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.

GRIK phosphorylates and activates KIN10 which also promotes its degradation.

The sensor kinase Sucrose Non-fermenting-1-Related Kinase 1 (SnRK1) plays a central role in energy and metabolic homeostasis. KIN10 is a major catalytic (α) kinase subunit of SnRK1 regulated by transcription, posttranslational modification, targeted protein degradation, and its subcellular localization. Geminivirus Rep Interacting Kinase 1 and 2 (GRIK1 and 2) are immediate upstream kinases of KIN10. In the transient protein expression assays carried out in Nicotiana benthamiana (N. benthamiana) leaves, GRIK1 not only phosphorylates KIN10 but also simultaneously initiates its degradation. Posttranslational GRIK-mediated KIN10 degradation is dependent on both GRIK kinase activity and phosphorylation of the KIN10 T-loop. KIN10 proteins are significantly enriched in the grik1-1 grik2-1 double mutant, consistent with the transient assays in N. benthamiana. Interestingly. Among the enriched KIN10 proteins from grik1-1 grik2-1, is a longer isoform, putatively derived by alternative splicing which is barely detectable in wild-type plants. The reduced stability of KIN10 upon phosphorylation and activation by GRIK represents a mechanism that enables the KIN10 activity to be rapidly reduced when the levels of intracellular sugar/energy are restored to their set point, representing an important homeostatic control that prevents a metabolic overreaction to low-sugar conditions. Since GRIKs are activating kinases of KIN10, KIN10s in the grik1 grik2 double null mutant background remain un-phosphorylated, with only their basal level of activity, are more stable, and therefore increase in abundance, which also explains the longer isoform KIN10L which is a minor isoform in wild type is clearly detected in the grik1 grik2 double mutant.

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.

CYCLIN-DEPENDENT KINASE 8 positively regulates oil synthesis by activating WRINKLED1 transcription.

CYCLIN-DEPENDENT KINASE 8 (CDK8), a component of the kinase module of the Mediator complex in Arabidopsis, is involved in many processes, including flowering, plant defense, drought, and energy stress responses. Here, we investigated cdk8 mutants and CDK8-overexpressing lines to evaluate whether CDK8 also plays a role in regulating lipid synthesis, an energy-demanding anabolism. Quantitative lipid analysis demonstrated significant reductions in lipid synthesis rates and lipid accumulation in developing siliques and seedlings of cdk8, and conversely, elevated lipid contents in wild-type seed overexpressing CDK8. Transactivation assays show that CDK8 is necessary for maximal transactivation of the master seed oil activator WRINKLED1 (WRI1) by the seed maturation transcription factor ABSCISIC ACID INSENSITIVE3, supporting a direct regulatory role of CDK8 in oil synthesis. Thermophoretic studies show GEMINIVIRUS REP INTERACTING KINASE1, an activating kinase of KIN10 (a catalytic subunit of SUCROSE NON-FERMENTING1-RELATED KINASE1), physically interacts with CDK8, resulting in its phosphorylation and degradation in the presence of KIN10. This work defines a mechanism whereby, once activated, KIN10 downregulates WRI1 expression and suppresses lipid synthesis via promoting the degradation of CDK8. The KIN10-CDK8-dependent regulation of lipid synthesis described herein is additional to our previously reported KIN10-dependent phosphorylation and degradation of WRI1.

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.

Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

Ectopic Expression of OLEOSIN 1 and Inactivation of GBSS1 Have a Synergistic Effect on Oil Accumulation in Plant Leaves.

During the transformation of wild-type (WT) Arabidopsis thaliana, a T-DNA containing OLEOSIN-GFP (OLE1-GFP) was inserted by happenstance within the GBSS1 gene, resulting in significant reduction in amylose and increase in leaf oil content in the transgenic line (OG). The synergistic effect on oil accumulation of combining gbss1 with the expression of OLE1-GFP was confirmed by transforming an independent gbss1 mutant (GABI_914G01) with OLE1-GFP. The resulting OLE1-GFP/gbss1 transgenic lines showed higher leaf oil content than the individual OLE1-GFP/WT or single gbss1 mutant lines. Further stacking of the lipogenic factors WRINKLED1, Diacylglycerol O-Acyltransferase (DGAT1), and Cys-OLEOSIN1 (an engineered sesame OLEOSIN1) in OG significantly elevated its oil content in mature leaves to 2.3% of dry weight, which is 15 times higher than that in WT Arabidopsis. Inducible expression of the same lipogenic factors was shown to be an effective strategy for triacylglycerol (TAG) accumulation without incurring growth, development, and yield penalties.

