Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue

In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl‐constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER‐to‐chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant.     

Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type‐2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter

When cultivated under stress conditions, many plants and algae accumulate oil. The unicellular green microalga Chlamydomonas reinhardtii accumulates neutral lipids (triacylglycerols; TAGs) during nutrient stress conditions. Temporal changes in TAG levels in nitrogen (N)‐ and phosphorus (P)‐starved cells were examined to compare the effects of nutrient depletion on TAG accumulation in C. reinhardtii. TAG accumulation and fatty acid composition were substantially changed depending on the cultivation stage before nutrient starvation. Profiles of TAG accumulation also differed between N and P starvation. Logarithmic‐growth‐phase cells diluted into fresh medium showed substantial TAG accumulation with both N and P deprivation. N deprivation induced formation of oil droplets concomitant with the breakdown of thylakoid membranes. In contrast, P deprivation substantially induced accumulation of oil droplets in the cytosol and maintaining thylakoid membranes. As a consequence, P limitation accumulated more TAG both per cell and per culture medium under these conditions. To enhance oil accumulation under P deprivation, we constructed a P deprivation‐dependent overexpressor of a Chlamydomonas type‐2 diacylglycerol acyl‐CoA acyltransferase (DGTT4) using a sulphoquinovosyldiacylglycerol 2 (SQD2) promoter, which was up‐regulated during P starvation. The transformant strongly enhanced TAG accumulation with a slight increase in 18 : 1 content, which is a preferred substrate of DGTT4. These results demonstrated enhanced TAG accumulation using a P starvation–inducible promoter.     

Metabolic engineering of biomass for high energy density: oilseed‐like triacylglycerol yields from plant leaves

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co‐expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild‐type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil‐processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.     

N‐glycan maturation is crucial for cytokinin‐mediated development and cellulose synthesis in Oryza sativa

To explore the physiological significance of N‐glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N‐acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N‐glycan maturation and accumulated high‐mannose N‐glycans. Phenotypic analyses revealed that gnt1 shows defective post‐seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark‐induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A‐type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N‐glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.     

Properties of capsaicinoids for the control of fungi and oomycetes pathogenic to pepper

Capsaicinoids are pungent compounds found in pepper (Capsicum spp.) fruits. Capsaicin showed antimicrobial activity in plate assays against seven isolates of five species of fungi and nine isolates of two species of oomycetes. The general trend was that oomycetes were more inhibited than fungi. Assays of capsaicin biosynthetic precursors suggest that the lateral chain of capsaicinoids has more inhibitory activity than the phenolic part. In planta tests of capsaicinoids (capsaicin and N‐vanillylnonanamide) applied to the roots demonstrated that these compounds conferred protection against the pathogenic fungus Verticillium dahliae and induced both chitinase activity and expression of several defence‐related genes, such as CASC1, CACHI2 and CABGLU. N‐Vanillylnonanamide infiltrated into cotyledons confers systemic protection to the upper leaves of pepper against the fungal pathogen Botrytis cinerea. In wild‐type tomato plants such cotyledon infiltration has no protective effect, but is effective in the Never‐ripe tomato mutant impaired in ethylene response. A similar effect was observed in tomato after salicylic acid infiltration.     

Development of a Saccharomyces cerevisiae strain for increasing the accumulation of triacylglycerol as a microbial oil feedstock for biodiesel production using glycerol as a substrate

Triacylglycerol (TAG) is a microbial oil feedstock for biodiesel production that uses an inexpensive substrate, such as glycerol. Here, we demonstrated the overproduction of TAG from glycerol in engineered Saccharomyces cerevisiae via the glycerol‐3‐phosphate (G3P) pathway by overexpressing the major TAG synthesis. The G3P accumulation was increased 2.4‐fold with the increased glycerol utilization gained by the overexpression of glycerol kinase (GUT1). By overexpressing diacylglycerol acyltransferase (DGA1) and phospholipid diacylglycerol acyltransferase (LRO1), the engineered YPH499 (pGutDgaLro1) strain produced 23.0 mg/L lipids, whereas the YPH499 (pESC‐TRP) strain produced 6.2 mg/L total lipids and showed a lipid content that was increased 1.4‐fold compared with 3.6% for the wild‐type strain after 96 h of cultivation. After 96 h of cultivation using glycerol, the overall content of TAG in the engineered strain, YPH499 (pGutDgaLro1), yielded 8.2% TAG, representing a 2.3‐fold improvement, compared with 3.6% for the wild‐type strain. The results should allow a reduction of costs and a more sustainable production of biodiesel. Biotechnol. Bioeng. 2013; 110: 343–347. © 2012 Wiley Periodicals, Inc.     

Phospholipid:diacylglycerol acyltransferase‐mediated triacylglycerol biosynthesis is crucial for protection against fatty acid‐induced cell death in growing tissues of Arabidopsis

Phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol:acyl CoA acyltransferase play overlapping roles in triacylglycerol (TAG) assembly in Arabidopsis, and are essential for seed and pollen development, but the functional importance of PDAT in vegetative tissues remains largely unknown. Taking advantage of the Arabidopsis tgd1–1 mutant that accumulates oil in vegetative tissues, we demonstrate here that PDAT1 is crucial for TAG biosynthesis in growing tissues. We show that disruption of PDAT1 in the tgd1–1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves. Lipid analysis data indicated that knockout of PDAT1 results in increases in the levels of free fatty acids (FFAs) and diacylglycerol. In vivo ¹⁴C‐acetate labeling experiments showed that, compared with wild‐type, tgd1–1 exhibits a 3.8‐fold higher rate of fatty acid synthesis (FAS), which is unaffected by disruption or over‐expression of PDAT1, indicating a lack of feedback regulation of FAS in tgd1–1. We also show that detached leaves of both pdat1–2 and tgd1–1 pdat1–2 display increased sensitivity to FFA but not to diacylglycerol. Taken together, our results reveal a critical role for PDAT1 in mediating TAG synthesis and thereby protecting against FFA‐induced cell death in fast‐growing tissues of plants.     

Elevated acetyl‐CoA by amino acid recycling fuels microalgal neutral lipid accumulation in exponential growth phase for biofuel production

Microalgal neutral lipids [mainly in the form of triacylglycerols (TAGs)], feasible substrates for biofuel, are typically accumulated during the stationary growth phase. To make microalgal biofuels economically competitive with fossil fuels, generating strains that trigger TAG accumulation from the exponential growth phase is a promising biological approach. The regulatory mechanisms to trigger TAG accumulation from the exponential growth phase (TAEP) are important to be uncovered for advancing economic feasibility. Through the inhibition of pyruvate dehydrogenase kinase by sodium dichloroacetate, acetyl‐CoA level increased, resulting in TAEP in microalga Dunaliella tertiolecta. We further reported refilling of acetyl‐CoA pool through branched‐chain amino acid catabolism contributed to an overall sixfold TAEP with marginal compromise (4%) on growth in a TAG‐rich D. tertiolecta mutant from targeted screening. Herein, a three‐step α loop‐integrated metabolic model is introduced to shed lights on the neutral lipid regulatory mechanism. This article provides novel approaches to compress lipid production phase and heightens lipid productivity and photosynthetic carbon capture via enhancing acetyl‐CoA level, which would optimize renewable microalgal biofuel to fulfil the demanding fuel market.