B and 3C). The presence of long and VLCFA-containing TAG molecular species in the seedling extracts of line 57 was further confirmed by electrospray ionization–tandem mass spectrometry (ESI-MS/MS) (Figure 3D), which produced product ions with masses consistent with the loss of VLCFAs. These spectra were consistent with each TAG molecule containing only one VLCFA.

Figure 3.
Figure 3.

DGTT2 Leads to the Accumulation of TAG with VLCFAs in Arabidopsis Seedlings (15 d Old) and Soil-Grown Plants (6 Weeks Old).

(A) ESI-MS quantification of TAGs in neutral lipid extracts of seedlings and soil-grown plants of wild-type (WT) and homozygous transgenic plants producing DGTT2 (n = 4; average ± sd).

(B) and (C) Positive-ion electrospray ionization mass spectra of neutral lipid extracts from seedlings of the wild type and transgenic line 57. Tritridecanoin (tri13:0) and tripentadecanoin (tri15:0) TAGs were added as internal standards.

(D) ESI-MS/MS analysis of neutral lipid extracts of line 57. Shown are the daughter fragment ions from TAGs with [M + NH4]+ adducts with mass-to-charge ratio values of 929, 957, and 983. The mass-to-charge values were rounded up to the nearest nominal mass.

We also used ESI-MS to analyze the TAG content in the leaves of 6-week-old soil-grown plants before bolting. As shown in Figure 3A, TAG levels in transgenic lines 22 and 57 were 0.23 and 0.36% of DW, an 11- and 18-fold increase relative to wild-type plants (0.02% TAG per DW), respectively. These increases were considerably lower compared with those observed with young seedlings and might reflect turnover of TAG in more mature leaves. The ESI-MS spectrum of neutral lipid extracts from 6-week-old leaves of line 57 confirmed the presence of TAG molecular species containing VLCFAs, which were not detected in the wild type (see Supplemental Figures 3A and 3B online). The presence of long and VLCFAs in neutral lipid extracts of soil-grown line 57 was also confirmed by ESI-MS/MS (see Supplemental Figure 3C online). Interestingly, the level of TAGs with VLCFAs (e.g., C24:0) in leaves of soil-grown plants of line 57 was considerably higher (∼8 to 10 mol %) than the wild type (∼2 mol %) (see Supplemental Figure 4 online). Using an independent method, we also confirmed the levels of TAG in transgenic soil-grown plants by transmethylation of thin layer chromatography (TLC)–separated TAG and gas chromatography–flame ionization detection analysis of the resulting FA methyl esters (FAMEs; see Supplemental Figure 5 online).

An Abundance of Oil Droplets in the Leaves of Transgenic Lines

Leaves of 6-week-old TAG-accumulating line 57 and of the wild type were compared by confocal microscopy following Nile Red staining and by transmission electron microscopy (TEM). Oil droplets were abundant in line 57 and were distributed in the proximity of the chloroplasts (Figures 4A to 4C). By contrast, few or no oil droplets were observed in the wild-type leaf sample (Figures 4D to 4F). We used TEM to analyze the location of oil droplets in leaf sections. In line 57, large and distinct electron-dense oil droplets were observed outside the chloroplast in mesophyll cells, most likely associated with the ER (Figure 4H). No oil droplets were observed in cells of wild-type sections (Figure 4G). The shape of chloroplasts and distribution of starch granules were similar in both wild-type and transgenic lines.

Figure 4.
Figure 4.

Oil Droplets Are Abundant in Leaves of DGTT2 Transgenic Line 57.

(A) to (C) Confocal fluorescence image of leaf mesophyll cells of line 57, showing chloroplasts (red) and oil droplets (OD; arrows, green) stained with Nile red. Bar = 5 μm.

(D) to (F) Confocal fluorescence image of leaf samples from the wild type of the same age as line 57 (6 weeks old, soil-grown). Bar = 5 μm.

(G) and (H) TEM analysis of the first leaf pair from 6-week-old wild type (G) and transgenic line 57 (H). Oil droplets (OD), chloroplasts (CH), and starch granules (S) are indicated. Bars = 500 μm.

Epidermal Surface Lipids Are Reduced in the Transgenic Lines

We suspected that a group of lipids specifically affected by the ectopic accumulation of TAGs with VLCFAs in leaves might be epidermal surface lipids. To test this hypothesis, leaf epidermal cell sections of DGTT2 transgenic line 57 and wild-type plants were examined by TEM. Plants of line 57 had leaves with a noticeably less osmium-dense cuticle than wild-type plants (Figures 5A and 5B). Line 57 also contained very distinct and large numbers of presumed oil droplets in epidermal and mesophyll cells, including guard cells. Similar oil droplet–like structures were not observed in epidermal cells of wild-type plants. Leaves of transgenic line 57 and wild-type plants were further examined by scanning electron microscopy (Figures 5C and 5D). By comparison, line 57 leaves produced relatively fewer rod-like wax crystals on the surface of epidermal cells (Figure 5D). To observe quantitative and qualitative changes in wax composition, the rosette leaf waxes of lines 14, 22, and 57 and the wild type (6 weeks old) were subjected to gas chromatography–mass spectrometry (GC-MS) analysis. We observed a considerable change in the wax composition, specifically a decrease in nonacosane (C29 alkane), which is the most abundant leaf wax constituent, and triacontanol (C30 1-alcohol) in the DGTT2 transgenic lines (Figure 5E). However, no statistically significant differences were detected between wild-type and DGTT2 transgenic lines in the total wax amounts (Table 1).

Figure 5.
Figure 5.

Effect of DGTT2 on Cuticular Wax Production in Arabidopsis.

(A) and (B) Transmission electron microscopy of leaf epidermal sections of the wild type (A) and DGTT2 transgenic line 57 (B). Oil droplets (OD) were only present in the transgenic line. The arrows point at the cuticle (CT), showing a layer of osmium dense material. The cell wall (CW) is indicated. Bars = 200 nm.

(C) and (D) Scanning electron microscopy of leaves of the wild type (C) and transgenic line 57 (D). Rod-like wax crystals are observed on the surface of wild-type plants near the base of trichomes but are much less abundant on the surface of cells of the transgenic line. Bars = 5 μm.

(E) Leaf epicuticular wax profile of wild-type (WT) plants and transgenic lines as indicated. The number of carbons in the respective compound class is indicated. Significant at *P < 0.05 or **P < 0.01 compared with the wild type by one-way ANOVA with post-hoc Dunnett’s test (n = 3; average ± sd).

