Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues.

BACKGROUND: Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. RESULTS: In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic "push pull and protect" factors correlated with biomass reduction. CONCLUSIONS: Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels.

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass.

BACKGROUND: The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production. RESULTS: Here, we report on the hyperaccumulation of triacylglycerol (TAG) in energycane following the overexpression of the lipogenic factors Diacylglycerol acyltransferase1-2 (DGAT1-2) and Oleosin1 (OLE1) in combination with RNAi suppression of SUGAR-DEPENDENT1 (SDP1) and Trigalactosyl diacylglycerol1 (TGD1). TAG accumulated up to 1.52% of leaf dry weight (DW,) a rate that was 30-fold that of non-modified energycane, in addition to almost doubling the total fatty acid content in leaves to 4.42% of its DW. Pearson's correlation analysis showed that the accumulation of TAG had the highest correlation with the expression level of ZmDGAT1-2, followed by the level of RNAi suppression for SDP1. CONCLUSIONS: This is the first report on the metabolic engineering of energycane and demonstrates that this resilient, high-biomass crop is an excellent target for the further optimization of the production of lipids from vegetative tissues.

Biosynthesis and accumulation of 20-hydroxyecdysone in individual male and female spinach plants during the reproductive stage.

The steroid 20-hydroxyecdysone (20E) is a major component of phytoecdysteroid in plants and may play a defensive role against insect pests in higher plants. In spinach, the biosynthesis and accumulation of 20E have been investigated during the vegetative stage; however, these processes have not been clearly studied during the reproductive stage, particularly in male and female individuals. In this study, we analyzed the level and distribution of 20E in individual male and female spinach plants during the reproductive stage via high performance liquid chromatography (HPLC). We found that 20E biosynthesis and accumulation were markedly different between male and female spinach during the late flowering stage. Compared with the male plant, biosynthesis of 20E in the leaves was more active and its accumulation in the floral parts was higher in female plants during the late flowering stage. These results indicate that the female reproductive organs at least in PE-positive plants could be effectively protected against harmful insects via active biosynthesis and accumulation of PE during the late flowering stage to protect floral parts from harmful insects for seed formation and store the available 20E in seeds for the next generation.