Expression of a Bacterial Trehalose-6-phosphate Synthase otsA Increases Oil Accumulation in Plant Seeds and Vegetative Tissues.

We previously demonstrated that exogenous trehalose 6-phosphate (T6P) treatment stabilized WRINKLED1 (WRI1), a master transcriptional regulator of fatty acid (FA) synthesis and increased total FA content in Brassica napus (B. napus) embryo suspension cell culture. Here, we explore Arabidopsis lines heterologously expressing the Escherichia coli T6P synthase (otsA) or T6P phosphatase (otsB) to refine our understanding regarding the role of T6P in regulating fatty acid synthesis both in seeds and vegetative tissues. Arabidopsis 35S:otsA transgenic seeds showed an increase of 13% in fatty acid content compared to those of wild type (WT), while seeds of 35:otsB transgenic seeds showed a reduction of 12% in fatty acid content compared to WT. Expression of otsB significantly reduced the level of WRI1 and expression of its target genes in developing seeds. Like Arabidopsis seeds constitutively expressing otsA, transient expression of otsA in Nicotiana benthamiana leaves resulted in strongly elevated levels of T6P. This was accompanied by an increase of 29% in de novo fatty acid synthesis rate, a 2.3-fold increase in triacylglycerol (TAG) and a 20% increase in total fatty acid content relative to empty vector (EV) controls. Taken together, these data support the heterologous expression of otsA as an approach to increasing TAG accumulation in plant seeds and vegetative tissues.

Arabidopsis SnRK1 negatively regulates phenylpropanoid metabolism via Kelch domain-containing F-box proteins.

Phenylpropanoid metabolism represents a substantial metabolic sink for photosynthetically fixed carbon. The evolutionarily conserved Sucrose Non-Fermenting Related Kinase 1 (SnRK1) is a major metabolic sensor that reprograms metabolism upon carbon deprivation. However, it is not clear if and how the SnRK1-mediated sugar signaling pathway controls phenylpropanoid metabolism. Here, we show that Arabidopsis SnRK1 negatively regulates phenylpropanoid biosynthesis via a group of Kelch domain-containing F-box (KFB) proteins that are responsible for the ubiquitination and degradation of phenylalanine ammonia lyase (PAL). Downregulation of AtSnRK1 significantly promoted the accumulation of soluble phenolics and lignin polymers and drastically increased PAL cellular accumulation but only slightly altered its transcription level. Co-expression of SnRK1α with PAL in Nicotiana benthamiana leaves resulted in the severe attenuation of the latter's protein level, but protein interaction assays suggested PAL is not a direct substrate of SnRK1. Furthermore, up or downregulation of AtSnRK1 positively affected KFBPALs gene expression, and energy starvation upregulated KFBPAL expression, which partially depends on AtSnRK1. Collectively, our study reveals that SnRK1 negatively regulates phenylpropanoid biosynthesis, and KFBPALs act as regulatory components of the SnRK1 signaling network, transcriptionally regulated by SnRK1 and subsequently mediate proteasomal degradation of PAL in response to the cellular carbon availability.

Trehalose 6-Phosphate Positively Regulates Fatty Acid Synthesis by Stabilizing WRINKLED1.

WRINKLED1 (WRI1), the transcriptional activator of fatty acid synthesis, was recently identified as a target of KIN10, a catalytic α-subunit of the SUCROSE-NON-FERMENTING1-RELATED PROTEIN KINASE1 (SnRK1). We tested the hypothesis that trehalose 6-phosphate (T6P), a signal of cellular sucrose status, can regulate fatty acid synthesis by inhibiting SnRK1. Incubation of Brassica napus suspension cells in medium containing T6P, or overexpression of the Escherichia coli T6P synthase, OtsA, in Nicotiana benthamiana, significantly increased T6P levels, WRI1 levels, and fatty acid synthesis rates. T6P directly bound to purified recombinant KIN10 with an equilibrium dissociation constant (K d) of 32 ± 6 µM based on microscale thermophoresis. GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1) bound to KIN10 (K d 19 ± 3 µM) and activated it by phosphorylation. In the presence of T6P, the GRIK1-KIN10 association was weakened by more than 3-fold (K d 68 ± 9.8 µM), which reduced both the phosphorylation of KIN10 and its activity. T6P-dependent inhibition of SnRK1 activity was reduced in extracts of individual Arabidopsis thaliana grik1 and grik2 mutants relative to the wild type, while SnRK1 activity in grik1 grik2 extracts was enhanced by T6P. These results indicate that the T6P sensitivity of SnRK1 in vivo is GRIK1/GRIK2 dependent. Based on our findings, we propose a mechanistic model that links sugar signaling and fatty acid homeostasis.