View this table:
Table 1. Total Amounts (nmol/mg DW) of Identified Leaf Wax and Cutin Constituents of DGTT2-Expressing Lines

Furthermore, we observed changes in cutin and cutin-associated features in the transgenic lines. Because alteration of cutin content and composition is often associated with changes in the cuticular ledge ultrastructure of guard cells (Kosma and Jenks, 2007; Li et al., 2007), we focused on the stomata of leaves of line 57 and the wild type using scanning electron microscopy and TEM. Scanning electron microscopy showed that leaves of line 57 had an altered stomatal structure, with wider stomatal openings (Figures 6A and 6B). TEM images revealed smaller and thinner cuticular ledges in guard cells of line 57 (Figures 6C and 6D). These observations led us to investigate leaf cutin monomer composition. Ectopic production of DGTT2 resulted in a 25 to 37% reduction in total identified cutin monomer content (Table 1). In particular, the α,ω-dicarboxylic acid (DCA) content of DGTT2 transgenic lines was lower, with slight reductions in 16:0 DCA and 18:1 DCA amounts (22 to 37% and 18 to 30% reductions, respectively, P < 0.001, n = 4) and particularly large reductions in 18:2 DCA content (30 to 44% reduction, P < 0.001, n = 4; Figure 6E).

Figure 6.
Figure 6.

Effect of DGTT2 on Cutin and Related Phenotypes in Arabidopsis.

(A) and (B) Scanning electron microscopy images of stomata from the adaxial surface of leaves of the wild type (A) and line 57 (B). Bars = 5 μm.

(C) and (D) TEM images of sections of leaf guard cells. Arrows indicate the cuticular ledge of the guard cells of the wild type (C) and line 57 (D). Bars = 2 μm.

(E) Leaf cutin monomer profile of the wild type (WT) and DGTT2 transgenic lines as indicated. Significant at ***P < 0.001 compared with the wild type by one-way ANOVA with post-hoc Dunnett’s test (n = 4; average ± sd).

Composition of Sphingolipids Is Altered in DGTT2 Lines

Given that DGTT2 accepts a broad range of acyl-CoA substrates, we hypothesized that expression of DGTT2 could result in reduced sphingolipid content in the leaves. We analyzed sphingolipid content in wild-type and transgenic lines using electrospray ionization–high-resolution/accurate mass spectrometry to identify glycosyl inositolphosphoceramide (GIPC) lipids. Normalization of GIPC lipid abundances against an internal standard (d18:1/12:0 lactosyl ceramide) demonstrated that in wild-type plants, the t18:1/h24:0 and t18:1/h24:1 molecular species were the most abundant (Figure 7A), constituting 46.2% ± 1.65% and 26.9% ± 0.05% of all GIPC lipids, respectively. Notably, line 57 (Figure 7A) exhibited 29.5 and 76.6% increases in levels of t18:1/h24:0 GIPC (P < 0.001, n = 3) and t18:1/h26:1 (P < 0.01, n = 3) relative to wild-type plants, respectively, and a concomitant 68.7% decrease in the level of t18:1/h24:1 GIPC (P < 0.001, n = 3). These data suggest that DGTT2 production in the leaves of transgenic plants reduces 24:1 FA content in GIPC lipids, while enriching the GIPC content of 24:0.

Figure 7.
Figure 7.

Analysis of Arabidopsis Sphingolipid Molecular Species by High-Resolution/Accurate Mass Spectrometry.

(A) Relative abundances of individual GIPC molecular species containing t18:1 long-chain bases in the wild type (WT) and line 57. t test significant at **P < 0.01, or ***P < 0.001 versus the wild type (n = 3; average ± sd).

(B) Relative abundances of individual GlcCer molecular species containing t18:1 long-chain bases in the wild type and line 57. t test significant at *P < 0.05, **P < 0.01, or ***P < 0.001 versus the wild type (n = 3; average ± sd).

To determine whether DGTT2-dependent modulation of sphingolipid 24:1 and 24:0 FA levels was specific to GIPC lipids or distributed across other sphingolipid classes, we also analyzed the less abundant glucosylceramide (GlcCer) and ceramide (Cer) lipids. As shown in Figure 7B, the presence of DGTT2 altered the content of t18:1 GlcCer lipids, as line 57 exhibited 39.8% (P < 0.05, n = 3) and 46.1% (P < 0.001, n = 3) higher levels of h22:0 and h24:0 FAs, respectively, compared with the wild type. As observed for the GIPC lipids, h24:1 species of t18:1 GlcCer were reduced in the line 57 by 33.5% (P < 0.01, n = 3) relative to the wild type. Only three molecular species of d18:1 GlcCer were detected (see Supplemental Figure 6D online), which contained h16:0, h24:0, and h24:1 FAs. Additionally, line 57 also contained 39.3% decreased levels (P < 0.01, n = 3) of h24:1 FA in t18:1 Cer relative to the wild type (see Supplemental Figure 6A online), while h26:0 increased 66%. However, no statistically significant differences were detected between the wild type and line 57 in the total abundances of these lipids (see Supplemental Figures 6A to 6D online). Together, these results suggest that decrease in C24:1 of sphingolipids reflects channeling of VLCFAs into TAG (Figure 3D; see Supplemental Figure 3C online).

Limited Changes in Global Expression of Genes in DGTT2 Lines

To evaluate possible compensatory mechanisms in the transgenic lines, we conducted microarray experiments with RNA extracts from the leaves of 6-week-old soil-grown plants. A two-factor mixed-model analysis of variance (ANOVA) analysis was employed to account for the effects of genotype and chip, and P values were calculated for the wild type versus line 57. Since no meaningful hits could be identified after a false discovery rate correction, 15 genes were selected using the unadjusted P value threshold of <0.002 and fold change >2 or <−2 (see Supplemental Table 1 online). Of the 15 genes selected from the microarray study, 10 were upregulated and five downregulated (Table 2). Two upregulated genes were predicted to encode enzymes with a known or possible involvement in TAG biosynthesis, including a bifunctional enzyme WSD1 (wax ester synthase and diacylglycerol acyltransferase), At5g37300 (Li et al., 2008), and a gene encoding an HXXXD-type acyltransferase, At1g65450. We also identified the qua-quine starch (QQS) regulatory gene, At3g30720, and cytosolic β-amylase encoding gene, At4g15210. Both proteins are involved in starch metabolism (Li et al., 2009). One of the downregulated genes with unknown function was predicted to encode a mini zinc finger protein, At3g28917, and another a UDP-glycosyltransferase superfamily protein, At2g23210.

