LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

Physaria fendleri and Ricinus communis lecithin:cholesterol acyltransferase-like phospholipases selectively cleave hydroxy acyl chains from phosphatidylcholine.

Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12-HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn-1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over-expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase-like PLAs (LCAT-PLAs) in HFA biosynthesis were characterized. The LCAT-PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2 , and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT-PLAs from the HFA-accumulating plant species Physaria fendleri (PfLCAT-PLA) and castor (RcLCAT-PLA) could cleave acyl chains at both the sn-1 and sn-2 positions of PC, and displayed substrate selectivity towards sn-2-ricinoleoyl-PC over sn-2-oleoyl-PC. Furthermore, co-expression of RcFAH12 with PfLCAT-PLA or RcLCAT-PLA, but not Arabidopsis AtLCAT-PLA, resulted in increased occupation of HFA at the sn-1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT-PLA and RcLCAT-PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops.

Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1.

The apparent bottleneck in the accumulation of oil during seed development in some oleaginous plant species is the formation of triacylglycerol (TAG) by the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol catalyzed by diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). Improving DGAT activity using protein engineering could lead to improvements in seed oil yield (e.g. in canola-type Brassica napus). Directed evolution of B. napus DGAT1 (BnaDGAT1) previously revealed that one of the regions where amino acid residue substitutions lead to higher performance in BnaDGAT1 is in the ninth predicted transmembrane domain (PTMD9). In this study, several BnaDGAT1 variants with amino acid residue substitutions in PTMD9 were characterized. Among these enzyme variants, the extent of yeast TAG production was affected by different mechanisms, including increased enzyme activity, increased polypeptide accumulation, and possibly reduced substrate inhibition. The kinetic properties of the BnaDGAT1 variants were affected by the amino acid residue substitutions, and a new kinetic model based on substrate inhibition and sigmoidicity was generated. Based on sequence alignment and further biochemical analysis, the amino acid residue substitutions that conferred increased TAG accumulation were shown to be present in the DGAT1-PTMD9 region of other higher plant species. When amino acid residue substitutions that increased BnaDGAT1 enzyme activity were introduced into recombinant Camelina sativa DGAT1, they also improved enzyme performance. Thus, the knowledge generated from directed evolution of DGAT1 in one plant species can be transferred to other plant species and has potentially broad applications in genetic engineering of oleaginous crops and microorganisms.

Turning Over a New Leaf in Lipid Droplet Biology.

Lipid droplets (LDs) in plants have long been viewed as storage depots for neutral lipids that serve as sources of carbon, energy, and lipids for membrane biosynthesis. While much of our knowledge of LD function in plants comes from studies of oilseeds, a recent surge in research on LDs in non-seed cell types has led to an array of new discoveries. It is now clear that both evolutionarily conserved and kingdom-specific mechanisms underlie the biogenesis of LDs in eukaryotes, and proteomics and homology-based approaches have identified new protein players. This review highlights some of these recent discoveries and other new areas of plant LD research, including their role in stress responses and as targets of metabolic engineering strategies aimed at increasing oil content in bioenergy crops.

Two Clades of Type-1 Brassica napus Diacylglycerol Acyltransferase Exhibit Differences in Acyl-CoA Preference.

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to produce triacylglycerol, which is the main component of the seed oil of Brassica oilseed species. Phylogenetic analysis of the amino acid sequences encoded by four transcriptionally active DGAT1 genes from Brassica napus suggests that the gene forms diverged over time into two clades (I and II), with representative members in each genome (A and C). The majority of the amino acid sequence differences in these forms of DGAT1, however, reside outside of motifs suggested to be involved in catalysis. Despite this, the clade II enzymes displayed a significantly enhanced preference for linoleoyl-CoA when assessed using in-vitro enzyme assays with yeast microsomes containing recombinant enzyme forms. These findings contribute to our understanding of triacylglycerol biosynthesis in B. napus, and may advance our ability to engineer DGAT1s with desired substrate selectivity properties.

In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase.

