Creating yellow seed Camelina sativa with enhanced oil accumulation by CRISPR-mediated disruption of Transparent Testa 8.

Camelina (Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well-organized seed coat mucilage layer. Transcriptomic analysis of CsTT8-edited seeds revealed significantly increased expression of the lipid-related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis-related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced-oil germplasm in camelina. The resulting lines may directly contribute to future net-zero carbon energy production or be combined with other traits to produce desired lipid-derived bioproducts at high yields.

The expression of genes encoding novel Sesame oleosin variants facilitates enhanced triacylglycerol accumulation in Arabidopsis leaves and seeds.

Triacylglycerols (TAG), accumulate within lipid droplets (LD), predominantly surrounded by OLEOSINs (OLE), that protect TAG from hydrolysis. We tested the hypothesis that identifying and removing degradation signals from OLE would promote its abundance, preventing TAG degradation and enhancing TAG accumulation. We tested whether mutating potential ubiquitin-conjugation sites in a previously reported improved Sesamum indicum OLE (SiO) variant, o3-3 Cys-OLE (SiCO herein), would stabilize it and increase its lipogenic potential. SiCOv1 was created by replacing all five lysines in SiCO with arginines. Separately, six cysteine residues within SiCO were deleted to create SiCOv2. SiCOv1 and SiCOv2 mutations were combined to create SiCOv3. Transient expression of SiCOv3 in Nicotiana benthamiana increased TAG by two-fold relative to SiCO. Constitutive expression of SiCOv3 or SiCOv5, containing the five predominant TAG-increasing mutations from SiCOv3, in Arabidopsis along with mouse DGAT2 (mD) increased TAG accumulation by 54% in leaves and 13% in seeds compared with control lines coexpressing SiCO and mD. Lipid synthesis rates increased, consistent with an increase in lipid sink strength that sequesters newly synthesized TAG, thereby relieving the constitutive BADC-dependent inhibition of ACCase reported for WT Arabidopsis. These OLE variants represent novel factors for potentially increasing TAG accumulation in a variety of oil crops.

Engineering triacylglycerol accumulation in duckweed (Lemna japonica).

Duckweeds are amongst the fastest growing of higher plants, making them attractive high-biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol-inducible cyan fluorescent protein-Arabidopsis WRINKLED1 fusion protein (CFP-AtWRI1), strong constitutive expression of a mouse diacylglycerol:acyl-CoA acyltransferase2 (MmDGAT), and a sesame oleosin variant (SiOLE(*)). Individual expression of each gene increased TAG accumulation by 1- to 7-fold relative to controls, while expression of pairs of these genes increased TAG by 7- to 45-fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45-fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108-fold to 8.7% of DW after incubation on medium containing 100 µm estradiol for 4 days. TAG accumulation was accompanied by an increase in total fatty acids of up to three-fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY-stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately seven-fold the yield of soybean, and similar to that of oil palm. These findings provide the foundation for optimizing TAG accumulation in duckweed and present a new opportunity for producing biofuels and lipidic bioproducts.

Alpha-arrestins Aly1/Art6 and Aly2/Art3 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis.

BACKGROUND INFORMATION: Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B-type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools. GIT1 gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasing GIT1 expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization. RESULTS: We find that the α-arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI-treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs in aly1∆ aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α-arrestin Aly1 by the protein phosphatase calcineurin improves steady-state and substrate-induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α-arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [3 H]-inositol-labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a-arrestins in this regulation. CONCLUSIONS: The α-arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance. SIGNIFICANCE: To our knowledge, this is the first example of α-arrestin regulation of phosphatidyliniositol-3-phosphate levels. In future studies it will be exciting to determine if other α-arrestins similarly alter PI and PIPs to change the cellular landscape.

Mobilizing Vacuolar Sugar Increases Vegetative Triacylglycerol Accumulation.

Photosynthetically derived sugars provide carbon skeletons for metabolism and carbon signals that favor anabolism. The amount of sugar available for fatty acid (FA) and triacylglycerol (TAG) synthesis depends on sugar compartmentation, transport, and demands from competing pathways. We are exploring the influence of sugar partitioning between the vacuole and cytoplasm on FA synthesis in Arabidopsis by building on our previous finding that reduced leaf sugar export in the sucrose-proton symporter2 (suc2) mutant, in combination with impaired starch synthesis in the ADP-glucose pyrophosphorylase (adg1) mutant, accumulates higher sugar levels and increased total FA and TAG compared to the wild type parent. Here we sought to relocalize sugar from the vacuole to the cytoplasm to drive additional FA/TAG synthesis and growth. Arabidopsis suc2 adg1 was therefore crossed with tonoplast monosaccharide transporter mutants tmt1 and tmt2 and overexpression of the sucrose/proton cotransporter SUC4 in which tmt1 tmt2 impairs sugar transport to the vacuole from the cytoplasm and SUC4 overexpression enhances sugar transport in the reverse direction from the vacuole to the cytoplasm. A resulting homozygous suc2 adg1 tmt1 tmt2 SUC4 line was used to test the hypothesis that increased intracellular carbon supply in the form of sugars would increase both FA and TAG accumulation. The data shows that relative to suc2 adg1, suc2 adg1 tmt1 tmt2 SUC4 significantly increases leaf total FA content by 1.29-fold to 10.9% of dry weight and TAG by 2.4-fold to 2.88%, supporting the hypothesis that mobilizing vacuolar sugar is a valid strategy for increasing vegetative oil accumulation.

