Expression of Rapeseed Microsomal Lysophosphatidic Acid Acyltransferase Isozymes Enhances Seed Oil Content in Arabidopsis

In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.With increasing environmental challenges and concerns, there is renewed interest in deriving plant-based sustainable alternatives for petroleum products, including carburants, lubricants, and industrial feed stocks. Modifying oilseed crops to produce oils of uniform composition containing fatty acids varying in chain length or possessing reactive functional groups is a primary objective (Jaworski and Cahoon, 2003), as is that of increasing the yield of seed oil (Lardizabal et al., 2008; Zheng et al., 2008). Early success in modifying seed oils to produce the more common fatty acids has been tempered by limited success in the production of high levels of unusual fatty acids (UFAs) in cultivated oilseeds (Thelen and Ohlrogge, 2002; Drexler et al., 2003). Such studies have led to the conclusion that in order to achieve levels of UFAs similar to those present in the oil of native species, enzymatic activities additional to fatty acid modification are necessary to optimize the synthesis (Mekhedov et al., 2001), stability (Eccleston and Ohlrogge, 1998), and channeling (Bafor et al., 1990) of the desired fatty acid into triacylglycerol (TAG).The synthesis of glycerolipids occurs in the cytoplasm using de novo-synthesized fatty acids exported from the plastid as acyl-CoA thioesters. The fatty acyl groups are incorporated into membrane and storage lipids by the sequential esterification of glycerol-3-phosphate by the action of glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) at sn-1 to form lysophosphatidic acid followed by lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51) at sn-2 to form phosphatidic acid (PA; Somerville et al., 2000). Dephosphorylation of PA results in the formation of diacylglycerol (DAG), which in developing seeds may be directed into the production of TAG by acyl-CoA-independent reactions or by diacylglycerol acyltransferase (DAGAT; EC 2.3.1.20; Roscoe, 2005). The substrate preferences for acyl-thioesters and the selectivities for the acceptor molecules displayed by the microsomal acyltransferases play a crucial role in establishing the acyl composition of lipids (Frentzen, 1998). The TAG synthesized in most oilseeds of agronomic importance contains fatty acids that are the same as those present in cytoplasmic membrane lipids. In contrast, the seeds of species that synthesize TAGs with exotic fatty acid compositions possess microsomal acyltransferases that facilitate the incorporation of UFAs into storage lipids because of their broad GPAT and/or their selective DAGAT specificities (Wiberg et al., 1994; Frentzen, 1998). Furthermore, oilseeds characterized by TAGs that contain UFAs at sn-2 possess additional seed-specific microsomal LPAATs (Brown et al., 1995; Hanke et al., 1995; Knutzon et al., 1995) that exhibit a wide variation in substrate preference and that serve to ensure the channeling of UFAs to this position, thereby segregating incompatible fatty acids away from membrane lipids.Cloning of cDNAs from cultivated and exotic plants and the availability of entirely sequenced genomes from plant and algal species have revealed that a minimum of two classes of genes encoding microsomal LPAATs exist (Frentzen, 1998) within a larger, LPAAT-like gene family containing acyltransferases as yet functionally uncharacterized but distinct from GPATs (Roscoe, 2005). The class A microsomal LPAATs defined by Frentzen (1998) possess substrate preferences for C18:1-CoA typical of enzymes involved in membrane lipid synthesis and are ubiquitously expressed in the plant. In contrast, individual members of the class B LPAATs display preferences for distinct, unusual saturated or unsaturated acyl groups and are normally expressed in storage organs. Although class B LPAATs have been exploited to alter the stereochemical composition of rapeseed (Brassica napus) oil to permit the incorporation of modified fatty acids at sn-2 (Lassner et al., 1995; Knutzon et al., 1999), a significant increase in the total amount of UFAs was not accomplished by the expression of the class B LPAATs alone. In contrast, the transformation of rapeseed and Arabidopsis (Arabidopsis thaliana) with a yeast gene encoding a variant LPAAT, SLC1-1, capable of accepting very long chain fatty acyl (VLCFA)-CoA substrates resulted in an increase in the total VLCFAs and, unexpectedly, in total oil content (Zou et al. 1997).In our efforts to modify the fatty acid composition of oil in rapeseed, in particular to increase the content of VLCFAs, we have addressed the question of optimizing the environment for the correct functioning of LPAATs encoded by transgenes. The above studies using the various LPAAT transgenes indicate that channeling of UFAs into sn-2 of oilseed species remains problematic. The ability to obtain oils with uniform composition strongly depends on the occupancy of sn-2 by UFAs, yet the level of occupancy of sn-2 by fatty acids corresponding to the selectivity of the introduced LPAAT is variable and relatively modest. Occupancy of sn-2 is determined in part by the ability of the LPAAT encoded by the transgene to compete with the endogenous enzyme, a function of the acyl-CoA substrates available to the enzymes and the relative efficiencies of the enzymes to compete for the donor and acceptor substrates. We argued that there is latitude for the reduction of competing activities using an antisense strategy, and although microsomal LPAATs have been cloned from rapeseed, there are no reports of the characterization of the enzyme. Our objectives in this work were to identify and evaluate the potential contribution of LPAAT isozymes to TAG biosynthesis in rapeseed, thereby discerning targets for optimizing efforts to modify oils for industrial purposes. In this study, we catalogue a previously undescribed complexity in microsomal LPAAT diversity and identify a LPAAT isozyme likely to play an important role in TAG synthesis in rapeseed. In contrast to diverged LPAATs of plant origin, we demonstrate a positive effect of the overexpression of microsomal LPAATs on oil content and seed weight.     

