Ectopic Expression of OLEOSIN 1 and Inactivation of GBSS1 Have a Synergistic Effect on Oil Accumulation in Plant Leaves

Abstract

During the transformation of wild-type (WT) Arabidopsis thaliana, a T-DNA containing OLEOSIN-GFP (OLE1-GFP) was inserted by happenstance within the GBSS1 gene, resulting in significant reduction in amylose and increase in leaf oil content in the transgenic line (OG). The synergistic effect on oil accumulation of combining gbss1 with the expression of OLE1-GFP was confirmed by transforming an independent gbss1 mutant (GABI_914G01) with OLE1-GFP. The resulting OLE1-GFP/gbss1 transgenic lines showed higher leaf oil content than the individual OLE1-GFP/WT or single gbss1 mutant lines. Further stacking of the lipogenic factors WRINKLED1, Diacylglycerol O-Acyltransferase (DGAT1), and Cys-OLEOSIN1 (an engineered sesame OLEOSIN1) in OG significantly elevated its oil content in mature leaves to 2.3% of dry weight, which is 15 times higher than that in WT Arabidopsis. Inducible expression of the same lipogenic factors was shown to be an effective strategy for triacylglycerol (TAG) accumulation without incurring growth, development, and yield penalties.

Keywords: fatty acids; granule bound starch synthase; metabolic engineering; oleosin; triacylglycerol.

Figure 1

Characterization of an Arabidopsis OLE1-GFP transgenic line (OG). (A) Phenotype of 1-month-old wild type (WT) and OG plants. (B) Bright light (Left) and GFP fluorescence (Right) of WT and OG by fluorescence image analyzer (ImageQuant LAS4000). (C) Leaf TAG contents of WT plants (WT) and OG. Values are means ± SE (n = 6) of measurements on mature leaves of 1-month-old soil grown plants. Asterisks denote statistically significant difference from WT (Student’s t test, **, p < 0.01). (D) Schematic illustration of the exon-intron structure of the GBSS1 gene. Exons are represented by blue boxes. Grey boxes represent the 5′ and 3′ UTRs. Translation start (ATG) and stop (TAG) codons are indicated. Open inverted triangles indicate T-DNA insertion sites for OG and gbss1 mutant respectively. Black arrowheads indicate primers for genotyping OG. Grey arrowheads represent primers used in quantitative real time PCR (qRT-PCR) for quantifying gene expression of GBSS1. (E) Genotyping of OG with primers in (D). M is DNA marker. (F) Analysis of GBSS1 expression in WT and OG plants. The values are means ± SE of measurements taken by qRT-PCR on three individual plants employing primers (gray arrow) shown in (D). F-box expression was used as a control for normalization (**, p < 0.01). (G) Iodine staining of leaves starch granules isolated from 0.5 g (FW) leaves of WT and OG plants.

Figure 2

Overexpression of OLE1-GFP and gbss1 mutation have synergistic roles in increasing TAG content in leaves. TAG quantification in leaves of varied genotypes as indicated. Values are means ± SE of measurements on mature leaves of 1-month-old soil grown plants. Levels indicated with different letters above histogram bars are significantly different (Student’s t test for all pairs of genotypes, n = 6, p < 0.05).

Figure 3

Stacking WRI1 and DGAT1 and Cys-OLE1 in GO significantly boosts its leaves oil content. (A) Schematic illustration of T-DNA construct designed for constitutive or ethanol inducible expression of Cys-OLE, WRI1, and DGAT1 (OWD and OWD.2). (B) Representative phenotype of constitutive or inducible OWD transgenic plants. Two representative 8-week-old soil grown transgenic lines for constitutive (OWD) or inducible expression (OWD.2) of OWD in WT or GO background were shown. (C) Leaves TAG quantification for varied genotypes in (B). C is a non-transgenic plant. Values are means ± SE (n = 6). For the OWD.2 plant, TAG was measured after 5 days of induction by irrigating with 2% of ethanol solution. Asterisks (*) denote statistically significant differences compared with C (Student’s t test, p < 0.01).