View this table:
Table 2. Changes in the Gene Expression in the Leaves of DGTT2 Plants

To validate the microarray analysis, we quantified the transcript levels of the four genes relevant to oil biosynthesis and starch metabolism using quantitative RT-PCR. For this experiment, RNA was isolated from an independent set of wild-type and line 57 plants. When normalized to wild-type values, the transcript levels for the WSD1-encoding gene were 3.5-fold higher, for the HXXXD-type acyltransferase protein-encoding gene 4.4-fold, for the QQS gene 4.3-fold, and for the cytosolic β-amylase–encoding gene 3.0-fold elevated in line 57, thus confirming the microarray results (see Supplemental Figure 7 online).

Growth, Starch, and Heating Value of DGTT2 Lines

The DW of the aerial parts of soil-grown transgenic line 57 and wild-type plants was measured on a weekly basis. The overall morphology of the leaves and shoots from the DGTT2 transgenic line was unchanged. Transgenic line 57 gained a considerably higher biomass than the wild type (105.6 and 87.6 mg DW per seedling, P < 0.001, n = 3) during the first 5 weeks after germination, and DW values continued to be higher until the end of the experiment (Figure 8A). The growth rate of transgenic line 57 and wild-type plants slowed after the 8th week.

Figure 8.
Figure 8.

Growth and Starch Content of the Wild Type and DGTT2 Transgenic Line 57.

(A) The DW of the aerial parts of Arabidopsis plants was measured on a weekly basis for a period of 8 weeks. Each time point represents the average value of at least six individual plants. Black symbols represent the wild type (WT) and gray symbols transgenic line 57. t test significant at *P < 0.05, **P < 0.01, or ***P < 0.001 versus the wild type (n = 6; average ± sd).

(B) Starch content of leaves of the wild type and line 57 (6 weeks old). t test significant at **P < 0.01 versus the wild type (n = 5; average ± sd).

The increased transcript levels of the QQS gene and cytosolic β-amylase–encoding gene in transgenic line 57 from the microarray experiments led us to investigate the starch content of DGTT2 transgenic plants. Contrary to expectation, given the increase in the expression of the genes encoding QQS and β-amylase, 6-week-old plants of line 57 accumulated 18.7% more starch than wild-type plants (58.0 and 47.1 µg Glu per mg DW [P < 0.01, n = 5], respectively) (Figure 8B). Heating values were calculated for 6-week-old line 57 and wild-type plants based on an elemental analysis of dry biomass in the presence of oxygen. Consistent with the increase in FA content, a slight increase was observed for line 57 (14.1 ± 0.2 mJ/kg DW, compared with the wild type 13.9 ± 0.4 mJ/kg DW; see Supplemental Table 2 online), but this difference was not statistically significant.

Caterpillars Feeding on DGTT2 Lines Gained More Weight

Increased TAG accumulation or decreased wax and cutin in vegetative tissue may result in an unusual diet for phytophagous organisms and may perhaps affect the nutritional value of the plants. To test this hypothesis, we conducted a “no choice” insect feeding assay, in which the generalist lepidopteran herbivore Spodoptera exigua was reared on the wild type and transgenic lines 22 and 57 (Figures 9A and 9B). Newly hatched larvae were caged in pots containing 6-week-old plants of two transgenic lines (22 and 57) and the wild type and were allowed to feed and grow for 14 d. At the end of the feeding trial, larvae were recovered and their body mass was determined. No visible difference in plant consumption was observed between DGTT2-producing lines and the wild type during the 14-d trial (Figure 9A). However, insects grown on DGTT2-producing lines were heavier (9.75 ± 3.42 mg larval weight) than larvae reared on wild-type plants (6.57 ± 1.39 mg larval weight) (P < 0.01, n = 95; Figures 9B and 9C).

Figure 9.
Figure 9.

S. exigua Feeding Assay and JA-Ile Measurements.

(A) and (B) Photographs of representative wild-type (WT) and DGTT2 transgenic plants (A) and larvae grown on each genotype (B) at the end of feeding trial.

(C) Average fresh weight of insects. t test significant at **P < 0.01 versus wild-type hosts (n = 95; average ± sd).

(D) JA-Ile accumulation in leaf tissue collected from undamaged control plants and those damaged during 14 d of insect feeding (n = 5; average ± sd). FW, fresh weight.

Active plant defense responses to insect herbivory are controlled by wound-induced production of the 18:3-derived phytohormone, jasmonoyl-l-isoleucine (JA-Ile) (Koo and Howe, 2012). The altered lipid content of DGTT2-producing plants raised the possibility that increased larvae performance was caused by reduced levels of JA-Ile in the transgenic lines. To test this idea, we quantified JA-Ile levels in leaf tissue from insect-fed and control undamaged plants (Figure 9D). Insect feeding increased the JA-Ile content in the damaged leaves of the wild type and two transgenic lines. In both control and damaged leaves, the amount of JA-Ile in DGTT2-producing lines was comparable to that observed in the wild type.

DISCUSSION

Different Roles for Different DGATs

High-throughput transcript profiling of C. reinhardtii under nitrogen deprivation showed that the expression levels and patterns of the five putative DGAT2-encoding genes varied considerably (Miller et al., 2010; Boyle et al., 2012). DGTT1 was the most highly regulated, being strongly upregulated following nitrogen deprivation. Based on the sequence analysis, DGTT2 and DGTT3 are the most similar. Additionally, they showed near-identical results in yeast assays. Although DGTT3 did show a change in expression during nitrogen deprivation, the overall change was less than twofold and statistically insignificant. For these reasons, it was assumed that their activity in Arabidopsis would also be similar and we focused only on one of the two isoforms.

Structural analysis of the DGAT2 candidates indicates that they are similar to previously identified DGAT2 proteins. The absence of predicted signal or targeting sequences may be explained by the fact that the programs were trained on land plant signal sequences, which could hinder the detection of such sequences in C. reinhardtii. Given the absence of obvious organellar targeting sequences, the most likely location is the ER membrane, which is the main site of TAG synthesis in plants (Lung and Weselake, 2006). Recently, however, the existence of a separate TAG assembly pathway in chloroplasts has been suggested (Fan et al., 2011; Goodson et al., 2011). It seems possible that some of the DGAT2 proteins are preferentially associated with the ER, whereas others are targeted preferentially to the chloroplast envelope by mechanisms that would not require classic targeting sequences.

Biochemically, DGTT2 possesses broad substrate specificity and is able to incorporate varying acyl-CoA species into TAG in vitro. However, it is unclear what its natural substrate in C. reinhardtii is. Both O. tauri DGAT2B and yeast Dga1 also have broad substrate specificity, in contrast with the more substrate specific type 2 DGATs found in land plants (Wagner et al., 2010). These, along with the relative abundance of type 2 DGATs in other green algae, suggest a more distinct role for these enzymes in algae, compared with plants. This difference may be related to the different roles and regulation of TAG synthesis, as microalgae produce oil in response to environmental stresses, whereas land plants produce oil primarily during seed development.

