Engineering Camelina sativa (L.) Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol-3-phosphate dehydrogenase expression

Abstract

Plant seed oil-based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum-derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproduct applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by co-expressing the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol-3-phosphate dehydrogenase (GPD1) genes under the control of seed-specific promoters. Plants co-expressing DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild-type plants. Further, DGAT1- and GDP1-co-expressing lines showed significantly higher seed and oil yields on a dry weight basis than the wild-type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1- and GPD1-co-expressing lines was almost twofold higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield.

Keywords: Camelina sativa; biofuels; lipid metabolism; metabolic engineering; triacylglycerols.

Figure 1

T‐DNA insertions used to transform Camelina sativa. Shown here are the pBnRGW RedSeed binary vectors containing seed‐specific cassettes for expression of the glycerol‐3‐phosphate dehydrogenase, GPD1 (a) and the diacylglycerol acyltransferase, DGAT1 and DGAT1m (b and c). DsRed fluorescence marker and the herbicide‐resistant bar gene (Basta containing phosphinothricin) for selection of transformants are shown in the constructs.

Figure 2

Analysis of DGAT1 and GPD1 transcript expression in Camelina developing seeds of T3 generation in the transgenic lines overexpressing AtDGAT1 (a), ScGPD1 (b) and DGAT1 + GPD1 (c). Values are the mean ± standard error (n = 3). Asterisks denote significance of differences between WT and transgenic lines (Student's t‐test): **P < 0.01; *P < 0.05.

Figure 3

Individual and combined effects of GPD1 and DGAT1 expression on average seed mass of homozygous T3 seeds in transgenic Camelina plants. (a) The seed weight (g/plant) of Camelina lines overexpressing AtDGAT1, ScGPD1 and WT controls. (b) Images of transgenic and WT seeds illuminated under DsRed fluorescence filter. Values in (a) are means ± SE on measurements on seeds from individual plants (n = 4–10) of each genotype grown in controlled conditions. Bar = 1 mm. Asterisks denote significance of differences between WT and transgenic lines (Student's t‐test): **P < 0.01; *P < 0.05.

Figure 4

Individual and combined effects of GPD1 and DGAT1 expression on average seed yields (a), % oil content (b), oil yield (c) and oil harvest index (d) of T3 homozygous seeds in transgenic Camelina plants. Values are means ± SE on measurements on seeds from individual plants (n = 4–10) of each genotype grown in controlled conditions. Asterisks denote significance of differences between WT and transgenic lines (Student's t‐test): **P < 0.01; *P < 0.05.

Figure 5

Individual and combined effects of GPD1 and DGAT1 expression on protein content in Camelina seeds. Values are means ±  SE on measurements on seeds from individual plants (n = 4) of each genotype grown under controlled conditions.

Figure 6

Relative change in FA composition in dried transgenic and WT Camelina seeds analysed by gas chromatography. The percentage relative increase or decrease in the levels of palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and eicosenoic acid (C20:1) in transgenic Camelina lines overexpressing AtDGAT1 and ScGPD1, individually or in a combination, as compared to nontransgenic WT are shown. Values are means ± SE (n = 4). WT values are normalized to the threshold 100. Asterisks denote significance of differences between WT and transgenic lines (Student's t‐test): **P < 0.01; *P < 0.05.

Figure 7

Early seedling growth rate of Camelina in the T3 transgenic and WT Plants. Values are means ± S.E. of measurements on seeds from individual plants (n = 12) of each genotype grown under controlled condition over germination papers.