Purple acid phosphatase2 stimulates a futile cycle of lipid synthesis and degradation, and mitigates the negative growth effects of triacylglycerol accumulation in vegetative tissues.

Storage lipids (mostly triacylglycerols, TAGs) serve as an important energy and carbon reserve in plants, and hyperaccumulation of TAG in vegetative tissues can have negative effects on plant growth. Purple acid phosphatase2 (PAP2) was previously shown to affect carbon metabolism and boost plant growth. However, the effects of PAP2 on lipid metabolism remain unknown. Here, we demonstrated that PAP2 can stimulate a futile cycle of fatty acid (FA) synthesis and degradation, and mitigate negative growth effects associated with high accumulation of TAG in vegetative tissues. Constitutive expression of PAP2 in Arabidopsis thaliana enhanced both lipid synthesis and degradation in leaves and led to a substantial increase in seed oil yield. Suppressing lipid degradation in a PAP2-overexpressing line by disrupting sugar-dependent1 (SDP1), a predominant TAG lipase, significantly elevated vegetative TAG content and improved plant growth. Diverting FAs from membrane lipids to TAGs in PAP2-overexpressing plants by constitutively expressing phospholipid:diacylglycerol acyltransferase1 (PDAT1) greatly increased TAG content in vegetative tissues without compromising biomass yield. These results highlight the potential of combining PAP2 with TAG-promoting factors to enhance carbon assimilation, FA synthesis and allocation to TAGs for optimized plant growth and storage lipid accumulation in vegetative tissues.

Triacylglycerol Metabolism, Function, and Accumulation in Plant Vegetative Tissues.

Oils in the form of triacylglycerols are the most abundant energy-dense storage compounds in eukaryotes, and their metabolism plays a key role in cellular energy balance, lipid homeostasis, growth, and maintenance. Plants accumulate oils primarily in seeds and fruits. Plant oils are used for food and feed and, increasingly, as feedstocks for biodiesel and industrial chemicals. Although plant vegetative tissues do not accumulate significant levels of triacylglycerols, they possess a high capacity for their synthesis, storage, and metabolism. The development of plants that accumulate oil in vegetative tissues presents an opportunity for expanded production of triacylglycerols as a renewable and sustainable bioenergy source. Here, we review recent progress in the understanding of triacylglycerol synthesis, turnover, storage, and function in leaves and discuss emerging genetic engineering strategies targeted at enhancing triacylglycerol accumulation in biomass crops. Such plants could potentially be modified to produce oleochemical feedstocks or nutraceuticals.

Dual role for phospholipid:diacylglycerol acyltransferase: enhancing fatty acid synthesis and diverting fatty acids from membrane lipids to triacylglycerol in Arabidopsis leaves.

There is growing interest in engineering green biomass to expand the production of plant oils as feed and biofuels. Here, we show that phospholipid:diacylglycerol acyltransferase1 (PDAT1) is a critical enzyme involved in triacylglycerol (TAG) synthesis in leaves. Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway. In the trigalactosyldiacylglycerol1-1 mutant, which is defective in eukaryotic thylakoid lipid synthesis, the combined overexpression of PDAT1 with oleosin increases leaf TAG content to 8.6% of the dry weight and total leaf lipid by fourfold. In the plastidic glycerol-3-phosphate acyltransferase1 mutant, which is defective in the prokaryotic glycerolipid pathway, PDAT1 overexpression enhances TAG content at the expense of thylakoid membrane lipids, leading to defects in chloroplast division and thylakoid biogenesis. Collectively, these results reveal a dual role for PDAT1 in enhancing fatty acid and TAG synthesis in leaves and suggest that increasing FAS is the key to engineering high levels of TAG accumulation in green biomass.

Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii.

Microalgal oils have attracted much interest as potential feedstocks for renewable fuels, yet our understanding of the regulatory mechanisms controlling oil biosynthesis and storage in microalgae is rather limited. Using Chlamydomonas reinhardtii as a model system, we show here that starch, rather than oil, is the dominant storage sink for reduced carbon under a wide variety of conditions. In short-term treatments, significant amounts of oil were found to be accumulated concomitantly with starch only under conditions of N starvation, as expected, or in cells cultured with high acetate in otherwise standard growth medium. Time-course analysis revealed that oil accumulation under N starvation lags behind that of starch and rapid oil synthesis occurs only when carbon supply exceeds the capacity of starch synthesis. In the starchless mutant BAFJ5, blocking starch synthesis results in significant increases in the extent and rate of oil accumulation. In the parental strain, but not the starchless mutant, oil accumulation under N starvation was strictly dependent on the available external acetate supply and the amount of oil increased steadily as the acetate concentration increased to the levels several-fold higher than that of the standard growth medium. Additionally, oil accumulation under N starvation is saturated at low light intensities and appears to be largely independent of de novo protein synthesis. Collectively, our results suggest that carbon availability is a key metabolic factor controlling oil biosynthesis and carbon partitioning between starch and oil in Chlamydomonas.

Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis.

Phosphatidate (PA) is a central metabolite of lipid metabolism and a signaling molecule in many eukaryotes, including plants. Mutations in a permease-like protein, TRIGALACTOSYLDIACYLGLYCEROL1 (TGD1), in Arabidopsis thaliana caused the accumulation of triacylglycerols, oligogalactolipids, and PA. Chloroplast lipids were altered in their fatty acid composition consistent with an impairment of lipid trafficking from the endoplasmic reticulum (ER) to the chloroplast and a disruption of thylakoid lipid biosynthesis from ER-derived precursors. The process mediated by TGD1 appears to be essential as mutation of the protein caused a high incidence of embryo abortion. Isolated tgd1 mutant chloroplasts showed a decreased ability to incorporate PA into galactolipids. The TGD1 protein was localized to the inner chloroplast envelope and appears to be a component of a lipid transporter. As even partial disruption of TGD1 function has drastic consequences on central lipid metabolism, the tgd1 mutant provides a tool to explore regulatory mechanisms governing lipid homeostasis and lipid trafficking in plants.

Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth.

The sulfolipid sulfoquinovosyldiacylglycerol is one of the three nonphosphorous glycolipids that provide the bulk of the structural lipids in photosynthetic membranes of seed plants. Unlike the galactolipids, sulfolipid is anionic at physiological pH because of its 6-deoxy-6-sulfonate-glucose (sulfoquinovose) head group. The biosynthesis of this lipid proceeds in two steps: first, the assembly of UDP-sulfoquinovose from UDP-glucose and sulfite, and second, the transfer of the sulfoquinovose moiety from UDP-sulfoquinovose to diacylglycerol. The first reaction is catalyzed by the SQD1 protein in Arabidopsis. Here we describe the identification of the SQD2 gene of Arabidopsis. We propose that this gene encodes the sulfoquinovosyltransferase catalyzing the second step of sulfolipid biosynthesis. Expression of SQD1 and SQD2 in Escherichia coli reconstituted plant sulfolipid biosynthesis in this bacterium. Insertion of a transfer DNA into this gene in Arabidopsis led to complete lack of sulfolipid in the respective sqd2 mutant. This mutant showed reduced growth under phosphate-limited growth conditions. The results support the hypothesis that sulfolipid can function as a substitute of anionic phospholipids under phosphate-limited growth conditions. Along with phosphatidylglycerol, sulfolipid contributes to maintaining a negatively charged lipid-water interface, which presumably is required for proper function of photosynthetic membranes.