Ectopic Production of DGTT2 Results in TAG Accumulation in Arabidopsis

Most of the DGTT2-producing Arabidopsis lines examined developed distinct seedling phenotypes, such as accelerated hypocotyl elongation and pale-green cotyledons. The increased hypocotyl length was presumed to be due to excess storage compounds, such as TAG in the seeds that may accelerate cell elongation compared with the wild type. The reduction in chlorophyll content in the DGTT2 seedlings could be related to the reduction in the major plastid lipids, limiting the extent of the photosynthetic membrane. The fact that this phenotype is only visible at the seedling stages, when leaves need to rapidly expand and demands on membrane lipid biosynthesis are high, supports this hypothesis. Thus, it seems likely that the acyl-group supply is limited and TAG accumulation in leaves diverts acyl groups away from other pathways, such as plastid glycerolipid biosynthesis, without a compensatory increase in acyl synthesis. This hypothesis is supported by the microarray results, which revealed no significant induction of genes encoding enzymes necessary for FA biosynthesis (Table 2; see Supplemental Table 1 online).

In general, plants accumulate TAG in seeds as stored energy that can be subsequently used to fuel germination and seedling establishment. We demonstrated that accumulation of TAG with VLCFA, such as C20:0 and longer, in green seedlings and leaves of soil-grown plants can be achieved by expression of DGTT2, even though VLCFA-containing glycerolipids do not normally accumulate in green tissues of Arabidopsis. This observation was unexpected because C. reinhardtii TAG primarily contains C16:0 and C18:1 FAs, but no TAG species containing VLCFAs (Fan et al., 2011). Following the ectopic production of DGTT2 in the leaves of Arabidopsis, most likely the availability of particular acyl-CoA species as well as the substrate preference of DGTT2 for VLCFAs leads to the observed composition of the TAG produced in the transgenic lines. It is interesting to note that the amount of TAG accumulated in DGTT2 transgenic seedlings was higher than that of soil-grown plants (Figure 3A), while the relative abundance of major chloroplast lipids, such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), was reduced (see Supplemental Figure 8 online). These seedlings had increased levels of 16:3-containing molecular species of MGDG but fewer 18:3-containing species, similar to mutants affected in ER-to-plastid lipid trafficking (Xu et al., 2003), suggesting that FA intermediates normally transported from the ER to the chloroplast as part of the eukaryotic pathway of thylakoid lipid biosynthesis are instead channeled into TAG. Compared with seedlings, the lower levels of TAG that accumulated in the leaves of soil-grown DGTT2 transgenic plants may perhaps be explained by FA catabolism in older leaves (Yang and Ohlrogge, 2009).

Acyl Group Diversion from Sphingolipids and Surface Lipids to TAG

In plants, FAs are synthesized in plastids, and following elongation and modification at the ER they provide the precursors for the synthesis of sphingolipids and surface lipids, such as cutin and waxes (Chen et al., 2009; Kunst and Samuels, 2009). Sphingolipids are composed of a Cer backbone that consists of a long-chain base (C18 long) to which a FA (C16-C26) is bound by an amide linkage. Sphingolipids are an important lipid component of the plasma membranes of plant cells. They are known to play roles in membrane structure, signal transduction, and programmed cell death (Chen et al., 2009). GlcCer and GIPC lipids are the two major classes of sphingolipids in plants (Chen et al., 2008; Li-Beisson et al., 2010; Chao et al., 2011). In Arabidopsis, the cutin polymer is comprised of DCAs, ω-hydroxy FAs, and glycerol monomers, which are embedded with and covered by waxes (Bonaventure et al., 2004; Franke et al., 2005). The increase in TAG molecular species containing VLCFA in the leaves of DGTT2 transgenic lines is consistent with a decreased carbon flux into the sphingolipid and surface lipid biosynthetic pathways. More specifically, the increased levels of 18:2 and 24:1 in TAG of DGTT2 lines and concomitant decrease in 18:2 DCA content of cutin, t18:1/h24:1 of sphingolipids and 18:3 of MGDG and DGDG (see Supplemental Figure 9 online), and the observed decrease in very-long-chain alkane content of surface waxes suggests a redirection of saturated and unsaturated acyl-chains from sphingolipids, surface lipids, and galactolipids into TAG, mediated by DGTT2 (Figure 10). Surface lipid metabolism is restricted to epidermal cells. The TEM analysis clearly demonstrated an alteration in epidermal cell lipid metabolism in DGTT2 lines, apparent by the accumulation of oil droplets in both pavement and guard cells, and altered cuticle and guard cell cuticular ledge ultrastructure.

Figure 10.
Figure 10.

A Schematic Overview of Altered Diversion of Acyl-CoA Groups from Other Pathways by DGTT2 in Arabidopsis Leaves.

The hatched arrow represents the activity of DGTT2 in channeling acyl-CoAs from wax/cutin/sphingolipids to the synthesis of TAGs with VLCFAs in the transgenic lines. CW, cell wall; DAG, diacylglycerol; FAE, fatty acid elongation; FAS, fatty acid synthesis; G3P, glycerol-3-phosphate; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; PA, phosphatidic acid; PM, plasma membrane.

[See online article for color version of this figure.]

The microarray results suggested no change in the expression of any known sphingolipid or surface lipid biosynthetic pathway genes other than WSD1. WSD1 is a bifunctional enzyme with both wax synthase and DGAT activities. However, WSD1 is known for its role in wax ester synthesis in inflorescence stems of Arabidopsis (Li et al., 2008). Wax esters are generally not present in the leaves of Arabidopsis, and our leaf wax analysis did not reveal the presence of wax esters in leaf waxes of DGTT2 transgenic lines. Whether or not WSD1 plays a role in the observed TAG accumulation of DGTT2 transgenic lines via its DGAT activity remains to be determined.