Seed oils of flax (Linum usitatissimum L.) and many other plant species contain substantial amounts of polyunsaturated fatty acids (PUFAs). Phosphatidylcholine (PC) is the major site for PUFA synthesis. The exact mechanisms of how these PUFAs are channeled from PC into triacylglycerol (TAG) needs to be further explored. By using in vivo and in vitro approaches, we demonstrated that the PC deacylation reaction catalyzed by the reverse action of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) can transfer PUFAs on PC directly into the acyl-CoA pool, making these PUFAs available for the diacylglycerol acyltransferase (DGAT)-catalyzed reaction for TAG production. Two types of yeast mutants were generated for in vivo and in vitro experiments, respectively. Both mutants provide a null background with no endogenous TAG forming capacity and an extremely low LPCAT activity. In vivo experiments showed that co-expressing flax DGAT1-1 and LPCAT1 in the yeast quintuple mutant significantly increased 18-carbon PUFAs in TAG with a concomitant decrease of 18-carbon PUFAs in phospholipid. We further showed that after incubation of sn-2-[(14)C]acyl-PC, formation of [(14)C]TAG was only possible with yeast microsomes containing both LPCAT1 and DGAT1-1. Moreover, the specific activity of overall LPCAT1 and DGAT1-1 coupling process exhibited a preference for transferring (14)C-labeled linoleoyl or linolenoyl than oleoyl moieties from the sn-2 position of PC to TAG. Together, our data support the hypothesis of biochemical coupling of the LPCAT1-catalyzed reverse reaction with the DGAT1-1-catalyzed reaction for incorporating PUFAs into TAG. This process represents a potential route for enriching TAG in PUFA content during seed development in flax.

Purification and properties of recombinant Brassica napus diacylglycerol acyltransferase 1.

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in the acyl-CoA-dependent triacylglycerol biosynthesis. Although the first DGAT1 gene was identified many years ago and the encoded enzyme catalyzes a key step in lipid biosynthesis, no detailed structure-function information is available on the enzyme due to difficulties associated with its purification. This study describes the purification of recombinant Brassica napus DGAT1 (BnaC.DGAT1.a) in active form through solubilization in n-dodecyl-ß-D-maltopyranoside, cobalt affinity chromatography, and size-exclusion chromatography. Different BnaC.DGAT1.a oligomers in detergent micelles were resolved during the size-exclusion process. BnaC.DGAT1.a was purified 126-fold over the solubilized fraction and exhibited a specific activity of 26 nmol TAG/min/mg protein. The purified enzyme exhibited substrate preference for α-linolenoyl-CoA>oleoyl-CoA=palmitoyl-CoA>linoleoyl-CoA>stearoyl-CoA.

Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase.

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol to produce triacylglycerol (TAG). This enzyme, which is critical to numerous facets of oilseed development, has been highlighted as a genetic engineering target to increase storage lipid production in microorganisms designed for biofuel applications. Here, four transcriptionally active DGAT1 genes were identified and characterized from the oil crop Brassica napus. Overexpression of each BnaDGAT1 in Saccharomyces cerevisiae increased TAG biosynthesis. Further studies showed that adding an N-terminal tag could mask the deleterious influence of the DGATs' native N-terminal sequences, resulting in increased in vivo accumulation of the polypeptides and an increase of up to about 150-fold in in vitro enzyme activity. The levels of TAG and total lipid fatty acids in S. cerevisiae producing the N-terminally tagged BnaDGAT1.b at 72 h were 53 and 28 % higher than those in cultures producing untagged BnaA.DGAT1.b, respectively. These modified DGATs catalyzed the synthesis of up to 453 mg fatty acid/L by this time point. The results will be of benefit in the biochemical analysis of recombinant DGAT1 produced through heterologous expression in yeast and offer a new approach to increase storage lipid content in yeast for industrial applications.

Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid.

MAIN CONCLUSION: Arabidopsis was engineered to produce 21.2 % punicic acid in the seed oil. Possible molecular factors limiting further accumulation of the conjugated fatty acid were investigated. Punicic acid (18:3Δ(9cis,11trans,13cis) ) is a conjugated linolenic acid isomer and is a main component of Punica granatum (pomegranate) seed oil. Medical studies have shown that punicic acid is a nutraceutical with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bonds in punicic acid are produced via the catalytic action of fatty acid conjugase (FADX), which is a homolog of the oleate desaturase. This enzyme catalyzes the conversion of the Δ(12)-double bond of linoleic acid (18:2Δ(9cis,12cis) ) into conjugated Δ(11trans) and Δ(13cis) -double bonds. Previous attempts to produce punicic acid in transgenic Arabidopsis thaliana seeds overexpressing P. granatum FADX resulted in a limited accumulation of punicic acid of up to 4.4 %, accompanied by increased accumulation of oleic acid (18:1∆(9cis) ), suggesting that production of punicic acid in some way inhibits the activity of oleate desaturase (Iwabuchi et al. 2003). In the current study, we applied a new strategy to enhance the production of punicic acid in a high linoleic acid A. thaliana fad3/fae1 mutant background using the combined expression of P. granatum FADX and FAD2. This approach led to the accumulation of punicic acid at the level of 21 % of total fatty acids and restored the natural proportion of oleic acid observed in the A. thaliana fad3/fae1 mutant. In addition, we provide new insights into the high oleate phenotype and describe factors limiting the production of punicic acid in genetically engineered plants.

A novel assay of DGAT activity based on high temperature GC/MS of triacylglycerol.