Overproduced <Emphasis Type="Italic">Brucella abortus</Emphasis> PdhS-mCherry forms soluble aggregates in <Emphasis Type="Italic">Escherichia coli</Emphasis>, partially associating with mobile foci of IbpA-YFP

Background  

When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.     

Effect of salinity on root-nodule conductance to the oxygen diffusion in the Cicer arietinum-Mesorhizobium ciceri symbiosis

Nodule conductance to O2 diffusion has been involved as a major factor of the inhibition of N2 fixation by soil salinity that severely reduces the production of grain legumes. In order to determine the effect of this constraint on the nodule conductance, oxygen uptake by the nodulated roots of Cicer arietinum was measured by recording the concentration of O2 as a function of pO2 in a gas-tight incubator. After germination and inoculation with the strain Mesorhizobium ciceri UPMCa7, the varieties Amdoun 1 and INRAT 93-1 were hydroponically grown in a glasshouse on 1L glass bottles filled with nutrient solution containing 25 mM NaCl. Salinity induced a marked decrease in shoot (30% versus 14%), root (43% versus 20%), and nodule biomass (100% versus 43%) for Amdoun 1 relative to INRAT 93-1. Although salinity completely prevented nodule formation in the sensitive variety Amdoun 1, nodule number and biomass were higher in the first than in the second variety in the absence of salt. This effect was associated with a significantly higher O2 uptake by nodulated root (510 versus 255 micromol O2 plant(-1)h(-1)) and nodule conductance (20 versus 5 microm s(-1)) in Amdoun 1 than in INRAT 93-1. Salinity did not significantly change the nodule conductance and nodule permeability for INRAT 93-1. Thus, the salt tolerance of this variety appears to be associated with stability in nodule conductance and the capacity to form nodules under salt constraint.     

Effects of information and 50 Hz magnetic fields on cognitive performance and reported symptoms

The aim of this study was to explore the role of expectancies and beliefs about the potential effects of electromagnetic fields (EMFs) (what the subject thought the effect was going to be) and the effects of 50 Hz magnetic fields (400 microT(rms)) acute exposure on cognitive performance, the reporting of physical symptoms and some psychological and physiological parameters. Seventy-four healthy male volunteers aged between 40 and 60 years of age were randomly assigned to one of five groups, which differed in (1) the type of information they were given concerning the expected magnetic field effect on performance in cognitive tests (positive = enhancement of the performance; negative = impairment of the performance; neutral) and (2) the type of exposure (real or sham). Three groups were sham exposed with positive (group+), negative (group-) and neutral information (group+/-); one group was really exposed with neutral information (group expo) and one group was not exposed, though they wore the helmet, and did not receive any field-related information (control group). All the volunteers, except the control group, were led to believe that they would be exposed to a magnetic field of 400 microT(rms). The experimental design respected a double blind procedure and the experimental session involved three steps (pre-testing, exposure, and post-testing). Various measurements were taken, including cognitive performance, psychological parameters such as mood, vigilance, and reporting of symptoms. Physiological parameters such as blood pressure and pulse rate were also recorded. The information given did not significantly modify beliefs. No significant difference was found among the five groups depending on the type of information and the type of exposure in cognitive performance, psychological and physiological parameters. In the context of the study, with our population, the type of information given failed to induce expected changes in parameters measured. Our results do not support the hypothesis that an acute exposure to extremely low frequency magnetic fields (50 Hz, 400 microT(rms)) affects the parameters measured.     