In agreement with Wagner et al. (2010), in vitro assays demonstrated that DGTT2 was capable of using both saturated and unsaturated acyl-CoAs of several different carbon chain lengths. Interestingly, the ESI-MS/MS spectra of the VLCFA-containing TAGs from the transgenic lines are consistent with these molecular species only containing one VLCFA (see Supplemental Figure 3C online; Figure 3D). This observation is consistent with DGTT2 adding a VLCFA to a DAG acceptor molecule. As wild-type Arabidopsis leaves typically do not possess VLCFAs in the TAG fraction (Li-Beisson et al., 2010), we can assume that the DAG-derived portion of TAG in DGTT2 lines did not contribute the VLCFA. This is further supported by the lack of VLCFAs in DAG and phosphatidic acid pools of DGTT2 transgenic plants (see Supplemental Figure 10 online). Thus, it is unlikely that the supply of the acyl acceptor for TAG biosynthesis by the Kennedy pathway is limiting. Rather, the observed diversion of acyl groups from other pathways (Figure 10), such as sphingolipid, surface lipid, or thylakoid lipid biosynthesis, suggests that the supply of acyl donors (acyl-CoAs) is limiting and that the broad substrate specificity of DGTT2 is responsible for the observed TAG molecular compositions.

However, the increased transcript level of WSD1 in the transgenic lines opens the possibility that this enzyme is involved in the elevated TAG levels observed in DGTT2-producing leaves as well. It is similar to a unique type of long-chain acyltransferase exhibiting both WS and DGAT activities (ADP1), which was identified in A. calcoaceticus (Kalscheuer and Steinbüchel, 2003). It has been shown that the WS reaction of ADP1 accepts a wide range of different chain length acyl-CoAs as substrates. However, the DGAT reaction preferred acyl-CoAs of greater chain lengths (Kalscheuer and Steinbüchel, 2003). Heterologous expression of ADP1 in a TAG synthesis–deficient quadruple mutant of yeast restored TAG synthesis (Kalscheuer et al., 2004). The petunia homolog of ADP1 failed to restore TAG synthesis in yeast due to lack of DGAT activity (King et al., 2007), but the Arabidopsis homolog, WSD1, had WS and DGAT activities (Li et al., 2008). Why the WSD1 gene is induced in the transgenic lines and whether WSD1 plays a role in TAG accumulation in the DGTT2 transgenic lines remains to be seen, especially since it is unclear whether WSD1 has an acyl chain length preference for its DGAT activity. Because DGTT2 showed affinity for C22:1 acyl-CoA in the in vitro assays, it is reasonable to assume that the activity of DGTT2 contributes to channeling long-chain CoAs from sphingolipids/surface lipids to the synthesis of TAGs with VLCFAs in the transgenic lines (Figure 10).

Enhancing the Nutritional Content of Vegetative Biomass

The microarray results suggested that in the DGTT2 transgenic lines, nonlipid pathways, especially starch biosynthesis, may be affected, which was confirmed by observing an increase in starch content (Figure 8B). The two starch-related genes upregulated in transgenic lines encode the regulatory protein QQS and a cytosolic β-amylase. Transgenic lines in which QQS levels are reduced have been shown to accumulate more leaf starch content (Li et al., 2009), seemingly in contradiction with the phenotypes observed for the DGTT2 transgenic lines. Similarly, in vitro studies suggested that β-amylase could play a role in starch degradation, even though a mutation in the respective gene did not alter leaf starch content (Laby et al., 2001). One explanation is that these two genes could be induced to compensate for the increase in starch content, rather than being responsible for the increase. Alternatively, the increased accumulation of these two transcripts may suggest a connection between lipid and carbohydrate metabolism. Whether the observed effects on starch metabolism and related genes have to be considered a pleiotropic effect or need to be interpreted in the context of altered carbon flow in the DGTT2 transgenic lines remains to be determined.

In principle, the enhanced TAG content should lead to increased heating values reflecting an increase in energy density, assuming the gains are not negated by other compositional changes. Only slight differences in growth rates of transgenic lines and the wild type implied that the ectopic introduction of DGTT2 activity in Arabidopsis had no detrimental effects under the employed laboratory conditions. The observed subtle increase in total heating value in the DGTT2 transgenic line is consistent with the expected contribution of total FAs/TAG toward total heating values in the leaves, which we calculated to be ∼0.7%. Our results also indicate that metabolic changes caused by DGTT2 activity do not negatively affect the growth and development of the insect herbivore S. exigua. The growth of S. exigua larvae was positively correlated with the increased levels of TAG and starch in the transgenic lines. A variety of factors may contribute to increased herbivore performance, including increased energy content (e.g., essential dietary FAs), increased digestibility of the biomass due to altered surface lipids, or decreased production of anti-insect compounds, such as glucosinolates. Either way, this study demonstrates the potential benefits of engineering TAG content in transgenic crops to increase energy density for biofuel production or enhanced nutritional value for animal feed.

METHODS

Phylogenetic Analysis

The protein sequences for the Chlamydomonas reinhardtii DGTT1-5 genes were retrieved by sequence comparison with Arabidopsis thaliana DGAT2 using BLAST (Altschul et al., 1997). Sequences of DGTT1-5 and other DGAT2 proteins were aligned using the ClustalW software in MEGA5 (Tamura et al., 2011). The alignment was then used to construct a phylogenetic tree using the neighbor-joining method in MEGA5, with the tree being tested by bootstrapping with 1000 replicates.

Plasmid Construction

C. reinhardtii strain dw15.1 (cw15, nit1, mt+), provided by Arthur Grossman, was grown under continuous light (∼80 µm/m2/s) and at 22°C in liquid Tris-Acetate Phosphate (TAP) media (Harris, 1989) until mid-log phase and then pelleted. Total RNA was extracted from the cells using a Qiagen RNeasy Plant Mini kit. cDNA was synthesized with Invitrogen SuperScript III and oligo(dT) primer and used as a template for PCR. The primers used are listed in Supplemental Table 3 online. The amplified regions were digested with HindIII and SphI, for DGTT2 and DGTT4, and HindIII and XhoI, for DGTT3 and DGTT5. The gene sequences were then subcloned into the Invitrogen pYES2 vector to form pYES2-DGTT2-5 for expression in yeast Saccharomyces cerevisiae.

DGTT2 cDNA was amplified from pYES2-DGTT2 by PCR using gene-specific primers (see Supplemental Table 3 online). A fragment of 975 bp containing the complete open reading frame was digested with BamHI and EcoRI. This fragment was then placed between the cauliflower mosaic virus 35S promoter and OCS terminator of vector p9-35S-OCS (DNA Cloning Service) to form 35S:DGTT2.

Yeast Expression

Yeast strain H1266 (are2Δ lro1Δ dga1Δ) (Sandager et al., 2002) was grown to mid-log phase in yeast extract peptone dextrose (YPD) media (Sherman, 2002) and transformed with the DGTT constructs, along with an empty pYES2 vector as a negative control, according to Gietz et al. (1995). The transformants were selected on Synthetic Complete (SC) medium (Sherman, 2002) with 2% Glc and the uracil omitted (SC-U). Colonies were picked and grown overnight in SC-U + 2% Glc, before being transferred to SC-U + 2% Gal and 1% raffinose. After 48 h, 30 mL of the cultures were collected and pelleted by centrifugation for 5 min at 3000g. Lipids were extracted and analyzed as described below.