Identification of bottlenecks in the accumulation of cyclic fatty acids in camelina seed oil.

Modified fatty acids (mFA) have diverse uses; for example, cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics and cosmetics. The expression of mFA-producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural-occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co-expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA-accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon co-expression of SfLPAT with EcCPS, di-CPA-PC increased by ~50% relative to lines expressing EcCPS alone with the di-CPA-PC primarily observed in the embryonic axis and mono-CPA-PC primarily in cotyledon tissue. EcCPS-SfLPAT lines revealed a redistribution of CPA from the sn-1 to sn-2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.

Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis.

WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors, positively regulates glycolysis and lipid biosynthesis in Arabidopsis thaliana Here, we identify mechanistic links between KIN10, the major SUCROSE NON-FERMENTATION1-RELATED KINASE1 involved in sugar/energy homeostasis, and the posttranslational regulation of WRI1. Transient expression of WRI1 with OLEOSIN1 in Nicotiana benthamiana stimulates triacylglycerol accumulation, but their coexpression with KIN10 abrogates this effect by inducing proteasomal degradation of WRI1. While WRI1 lacks canonical KIN10 target sequences, we demonstrated direct KIN10-dependent phosphorylation of WRI1 using purified Escherichia coli-expressed components. The resulting phosphorylated WRI1 was more rapidly degraded than native WRI1 in cell-free degradation assays. WRI1 phosphorylation was localized to two variants of the canonical KIN10 recognition sequence, one in each of its two AP2 DNA binding domains. Conversion of the phosphorylation sites at Thr-70 and Ser-166 to Ala resulted in a loss of KIN10-dependent phosphorylation, and when coexpressed with KIN10 the WRI1 double mutant accumulated to 2- to 3-fold higher levels than native WRI1. KIN10-dependent degradation of WRI1 provides a homeostatic mechanism that favors lipid biosynthesis when intracellular sugar levels are elevated and KIN10 is inhibited; conversely, glycolysis and lipid biosynthesis are curtailed as sugar levels decrease and KIN10 regains activity.

Arabidopsis lipins, PDAT1 acyltransferase, and SDP1 triacylglycerol lipase synergistically direct fatty acids toward ß-oxidation, thereby maintaining membrane lipid homeostasis.

Triacylglycerol (TAG) metabolism is a key aspect of intracellular lipid homeostasis in yeast and mammals, but its role in vegetative tissues of plants remains poorly defined. We previously reported that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is crucial for diverting fatty acids (FAs) from membrane lipid synthesis to TAG and thereby protecting against FA-induced cell death in leaves. Here, we show that overexpression of PDAT1 enhances the turnover of FAs in leaf lipids. Using the trigalactosyldiacylglycerol1-1 (tgd1-1) mutant, which displays substantially enhanced PDAT1-mediated TAG synthesis, we demonstrate that disruption of SUGAR-DEPENDENT1 (SDP1) TAG lipase or PEROXISOMAL TRANSPORTER1 (PXA1) severely decreases FA turnover, leading to increases in leaf TAG accumulation, to 9% of dry weight, and in total leaf lipid, by 3-fold. The membrane lipid composition of tgd1-1 sdp1-4 and tgd1-1 pxa1-2 double mutants is altered, and their growth and development are compromised. We also show that two Arabidopsis thaliana lipin homologs provide most of the diacylglycerol for TAG synthesis and that loss of their functions markedly reduces TAG content, but with only minor impact on eukaryotic galactolipid synthesis. Collectively, these results show that Arabidopsis lipins, along with PDAT1 and SDP1, function synergistically in directing FAs toward peroxisomal ß-oxidation via TAG intermediates, thereby maintaining membrane lipid homeostasis in leaves.

Survey of the total fatty acid and triacylglycerol composition and content of 30 duckweed species and cloning of a Δ6-desaturase responsible for the production of γ-linolenic and stearidonic acids in Lemna gibba.