Technical note: compatibility of microtomographic imaging systems for dental measurements

Modern micro-computed tomography techniques allow the accurate visualization of internal dental structures, and are becoming widely used within (paleo-) anthropological dental studies. There exist several types and name brands of microtomographic systems, however, which have been demonstrated to produce images that vary in resolution and signal-to-noise ratio. As a growing body of dental research using disparate microtomographic techniques is likely to continue accumulating, it is imperative that different systems are compared to ensure that results are comparable and not machine-dependent. In the present study, we compare volume, surface area, and linear measurements recorded on a sample of modern and fossil teeth using four microtomographic systems (three laboratory scanners, and the ID19 beamline of the European Synchrotron Radiation Facility). Results indicate that measurements are comparable between systems (within 3%), but that synchrotron radiation is superior to the other systems because its monochromatic X-rays prevent beam hardening and its parallel beam prevents geometric artifacts in the resultant images, making it easier to record measurements and see fine details at the enamel cervix or dentine horn tips. Although the synchrotron produces higher resolution images with less artifacts, results indicate that for gross morphological measurements (e.g., enamel cap volume, intercuspal distances), each of the scanners produces approximately the same measurements. Combining measurements of teeth from multiple microCT systems presupposes that measurements from each system are comparable; the research presented here indicates that this is the case when teeth are not severely diagenetically remineralized.     

Geometry of the superior colliculus mapping and efficient oculomotor computation

Numerous brain regions encode variables using spatial distribution of activity in neuronal maps. Their specific geometry is usually explained by sensory considerations only. We provide here, for the first time, a theory involving the motor function of the superior colliculus to explain the geometry of its maps. We use six hypotheses in accordance with neurobiology to show that linear and logarithmic mappings are the only ones compatible with the generation of saccadic motor command. This mathematical proof gives a global coherence to the neurobiological studies on which it is based. Moreover, a new solution to the problem of saccades involving both colliculi is proposed. Comparative simulations show that it is more precise than the classical one.     

Fucosylated pentaerythrityl phosphodiester oligomers (PePOs): automated synthesis of DNA-based glycoclusters and binding to Pseudomonas aeruginosa lectin (PA-IIL)

The synthesis of propargylated pentaerythrityl phosphodiester oligomers (PePOs) was achieved using a DNA synthesizer with a bis-propargylated pentaerythritol-based phosphoramidite. An azido fucose derivative was reacted under "click" chemistry conditions activated by microwaves to construct a series of glycosylated PePOs bearing 4, 6, 8, and 10 L-fucose residues. Binding to the fucose-specific bacterial lectin (PA-IIL) was determined for the fucosylated PePOs through an enzyme-linked lectin amplification competition assay. The IC50 values measured are 10-20 times better than for monovalent l-fucose and denotate for a "macromolecular" effect rather than a "cluster" effect.     

Comparative maps of motion and assembly of filamentous actin and myosin II in migrating cells

To understand the mechanism of cell migration, one needs to know how the parts of the motile machinery of the cell are assembled and how they move with respect to each other. Actin and myosin II are thought to be the major structural and force-generating components of this machinery (Mitchison and Cramer, 1996; Parent, 2004). The movement of myosin II along actin filaments is thought to generate contractile force contributing to cell translocation, but the relative motion of the two proteins has not been investigated. We use fluorescence speckle and conventional fluorescence microscopy, image analysis, and computer tracking techniques to generate comparative velocity and assembly maps of actin and myosin II over the entire cell in a simple model system of persistently migrating fish epidermal keratocytes. The results demonstrate contrasting polarized assembly patterns of the two components, indicate force generation at the lamellipodium-cell body transition zone, and suggest a mechanism of anisotropic network contraction via sliding of myosin II assemblies along divergent actin filaments.