Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1^cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2^cisΔ9,12; 17.9%–44.4% and 7%–13.2%, respectively) and decreases in 20:1^cisΔ11 (38.7%–60.7% and 13.8%–16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3^cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%–48.9% and 5.3%–10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil.Cytosolic low-molecular mass (approximately 10 kD) acyl-coenzyme A-binding protein (ACBP) consists of a four-α-helix domain capable of binding acyl-CoAs with high affinity in a wide range of eukaryotic organisms (Faergeman et al., 2007). It is believed to serve a housekeeping function of maintaining free acyl-CoA concentrations at low nanomolar levels and, thus, prevents micelle formation and the partitioning of acyl-CoA into membranes (Knudsen et al., 1999). This protein is also considered to contribute to another facet of acyl-CoA pool maintenance via its role in the intracellular transport of acyl-CoAs in the aqueous environment of the cytosol (Rasmussen et al., 1994). Moreover, it has also been shown to exhibit more specialized functions in metabolic processes in which acyl-CoA is actively involved, depending on the tissue and physiological state (Guerrero et al., 2006; Xiao and Chye 2011; Yurchenko and Weselake, 2011).In the developing seeds of oleaginous plants, fatty acids (FAs) are synthesized de novo in plastids and are activated to acyl-CoAs upon their transfer to the cytosol, after which time they can undergo additional modifications (e.g. elongation and desaturation) on the membranes of the endoplasmic reticulum (ER; for review, see Rawsthorne, 2002). While FA elongation is performed on the acyl-CoA substrate, the introduction of the second and third double bonds requires the acyl group to be esterified to phosphatidylcholine (PC; Jaworski, 1987). The composition of the acyl-CoA pool, therefore, is highly dynamic and represents a net result of both de novo synthesis and acyl-editing processes (Bates et al., 2009).The acyl-CoA pool provides substrate for acyltransferases involved in the biosynthesis of triacylglycerol (TAG), which is a major component of seed oil (Weselake et al., 2009). More specifically, TAG synthesis typically occurs via a series of acyl-CoA-dependent acylations of a glycerol backbone derived from sn-glycerol-3-phosphate in a pathway known as the sn-glycerol-3-phosphate or Kennedy pathway (for review, see Snyder et al., 2009; Weselake et al., 2009), although acyl-CoA-independent reactions can also be involved in the production of TAG (Stobart et al., 1997; Banaś et al., 2000; Dahlqvist et al., 2000) and thus contribute to its final composition. Low-molecular mass ACBPs have been demonstrated to modulate the activities of Kennedy pathway acyltransferases in a manner dependent upon the ratio of ACBP to acyl-CoA, stimulating TAG biosynthesis under conditions of acyl-CoA excess and inhibiting acyltransferase activities when relative amounts of acyl-CoA are low compared with ACBP, thus regulating the size of the acyl-CoA pool (for review, see Yurchenko and Weselake, 2011).The acyl-CoA pool in seeds is also influenced through a distinct route involving lysophosphatidylcholine acyltransferase (LPCAT), which catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine at the sn-2 position to form PC (Ichihara et al., 1995). Acyl groups esterified to PC become substrates for FA desaturation and other modifications (Miquel and Browse, 1992; Broun et al., 1998) and can then be returned back to the acyl-CoA pool or channeled into TAG through acyl-CoA-independent mechanisms (Stymne and Stobart, 1984; Weselake, 2005; Lager et al., 2013). The efficiency of this acyl group channeling to and from PC is an important determinant of the overall composition of FAs in the acyl-CoA pool and, subsequently, in seed oil.Previously, we demonstrated that the expression of the Brassica napus low-molecular mass ACBP (hereafter referred to as BnACBP) in the presence of Arabidopsis (Arabidopsis thaliana) LPCAT isoforms in an in vitro system enhanced the incorporation of oleic acid (18:1^cisΔ9; hereafter referred to as 18:1) into PC and the release of linoleic acid (18:2^cisΔ9,12; hereafter referred to as 18:2) from PC into acyl-CoA (Yurchenko et al., 2009). In line with these results, the expression of BnACBP complementary DNA (cDNA) in Arabidopsis developing seeds was also shown to result in elevated levels of the polyunsaturated fatty acids (PUFAs) 18:2 and α-linolenic acid (18:3^cisΔ9,12,15; hereafter referred to as 18:3) in seed oil, mainly at the expense of eicosenoic acid (20:1^cisΔ11; hereafter referred to as 20:1) and saturated fatty acids (SFAs; Yurchenko et al., 2009). Based on these findings, BnACBP was proposed to be involved in acyl exchange between acyl-CoA and PC pools, which may affect the rate of FA modifications and, ultimately, the FA composition of seed oil (Yurchenko et al., 2009).In this study, we endeavored to provide further evidence that low-molecular mass ACBP functions in acyl trafficking by investigating whether changes in the FA composition of TAG in Arabidopsis seeds expressing BnACBP were correlated with modifications in the composition of the acyl-CoA pool. In addition, since FA modifications such as elongation and desaturation as well as TAG synthesis occur on ER membranes, we also examined the effect of changing the subcellular localization of BnACBP (from the cytosol to the ER) on the acyl composition of TAG and the acyl-CoA pool in transgenic Arabidopsis. Consequently, we generated localized pools of acyl-CoAs that could be readily accessed by acyltransferases involved in seed oil biosynthesis. Taken together, our findings provide insight into the role of low-molecular mass ACBP in seed oil metabolism and suggest that ACBP (either in its native cytosolic form or as an ER-targeted fusion protein) may serve as a useful tool in biotechnological modifications of FA composition in oil crops.     