Microsome DGTT2 Assays

Transformed yeast colonies were picked and grown in SC-U + 2% Glc overnight and then transferred to SC-U + 2% Gal and 1% raffinose. The cells were harvested after 12 h, and the microsomes were prepared as described (Milcamps et al., 2005). The total protein concentration was measured using Bio-Rad Protein Assay Dye Reagent by adding 900 μL of the reagent to 20 μL of BSA standards or microsome samples. Fifty nanograms of microsome were added to a mix containing 100 mM Tris, 8 mM MgCl2, 1 mg/mL BSA, 20% glycerol, 0.25 mg/mL DAG, and 1.725 nmol [1-14C]-16:0-acyl-CoA or [1-14C]-18:1-acyl-CoA (Moravek Biochemicals). The reaction was incubated at room temperature for 1 h. The lipids were extracted with chloroform:methanol (1:1 [v/v]) and phase separated with 0.2 M H3PO4 and 1 M KCl. The organic layer was extracted and separated on a silica TLC plate using 80:20:1 (v/v/v) petroleum ether:ethyl ether:acetic acid as the solvent. The TLC plate was exposed to film for 72 h to visualize the radiolabeled lipids.

For the competition assay, 1.725 nmol unlabeled 16:0-acyl-CoA, 18:1-acyl-CoA, or 22:1-acyl-CoA was added. The separated TAG bands were scraped from the plate and counted in a scintillation counter to quantify the amount of radioactivity incorporated into the lipid.

Plant Material and Generation of Arabidopsis Transgenic Plants

Arabidopsis wild-type (Columbia 2) seeds or transgenic seeds were surface sterilized and grown on half-strength Murashige and Skoog (Murashige and Skoog, 1962) agar plates containing 1% Suc in a growth chamber adjusted to 16 h light/8 h dark (100 µm/m2/s) at 22°C after 3 d of stratification at 4°C. Fifteen-day-old wild-type and transgenic plants were transferred onto soil and grown in a growth chamber at 16 h light/8 h dark (100 µm/m2/s) and 22°C. These plants were harvested at 6 weeks for lipid and metabolic assays. Starch analysis was performed using a Megazyme kit as per the manufacturer’s instructions (Megazyme). For insect assays, transgenic lines and the wild type were grown in a short-day growth chamber at 12 h light/12 h dark (100 µm/m2/s) at 22°C. The binary vector containing DGTT2 was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. Arabidopsis was transformed using the flower dip method (Clough and Bent, 1998). Transgenic plants (T1) were selected on half-strength Murashige and Skoog agar plates containing 1% Suc and 100 mg/L kanamycin. Wild-type and homozygous transgenic seedlings were grown on the same shelf of the growth chamber when used for lipid, metabolite, and insect assays.

Lipid Isolation and Quantification

Soil-grown 6-week-old and 15-d-old (grown on half-strength Murashige and Skoog agar plates containing 1% Suc) wild-type and transgenic Arabidopsis plants were freeze dried. Neutral lipids were extracted from dried samples using chloroform:methanol (1:1 [v/v]) with 100 μM internal standard tri15:0 TAG and separated on a silica TLC plate using a mixture of solvents consisting of petroleum ether:ethyl ether:acetic acid (80:20:1, by volume). TAG bands were isolated from the TLC plate after separation, dissolved in toluene with 10 μM tri13:0 TAG internal standard, and assayed using ESI-MS as previously described (Durrett et al., 2010). To quantify the amount of TAG accumulating in yeast expressing the DGTT constructs, neutral lipids were extracted from the yeast pellets and submitted for ESI-MS, following the method described by Durrett et al. (2010).

Distinct lipid and TAG bands were scraped from the TLC plates and used to prepare FAMEs by acid-catalyzed transmethylation. Identification and quantification of FAMEs was performed as previously described (Xu et al., 2003). The amounts of lipids were calculated based on the content of FAs derived from GC using C15:0 as an internal standard.

Microscopy

For oil droplet visualization and TEM, the leaf samples from 6-week-old soil-grown transgenic and wild-type plants were used. Whole leaf samples for TEM were fixed in a mixture of 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M cacodylate buffer at 4°C for 24 h, postfixed in 1% osmium tetroxide, and dehydrated in a graded acetone series. Samples were infiltrated and embedded in Spurr resin (Polysciences). Thin sections were imaged using a JEOL 100CX transmission electron microscope at a 100-kV accelerating voltage. Freshly harvested leaf samples were used for oil droplet visualization by confocal microscopy as previously described (Sanjaya et al., 2011). For scanning electron microscopy, leaves from 6-week-old transgenic line 57 and wild-type plants were plunge frozen in liquid nitrogen, freeze dried with an EMS750X Turbo Freeze Drier (Electron Microscopy Sciences), and mounted on aluminum stubs using carbon tape (Ted Pella). These samples were coated with gold (∼20-nm thickness) for 3.5 min in an Emscope Sputter Coater model SC 500 (Ashford) purged with argon gas and coated with osmium (∼10-nm thickness) in an NEOC-AT osmium coater (Meiwafosis) for 35 s. The samples were examined in a JEOL JSM-6400V (Lanthanum Hexaboride electron emitter) scanning electron microscope. Digital images were acquired using Analysis Pro software version 3.2 (Olympus Soft Imaging Solution) at the Center for Advanced Microscopy, Michigan State University.

Analysis and Quantification of Sphingolipid Classes

Leaves of soil-grown (6-week-old) plants were used for the extraction of sphingolipids, as previously described (Markham et al., 2006). Sphingolipid identification and quantification from wild-type and line 57 samples was performed by electrospray ionization–high-resolution/accurate mass spectrometry operating in positive ion mode. All solvents used were HPLC grade or the highest grade available. Isopropanol, methanol, and chloroform were purchased from Macron Chemical. Hexane and HPLC water were from Fisher. Ammonium formate was purchased from Alfa Aesar. The sphingolipid internal standard mixture used for mass spectrometry analysis was Avanti Sphingolipid Mix II (Avanti Polar Lipids). Around 30-μL aliquots of total lipid extract were dried under nitrogen and subjected to mild alkaline hydrolysis of glycerolipids by the addition of 500 μL of methanol, 250 μL of chloroform, and 75 μL of 1 M KOH. Samples were incubated for 2 h at 37°C in a shaking water bath and then neutralized with 6 μL of glacial acetic acid (Merrill et al., 2005). For Cer and GlcCer analysis, samples were reextracted by a modified Folch method (Busik et al., 2009) and resuspended in 300 μL of isopropanol/methanol/chloroform (4:2:1 [v/v/v]). For GIPC lipid analysis, dried samples were extracted into 300 μL of isopropanol/hexane/water (3:1:1 [v/v/v]) (Markham et al., 2006), centrifuged to remove particulates, and the supernatants saved for analysis. Samples were further diluted 30-fold in isopropanol/methanol/chloroform (4:2:1 [v/v/v], for Cer and GlcCer analysis) or isopropanol/hexane/water (3:1:1 [v/v/v], for GIPC analysis) containing a final concentration of 20 mM ammonium formate and the sphingolipid internal standard mixture diluted to 100 nM of each sphingolipid species. Samples were centrifuged and loaded into Whatman Multichem 96-well plates (Sigma Aldrich) sealed with Teflon Ultra Thin Sealing Tape (Analytical Sales and Services).