BACKGROUND: Duckweeds, i.e., members of the Lemnoideae family, are amongst the smallest aquatic flowering plants. Their high growth rate, aquatic habit and suitability for bio-remediation make them strong candidates for biomass production. Duckweeds have been studied for their potential as feedstocks for bioethanol production; however, less is known about their ability to accumulate reduced carbon as fatty acids (FA) and oil. RESULTS: Total FA profiles of thirty duckweed species were analysed to assess the natural diversity within the Lemnoideae. Total FA content varied between 4.6% and 14.2% of dry weight whereas triacylglycerol (TAG) levels varied between 0.02% and 0.15% of dry weight. Three FA, 16:0 (palmitic), 18:2Δ9,12 (Linoleic acid, or LN) and 18:3Δ9,12,15 (α-linolenic acid, or ALA) comprise more than 80% of total duckweed FA. Seven Lemna and two Wolffiela species also accumulate polyunsaturated FA containing Δ6-double bonds, i.e., GLA and SDA. Relative to total FA, TAG is enriched in saturated FA and deficient in polyunsaturated FA, and only five Lemna species accumulate Δ6-FA in their TAG. A putative Δ6-desaturase designated LgDes, with homology to a family of front-end Δ6-FA and Δ8-spingolipid desaturases, was identified in the assembled DNA sequence of Lemna gibba. Expression of a synthetic LgDes gene in Nicotiana benthamiana resulted in the accumulation of GLA and SDA, confirming it specifies a Δ6-desaturase. CONCLUSIONS: Total accumulation of FA varies three-fold across the 30 species of Lemnoideae surveyed. Nine species contain GLA and SDA which are synthesized by a Δ6 front-end desaturase, but FA composition is otherwise similar. TAG accumulates up to 0.15% of total dry weight, comparable to levels found in the leaves of terrestrial plants. Polyunsaturated FA is underrepresented in TAG, and the Δ6-FA GLA and SDA are found in the TAG of only five of the nine Lemna species that produce them. When present, GLA is enriched and SDA diminished relative to their abundance in the total FA pool.

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.

Male Sterile2 encodes a plastid-localized fatty acyl carrier protein reductase required for pollen exine development in Arabidopsis.

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. In addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30°C), MS2 exhibits a K(m) for 16:0-Acyl Carrier Protein of 23.3 ± 4.0 µm, a V(max) of 38.3 ± 4.5 nmol mg⁻¹ min⁻¹, and a catalytic efficiency/K(m) of 1,873 M⁻¹ s⁻¹. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.

Characterization and analysis of the cotton cyclopropane fatty acid synthase family and their contribution to cyclopropane fatty acid synthesis.

BACKGROUND: Cyclopropane fatty acids (CPA) have been found in certain gymnosperms, Malvales, Litchi and other Sapindales. The presence of their unique strained ring structures confers physical and chemical properties characteristic of unsaturated fatty acids with the oxidative stability displayed by saturated fatty acids making them of considerable industrial interest. While cyclopropenoid fatty acids (CPE) are well-known inhibitors of fatty acid desaturation in animals, CPE can also inhibit the stearoyl-CoA desaturase and interfere with the maturation and reproduction of some insect species suggesting that in addition to their traditional role as storage lipids, CPE can contribute to the protection of plants from herbivory. RESULTS: Three genes encoding cyclopropane synthase homologues GhCPS1, GhCPS2 and GhCPS3 were identified in cotton. Determination of gene transcript abundance revealed differences among the expression of GhCPS1, 2 and 3 showing high, intermediate and low levels, respectively, of transcripts in roots and stems; whereas GhCPS1 and 2 are both expressed at low levels in seeds. Analyses of fatty acid composition in different tissues indicate that the expression patterns of GhCPS1 and 2 correlate with cyclic fatty acid (CFA) distribution. Deletion of the N-terminal oxidase domain lowered GhCPS's ability to produce cyclopropane fatty acid by approximately 70%. GhCPS1 and 2, but not 3 resulted in the production of cyclopropane fatty acids upon heterologous expression in yeast, tobacco BY2 cell and Arabidopsis seed. CONCLUSIONS: In cotton GhCPS1 and 2 gene expression correlates with the total CFA content in roots, stems and seeds. That GhCPS1 and 2 are expressed at a similar level in seed suggests both of them can be considered potential targets for gene silencing to reduce undesirable seed CPE accumulation. Because GhCPS1 is more active in yeast than the published Sterculia CPS and shows similar activity when expressed in model plant systems, it represents a strong candidate gene for CFA accumulation via heterologous expression in production plants.

