The Peanut (<i>Arachis hypogaea</i> L.) Gene <i>AhLPAT2</i> Increases the Lipid Content of Transgenic Arabidopsis Seeds

<div><p>Lysophosphatidic acid acyltransferase (LPAT), which converts lysophosphatidic acid (LPA) to phosphatidic acid (PA), catalyzes the addition of fatty acyl moieties to the <i>sn</i>-2 position of the LPA glycerol backbone in triacylglycerol (TAG) biosynthesis. We recently reported the cloning and temporal-spatial expression of a peanut (<i>Arachis hypogaea</i>) <i>AhLPAT2</i>gene, showing that an increase in <i>AhLPAT2</i> transcript levels was closely correlated with an increase in seed oil levels. However, the function of the enzyme encoded by the <i>AhLPAT2</i> gene remains unclear. Here, we report that <i>AhLPAT2</i> transcript levels were consistently higher in the seeds of a high-oil cultivar than in those of a low-oil cultivar across different seed developmental stages. Seed-specific overexpression of <i>AhLPAT2</i> in Arabidopsis results in a higher percentage of oil in the seeds and greater-than-average seed weight in the transgenic plants compared with the wild-type plants, leading to a significant increase in total oil yield per plant. The total fatty acid (FA) content and the proportion of unsaturated FAs also increased. In the developing siliques of <i>AhLPAT2</i>-overexpressing plants, the expression levels of genes encoding crucial enzymes involved in <i>de novo</i> FA synthesis, acetyl-CoA subunit (<i>AtBCCP2</i>) and acyl carrier protein 1 (<i>AtACP1</i>) were elevated. <i>AhLPAT2</i> overexpression also promoted the expression of several key genes related to TAG assembly, sucrose metabolism, and glycolysis. These results demonstrate that the expression of <i>AhLPAT2</i> plays an important role in glycerolipid production in peanuts.</p></div

Effect of <i>AhLPAT2</i> Overexpression on Seed Storage Reserve Content of Independent Homozygous T3 Lines.

<p>A. Seeds per silique. B. Numbers of siliques per plant. C. 100-seed weight. D. Seed yield per plant. Values are means ± SE (<i>n</i> = 10). Asterisks indicate significant differences between the wild-type and transgenic lines at p < 0.01 (**).</p

Altered Expression of Genes Involved in the FAs and TAG Biosynthesis Pathway.

<p>Values are means ± SE (<i>n</i> = 3). Total RNA was prepared from developing siliques of transgenic lines. Gene expression levels are shown relative to the expression of <i>AtActin7</i> in each sample. The transcription level of each gene in the wildtype was set as 1.0.Asterisks indicate significant differences between the wild-type and transgenic lines at p < 0.01 (**) and p < 0.05 (*).</p

Effect of <i>AhLPAT2</i> Overexpression on FA Content and Composition in Independent Homozygous Seeds.

<p>A. Total FA content. B. Main FA profiles. C. The ratio of unsaturated to saturated FAs. D. The ratio of 18- to 20-carbon FA. Values are means ± SE (<i>n</i> = 10). Asterisks indicate a significant differences between the wild-type and transgenic lines at p < 0.01 (**) and p < 0.05 (*).</p

Altered Expression of Genes Involved in Sucrose Metabolism and Glycolysis.

<p>Values are means ± SE (<i>n</i> = 3). Total RNA was prepared from developing siliques of transgenic lines. Gene expression levels are shown relative to the expression of <i>AtActin7</i> in each sample. The transcription level of each gene in the wild type was set as 1.0.Asterisks indicate significant differences between the wild-type and transgenic lines at p < 0.01 (**) and p < 0.05 (*).</p

Effect of <i>AhLPAT2</i> Overexpression on Seed Oil and Protein Contents of Independent Homozygous T3 Lines.

<p>A. Seed oil content as a percentage. B. Oil yield per plant. C. Seed protein content. Values are means ± SE of measurements of individual plants (<i>n</i> = 10). Asterisks indicate a significant difference between the wild-type and transgenic lines at p < 0.01 (**)and p < 0.05 (*).</p

<i>AhLPAT2</i> Gene Expression in Young Siliques of the Selected Transgenic Lines Overexpressing <i>AhLPAT2</i>.

<p>Two transgenic T3 lines, FNH1-21 and FNH2-2, were used. Total RNA was prepared from developing siliques of transgenic lines. Gene expression levels are shown relative to the expression of <i>AtActin7</i> in each sample. Values are means ± SE (<i>n</i> = 3).</p

Subcellular Localization of the AhLPAT2–GFP Fusion Protein in Onion Epidermal Cells.

<p>The top panels show the expression vector containing <i>AhLPAT2</i> and the <i>GFP</i> reporter gene. <i>AhLPAT2</i> tagged with <i>GFP</i> in the C-terminus was inserted into the pEGFP vector between the <i>Kpn</i>I and <i>Xma</i>I restriction sites. LB, left border; RB, right border; PNOS and TNOS are the promoter and polyadenylation signal of the nopaline synthase gene, respectively; NptII neomycin phosphotransferase II, CaMV 35S promoter. Bottom panels show the expression of <i>GFP</i> and <i>AhLPAT2</i>-<i>GFP</i>. (A) GFP fluorescence of onion epidermal cells expressing <i>GFP</i>. (B) Bright field image of (A). (C) Cell nuclei counterstained with DAPI. (D) Merged image of (A), (B) and (C). (E) GFP fluorescence of onion epidermal cells expressing AhLPAT2-GFP. (F) Bright field image of (E). (G) Cell nuclei counterstained with DAPI. (H) Merged image of (E), (F) and (G). Scale bars = 50 μm.</p