Samples were directly infused into a Thermo Scientific model LTQ Orbitrap Velos high-resolution/accurate mass spectrometer using an Advion Triversa Nanomate nano-electrospray ionization source (Advion) with a spray voltage of 1.4 kV and a gas pressure of 0.3 p.s.i. The ion source interface settings (inlet temperature of 100°C and S-Lens value of 50%) were optimized to maximize the sensitivity for precursor ions while minimizing in-source fragmentation. High-resolution mass spectra were acquired using the FT analyzer operating at 100,000 resolving power, across the range of mass-to-charge ratio from 400 to 2000, and were signal averaged for 2 min. All mass spectra were recalibrated offline using XCalibur software (Thermo Scientific) and the exact masses of the sphingolipid internal standards d18:1/12:0 Cer, d18:1/12:0 glucosyl/galactosyl ceramide, d18:1/12:0 sphingomyelin, and d18:1/12:0 lactosyl ceramide. Automated peak finding, correction for 13C isotope effects, and quantitation of lipid molecular species were performed using Lipid Mass Spectrum Analysis software version 1.0 (Haimi et al., 2006) linear fit algorithm. Sphingolipids were quantitated as the sum of their [M+H]+ and [M+NH4]+ (when present) ion abundances against the d18:1/12:0 lactosyl ceramide internal standard. As no attempts were made to correct for differences in ionization efficiency among individual molecular species of Cer, glucosyl ceramide, and GIPC lipids, as well as the lack of an ideal internal standard for quantitation of GIPC lipids, sphingolipid molecular species are presented only as a fraction of the total normalized abundance of each lipid class.

Surface Wax and Cutin Analysis

Rosette leaves of 6-week-old soil-grown transgenic and wild-type plants were used for cutin and wax analysis, using slight modifications of methods previously described (Molina et al., 2006; Kosma et al., 2009). Briefly, surface waxes were extracted from leaves by a 30-s submersion in hexane. Internal standards pentadecanoic acid (C15:0), tricosanol (C23:0), and octacosane (C28:0) were added to the hexane extracts, and the solvent was removed by evaporation under nitrogen gas. Dried extracts were derivatized with 100 μL each of pyridine and N,O-bis(trimethylsilyl trifluoroacetamide) (BSTFA). Excess pyridine and BSTFA were removed by evaporation with N2, and samples were dissolved in heptane:toluene (1:1 [v/v]) for GC-MS analysis.

Thoroughly delipidated ground leaf tissues were used for cutin monomer analysis. Methyl heptadecanoate and pentadecalactone were used as internal standards. Base catalyzed transmethylation reactions (NaOMe/methanol) consisted of 4.5% sodium methoxide (NaOMe) and 7.5% methyl acetate in methanol in a total volume of 6 mL.

Reaction mixtures were heated overnight (∼16 h) at 60°C and then allowed to cool to room temperature. Reaction mixtures were acidified with glacial acetic acid to pH 4 to 5, 2 to 3 mL saline solution was added (0.5 M NaCl), and FAMEs were extracted with 7 mL CH2Cl2. The organic phase was washed twice with dilute saline solution (0.9% NaCl [w/v]) and dried over anhydrous Na2SO4. Extracts were evaporated to dryness under N2 and the product silylated to convert hydroxyl groups to their trimethylsilyl ethers using BSTFA and pyridine. Excess pyridine and BSTFA were evaporated under N2, and samples were dissolved in heptane:toluene (1:1 [v/v]) for GC-MS analysis. GC-MS temperature programs were as follows: wax, inlet 350°C, detector 320°C, oven 130°C for 3 min then increased at 5°C/min to 325°C, and 325°C for 10 min; cutin, inlet 330°C, detector 320°C, oven 140°C for 3 min and then increased at 5°C/min to 310°C and held at 310°C for 10 min.

Elemental Analysis

Rosette leaves of 6-week-old soil-grown transgenic line 57 and wild-type plants were harvested at the end of the day and thoroughly freeze-dried, ground into fine powder in a Retsch Mill at a frequency of 25 for 2 min, and used to measure carbon, hydrogen, sulfur, nitrogen, and ash content employing a commercial service (Elemental Analysis). Protocols are listed on the company website (http://www.elementalanalysis.com/). Calculation of heating value (mJ/kg DW) = 0.3491XC + 1.1783XH + 0.1005XS − 0.0151XN − 0.1034XO − 0.0211Xash; Xs are entered as mass percentages (Gaur and Reed, 1995).

Insect Feeding Assay and JA-Ile Quantification Using Mass Spectrometry

Insect feeding trials and JA-Ile analysis by liquid chromatography–tandem mass spectrometry were performed as previously described (Koo et al., 2011). Spodoptera exigua eggs (Benzon Research) were hatched at 30°C on an artificial insect diet (Southland Products) for 2 d and used for feeding experiments. Newly hatched neonates were allowed to grow on artificial diet for another 2 d before being transferred to 6-week-old plants. Ten larvae were reared per single pot, and each pot contained two plants of the same genotype. A total of 14 pots were used for each genotype. Larvae were caged in each pot using an inverted clear plastic cup with an opening on the top covered with Miracloth for air exchange. Plants with insects were grown in a growth chamber maintained at 21°C under a 12-h light (100 µm/m2/s) and 12-h dark cycle. Fresh weight of individual larvae was determined 14 d after the start of the feeding trial. For JA-Ile measurements, ∼350 mg of leaf tissue was harvested from insect damaged and undamaged control plants at the end of the trial. Jasmonate extraction and JA-Ile quantification by liquid chromatography–tandem mass spectrometry were performed as previously described (Koo et al., 2011).