Overproduction of Phospholipids by the Kennedy Pathway Leads to Hypervirulence in Candida albicans.

Candida albicans is an opportunistic human fungal pathogen that causes life-threatening systemic infections, as well as oral mucosal infections. Phospholipids are crucial for pathogenesis in C. albicans, as disruption of phosphatidylserine (PS) and phosphatidylethanolamine (PE) biosynthesis within the cytidine diphosphate diacylglycerol (CDP-DAG) pathway causes avirulence in a mouse model of systemic infection. The synthesis of PE by this pathway plays a crucial role in virulence, but it was unknown if downstream conversion of PE to phosphatidylcholine (PC) is required for pathogenicity. Therefore, the enzymes responsible for methylating PE to PC, Pem1 and Pem2, were disrupted. The resulting pem1Δ/Δ pem2Δ/Δ mutant was not less virulent in mice, but rather hypervirulent. Since the pem1Δ/Δ pem2Δ/Δ mutant accumulated PE, this led to the hypothesis that increased PE synthesis increases virulence. To test this, the alternative Kennedy pathway for PE/PC synthesis was exploited. This pathway makes PE and PC from exogenous ethanolamine and choline, respectively, using three enzymatic steps. In contrast to Saccharomyces cerevisiae, C. albicans was found to use one enzyme, Ept1, for the final enzymatic step (ethanolamine/cholinephosphotransferase) that generates both PE and PC. EPT1 was overexpressed, which resulted in increases in both PE and PC synthesis. Moreover, the EPT1 overexpression strain is hypervirulent in mice and causes them to succumb to system infection more rapidly than wild-type. In contrast, disruption of EPT1 causes loss of PE and PC synthesis by the Kennedy pathway, and decreased kidney fungal burden during the mouse systemic infection model, indicating a mild loss of virulence. In addition, the ept1Δ/Δ mutant exhibits decreased cytotoxicity against oral epithelial cells in vitro, whereas the EPT1 overexpression strain exhibits increased cytotoxicity. Taken altogether, our data indicate that mutations that result in increased PE synthesis cause greater virulence and mutations that decrease PE synthesis attenuate virulence.

The glycerophosphocholine acyltransferase Gpc1 is part of a phosphatidylcholine (PC)-remodeling pathway that alters PC species in yeast.

Phospholipase B-mediated hydrolysis of phosphatidylcholine (PC) results in the formation of free fatty acids and glycerophosphocholine (GPC) in the yeast Saccharomyces cerevisiae GPC can be reacylated by the glycerophosphocholine acyltransferase Gpc1, which produces lysophosphatidylcholine (LPC), and LPC can be converted to PC by the lysophospholipid acyltransferase Ale1. Here, we further characterized the regulation and function of this distinct PC deacylation/reacylation pathway in yeast. Through in vitro and in vivo experiments, we show that Gpc1 and Ale1 are the major cellular GPC and LPC acyltransferases, respectively. Importantly, we report that Gpc1 activity affects the PC species profile. Loss of Gpc1 decreased the levels of monounsaturated PC species and increased those of diunsaturated PC species, whereas Gpc1 overexpression had the opposite effects. Of note, Gpc1 loss did not significantly affect phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine profiles. Our results indicate that Gpc1 is involved in postsynthetic PC remodeling that produces more saturated PC species. qRT-PCR analyses revealed that GPC1 mRNA abundance is regulated coordinately with PC biosynthetic pathways. Inositol availability, which regulates several phospholipid biosynthetic genes, down-regulated GPC1 expression at the mRNA and protein levels and, as expected, decreased levels of monounsaturated PC species. Finally, loss of GPC1 decreased stationary phase viability in inositol-free medium. These results indicate that Gpc1 is part of a postsynthetic PC deacylation/reacylation remodeling pathway (PC-DRP) that alters the PC species profile, is regulated in coordination with other major lipid biosynthetic pathways, and affects yeast growth.

Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants: A NOVEL ENZYME IN PHOSPHATIDYLCHOLINE SYNTHESIS.

Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).