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG), a high-energy compound composed of three fatty acids esterified to a glycerol backbone. In vitro DGAT assays, which are usually conducted with radiolabeled substrate using microsomal fractions, have been useful in identifying compounds and genetic modifications that affect DGAT activity. Here, we describe a high-temperature gas chromatography (GC)/mass spectrometry (MS)-based method for monitoring molecular species of TAG produced by the catalytic action of microsomal DGAT. This method circumvents the need for radiolabeled or modified substrates, and only requires a simple lipid extraction prior to GC. The utility of the method is demonstrated using a recombinant type-1 Brassica napus DGAT produced in a strain of Saccharomyces cerevisae that is deficient in TAG synthesis. The GC/MS-based assay of DGAT activity was strongly correlated with the typical in vitro assay of the enzyme using [1-(14)C] acyl-CoA as an acyl donor. In addition to determining DGAT activity, the method is also useful for determining substrate specificity and selectivity properties of the enzyme.

Plant phospholipase A: advances in molecular biology, biochemistry, and cellular function.

Plant phospholipase As (PLAs) are a complex group of enzymes that catalyze the release of free fatty acids from phospholipids. Plant PLAs can be grouped into three families, PLA1, PLA2, and patatin-like PLA, that catalyze the hydrolysis of acyl groups from the sn-1 and/or sn-2 position. Each family is composed of multiple isoforms of phospholipases that differ in structural, catalytic, and physiological characteristics. In this review, recently acquired information on molecular, biochemical, and functional aspects of plant PLAs will be discussed.

Identification and characterization of an LCAT-like Arabidopsis thaliana gene encoding a novel phospholipase A.

A previously uncharacterized Arabidopsis lecithin:cholesterol acyltransferase (LCAT) family gene (At4g19860) was functionally expressed in yeast, where it was demonstrated to encode a novel cytosolic and calcium-independent phospholipase A with preferences for the sn-2 position. This enzyme shows optimal activity at pH 5.0, exhibits a headgroup specificity for phosphatidylcholine>phosphatidic acid>phosphatidylethanolamine>phosphatidylglycerol>phosphatidylserine and has an acyl chain specificity for oleoyl>linoleoyl>ricinoleoyl. The expression of AtLCAT-PLA inhibited yeast cell growth and fatty acid accumulation. AtLCAT-PLA transcript in Arabidopsis was detected at high levels in roots and siliques.

Management of chronic mesenteric vascular insufficiency: an endovascular approach.

BACKGROUND: In patients with chronic mesenteric insufficiency (CMI), traditional bypass surgery carries a high operative mortality. Endovascular therapy for reconstruction of the mesenteric vascular system has high technical success but poor long-term patency. Secondary procedures are often mandatory for recurrent disease. The purpose of this study was to evaluate an endovascular-first treatment strategy for CMI, reserving open reconstruction for complex disease patterns without an endovascular option. STUDY DESIGN: Data for consecutive adult patients (N = 107) initially treated with endovascular techniques for CMI were reviewed. The management algorithm consisted of postoperative and biannual ultrasound and clinical follow-up. RESULTS: A total of 107 patients with CMI were treated from April 2004 through June 2010. Technical success for endovascular reconstruction was 100%. Long-term follow-up data were available on 90% of patients. After the index procedure, 57% of patients (n = 55) had complete resolution of the preoperative symptoms. During the management phase, 83% of patients had elevated velocities on duplex evaluation. During this interval, 53% of patients required no further intervention after the index procedure, and the remaining patients required an additional 78 procedures. Five patients required open revascularization for recurrent disease, and only 2 patients died from complications of mesenteric insufficiency. CONCLUSIONS: Endovascular management for CMI has a high technical success rate with low morbidity and mortality. Regular follow-up is essential to optimize patient outcomes. Ultrasound findings alone are a poor predictor of recurrent disease. Long-term success requires adaptation of a management program to elicit recurrent symptoms and offer prompt treatment.

Ammonium nitrate improves direct somatic embryogenesis and biolistic transformation of Triticum aestivum.

Triticum aestivum is of major importance both nutritionally and economically. Introduction of new genes has been difficult to apply to elite wheat varieties mainly as a result of their recalcitrance to prerequisite tissue culture. We attempted to improve the frequency of wheat transformation by exposing plants to high level of ammonium nitrate. Our experiments showed that modification of the ammonium nitrate content in the direct somatic embryogenesis induction medium can increase the number of primary embryos produced over twofold in the elite hard red wheat cultivar Superb. The number of primary embryos that were capable of transitioning into shoot development also increased twofold. Biolistic transformation efficiency improved as much as sevenfold when targeted scutellar tissue was exposed to elevated ammonium nitrate levels. This simple approach could become extremely useful for increasing transformation efficiency in wheat.