Microarray and Data Analysis

Leaves from 6-week-old plants grown on soil at 22°C under 16 h light/8 h dark (100 µm/m2/s) were used for isolating total RNAs with the RNeasy plant mini kit (Qiagen). Subsequently, the total RNA samples were pretreated with RNase-free DNase I and cleaned with a Plant Total RNA isolation kit (Qiagen). Four biologically independent RNA samples from DGTT2 line 57 and the wild type each were used for the microarray experiments. Probe preparation, hybridization to the 4-plex GeneChip Arabidopsis ATH1 Genome Arrays (NimbleGen), and subsequent processing steps were performed according to the manufacturer’s instructions (Gene Expression Center, University of Wisconsin). Arrays were scanned at 5 μm on an Axon4000B scanner (Molecular Dynamics). Microarray data were analyzed using Partek Genomics Suite software (2009). RMA normalization (Irizarry et al., 2003a, 2003b) was performed by Partek upon data import, producing log2-transformed intensity values. Probe set log intensities had a bimodal distribution with a valley at ∼9 (data not shown). Such bimodality appears to be a common feature of oligonucleotide arrays, and the lower mode is thought to correspond to unexpressed genes (Irizarry et al., 2003b; https://stat.ethz.ch/pipermail/bioconductor/2006-June/013255.html). Therefore, probe sets whose maximal values across all arrays were <9 were taken to target genes that were not expressed at detectable levels in any of the samples and were therefore removed from further analysis. This decreased the number of probe sets in the analysis from 30,361 to 18,221.

The PCA plot of the filtered data set did not show clear separation between wild-type and transgenic plants (data not shown), indicating that the transgene expression did not have a strong effect on the global gene expression profile of the plants. Since the experiment was performed on two NimbleGen chips, with four arrays per chip, two wild type and two transgenic each, two-factor mixed-model ANOVA analysis was employed to account for the effects of genotype and chip, the latter being a random effect. P values were calculated for the wild type versus line 57 contrast. Multiple testing correction using the step-up false discovery rate method identified only one gene with adjusted P value < 0.05, encoding a transposable element. Therefore, putative hits were selected using the unadjusted P value threshold of 0.002 and fold change threshold of >2 or <−2. This identified 15 genes (see Supplemental Table 1 online), several of which could be expected to be false positives.

Quantitative RT-PCR

Total RNA was extracted from 15-d-old and 6-week-old DGTT2 transgenic and wild-type plants. cDNA synthesis, quantitative RT-PCR with gene-specific primers (see Supplemental Table 3 online), and data analysis were performed as previously described (Sanjaya et al., 2011).

Accession Numbers

The protein sequences used in the phylogenetic analysis were as follows: DGTT1, XP_001702848.1; DGTT2, XP_001694904.1; DGTT3, XP_001691447.1; DGTT4, XP_001693189.1; DGTT5, XP_001701667.1; Arabidopsis DGAT2, NP_566952.1; VfDGAT2, DQ356682.1; RcDGAT2, DQ923084.1; ScDGA1, NP_014888.1; and OtDGAT2B, XP_003083539.1. The microarray data set is deposited at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE38898.

Supplemental Data

The following materials are available in the online version of this article.

Acknowledgments

We thank Azrin Jamalruddin and Jeremy Letchford (Department of Biochemistry and Molecular Biology, Michigan State University) for technical assistance. We also thank Alicia Pastor for TEM sections, Melinda Frame for assistance with confocal microscopy and Carol Flegler for scanning electron microscopy (Center for Advanced Microscopy, Michigan State University), Kathy Richmond, Nick Santoro, and Shane Cantu (Enabling Technologies) for microarrays, starch, and sugars analysis at the Great Lakes Bioenergy Research Center, Michigan State University. We thank Christopher Saffron and Jonathan Bovee (Department of Biosystems and Agricultural Engineering, Michigan State University) for helpful discussion on elemental analysis and calculation of heating values. This work was funded by the Department of Energy Great Lakes Bioenergy Research Center under the Cooperative Agreement DE-FC02-07ER64494 and by a grant from the U.S. Air Force Office of Scientific Research (Grant FA9550-11-1-0264) to C.B. Insect feeding assays and jasmonate measurements were supported by a grant from the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (Grant DE-FG02-91ER20021).

AUTHOR CONTRIBUTIONS

Sanjaya, R.M., G.A.H., G.E.R., J.O., and C.B. designed research. Sanjaya, R.M., T.P.D., D.K.K., T.A.L., B.M., and A.J.K.K. performed research. Sanjaya, R.M., T.P.D., D.K.K., T.A.L., B.M., A.J.K.K., and Y.V.B. analyzed data. Sanjaya, R.M., and C.B. wrote the article.

Footnotes

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Christoph Benning (benning{at}cns.msu.edu).

  • www.plantcell.org/cgi/doi/10.1105/tpc.112.104752

  • ↵1 These authors contributed equally to this work.

  • ↵2 Current address: Department of Biochemistry, Kansas State University, Manhattan, KS 66506.

  • ↵3 Current address: Department of Biochemistry, University of Missouri, Columbia, MO 65211.

  • ↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.

  • ↵[W] Online version contains Web-only data.

Glossary

TAG
triacylglycerol
ER
endoplasmic reticulum
FA
fatty acid
DGAT
diacylglycerol acyltransferase
WS
wax ester synthase
VLCFA
very-long-chain fatty acid
ESI-MS
electrospray ionization–mass spectrometry
DW
dry weight
ESI-MS/MS
electrospray ionization–tandem mass spectrometry
TLC
thin layer chromatography
TEM
transmission electron microscopy
GC-MS
gas chromatography–mass spectrometry
DCA
α,ω-dicarboxylic acid
GIPC
glycosyl inositolphosphoceramide
GlcCer
glucosylceramide
Cer
ceramide
ANOVA
analysis of variance
JA-Ile
jasmonoyl-l-Ile
MGDG
monogalactosyldiacylglycerol
FAME
fatty acid methyl ester
BSTFA
N,O-bis(trimethylsilyl trifluoroacetamide)
  • Received September 12, 2012.
  • Revised January 4, 2013.
  • Accepted January 23, 2013.
  • Published February 15, 2013.

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Altered Lipid Composition and Enhanced Nutritional Value of Arabidopsis Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2
Sanjaya, Rachel Miller, Timothy P. Durrett, Dylan K. Kosma, Todd A. Lydic, Bagyalakshmi Muthan, Abraham J.K. Koo, Yury V. Bukhman, Gavin E. Reid, Gregg A. Howe, John Ohlrogge, Christoph Benning
The Plant Cell Feb 2013, 25 (2) 677-693; DOI: 10.1105/tpc.112.104752
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