Modulating seed beta-ketoacyl-acyl carrier protein synthase II level converts the composition of a temperate seed oil to that of a palm-like tropical oil.

beta-Ketoacyl-acyl carrier protein (ACP) synthase II (KASII) elongates 16:0-ACP to 18:0-ACP in the plastid, where it competes with three other enzymes at the first major branch point in fatty acid biosynthesis. Despite its key metabolic location, the influence of KASII in determining seed oil composition remains unclear, in part because the biochemical consequences of the fab1-1 mutation were unresolved. Thus, fab1-1, and a newly identified knockout allele, fab1-2, were analyzed in the context of the hypothesis that modulating KASII activity is sufficient to convert the composition of a temperate seed oil into that of a palm-like tropical oil. No homozygous fab1-2 individuals were identified in progeny of self-fertilized heterozygous fab1-2 plants, approximately 1/4 of which aborted before the torpedo stage, suggesting that fab1-2 represents a complete loss of function and results in lethality when homozygous. Consistent with this hypothesis, homozygous fab1-2 plants were identified when a fab1-1 transgene was introduced, demonstrating that fab1-1 encodes an active KASII. Strong seed-specific hairpin-RNAi reductions in FAB1 expression resulted in abortion of approximately 1/4 of the embryos in an apparent phenocopy of fab1-2 homozygosity. In less severe FAB1 hairpin-RNAi individuals, embryos developed normally and exhibited a 1:2:1 segregation ratio for palmitate accumulation. Thus, early embryo development appears sensitive to elevated 16:0, whereas at later stages, up to 53% of 16:0, i.e., a 7-fold increase over wild-type levels, is tolerated. These results resolve the role of KASII in seed metabolism and demonstrate that modulation of Arabidopsis KASII levels is sufficient to convert its temperate oilseed composition to that of a palm-like tropical oil.

Effect of substrate on the diiron(III) site in stearoyl acyl carrier protein delta 9-desaturase as disclosed by cryoreduction electron paramagnetic resonance/electron nuclear double resonance spectroscopy.

The diiron center in stearoyl-acyl carrier protein (ACP) desaturase (DS) from castor plant Ricinus communis catalyzes the dioxygen- and NADPH-dependent introduction of a cis double bond between C9 and C10 of stearoyl-ACP. Radiolytic reduction of diferric DS at 77 K produces an electron paramagnetic resonance (EPR)-detectable mixed-valence center (or [DS(ox)](mv)) that is trapped in the conformation of the diferric precursor and thus provides a sensitive EPR/electron nuclear double resonance (ENDOR) probe of the structure of the diamagnetic diiron(III) state. The cryoreduced DS shows two distinct EPR signals, suggesting the presence of two diiron(III) states: the mu-oxo (major)- and mu-hydroxo (minor)-bridged diiron centers. ENDOR studies show that in the dominant oxo-bridged diferric state each iron(III) coordinates a histidine and a water along with other ligands. Samples containing stoichiometric amounts of stearoyl-ACP show pronounced changes in the EPR and (1)H ENDOR spectra of cryoreduced DS. EPR spectra of the cryoreduced DS-substrate complex reveal two distinct substates of the parent. EPR and ENDOR studies show that both major conformers of the diferric cluster have a mu-oxo bridge. ENDOR shows that the major conformer has a histidine and a water bound to both Fe ions. In the minor conformer, one of the irons has lost the terminal water ligand. The structure of the trapped [DS(ox)](mv) state relaxes upon annealing to 170 K: the mu-oxo bridge in the major cryoreduced DS species protonates on annealing to 170 K; this does not occur for the major DS-substrate complex, even upon annealing to 230 K. The relaxed Fe(II)Fe(III) center in cryoreduced DS and DS-substrate show much less intense and resolved (14)N ENDOR spectra than those of the structurally similar cryoreduced diiron center in ribonucleotide reductase (RNR) protein R2. This difference may reflect some differences in His-Fe bonds. The alterations in the diferric site of DS induced by substrate are suggested to be mediated by conformational changes in the polypeptide chain produced by substrate binding. These structural alterations may provide DS with an additional mechanism for tuning the redox potential of the diferric site. The mixed-valence states of DS are unstable at temperatures above 230 K.

Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family.

Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site.