Mitigation of establishment of Brassica napus transgenes in volunteers using a tandem construct containing a selectively unfit gene

Transgenic oilseed rape ( Brassica napus ) plants may remain as 'volunteer' weeds in following crops, complicating cultivation and contaminating crop yield. Volunteers can become feral as well as act as a genetic bridge for the transfer of transgenes to weedy relatives. Transgenic mitigation using genes that are positive or neutral to the crop, but deleterious to weeds, should prevent volunteer establishment, as previously intimated using a tobacco ( Nicotiana tabacum ) model. A transgenically mitigated (TM), dwarf, herbicide-resistant construct using a gibberellic acid-insensitive (Δ gai ) gene in the B. napus crop was effective in offsetting the risks of transgene establishment in volunteer populations of B. napus . This may be useful in the absence of herbicide, e.g. when wheat is rotated with oilseed rape. The TM dwarf B. napus plants grown alone had a much higher yield than the non-transgenics, but were exceedingly unfit in competition with non-transgenic tall cohorts. The reproductive fitness of TM B. napus was 0% at 2.5-cm and 4% at 5-cm spacing between glasshouse-grown plants relative to non-transgenic B. napus . Under screen-house conditions, the reproductive fitness of TM B. napus relative to non-transgenic B. napus was less than 12%, and the harvest index of the TM plants was less than 40% of that of the non-transgenic competitors. The data clearly indicate that the Δ gai gene greatly enhances the yield in a weed-free transgenic crop, but the dwarf plants can be eliminated when competing with non-transgenic cohorts (and presumably other species) when the selective herbicide is not used.     

High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds

Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova?400.     

Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants - a review

The increasing effect of non-degradable plastic wastes is a growing concern. Polyhydroxyalkanoates (PHAs), macromolecule-polyesters naturally produced by many species of microorganisms, are being considered as a replacement for conventional plastics. Unlike petroleum-derived plastics that take several decades to degrade, PHAs can be completely bio-degraded within a year by a variety of microorganisms. This biodegradation results in carbon dioxide and water, which return to the environment. Attempts based on various methods have been undertaken for mass production of PHAs. Promising strategies involve genetic engineering of microorganisms and plants to introduce production pathways. This challenge requires the expression of several genes along with optimization of PHA synthesis in the host. Although excellent progress has been made in recombinant hosts, the barriers to obtaining high quantities of PHA at low cost still remain to be solved. The commercially viable production of PHA in crops, however, appears to be a realistic goal for the future.     

The influence of meteorology on the spread of influenza: survival analysis of an equine influenza (A/H3N8) outbreak

The influences of relative humidity and ambient temperature on the transmission of influenza A viruses have recently been established under controlled laboratory conditions. The interplay of meteorological factors during an actual influenza epidemic is less clear, and research into the contribution of wind to epidemic spread is scarce. By applying geostatistics and survival analysis to data from a large outbreak of equine influenza (A/H3N8), we quantified the association between hazard of infection and air temperature, relative humidity, rainfall, and wind velocity, whilst controlling for premises-level covariates. The pattern of disease spread in space and time was described using extraction mapping and instantaneous hazard curves. Meteorological conditions at each premises location were estimated by kriging daily meteorological data and analysed as time-lagged time-varying predictors using generalised Cox regression. Meteorological covariates time-lagged by three days were strongly associated with hazard of influenza infection, corresponding closely with the incubation period of equine influenza. Hazard of equine influenza infection was higher when relative humidity was <60% and lowest on days when daily maximum air temperature was 20-25°C. Wind speeds >30 km hour(-1) from the direction of nearby infected premises were associated with increased hazard of infection. Through combining detailed influenza outbreak and meteorological data, we provide empirical evidence for the underlying environmental mechanisms that influenced the local spread of an outbreak of influenza A. Our analysis supports, and extends, the findings of studies into influenza A transmission conducted under laboratory conditions. The relationships described are of direct importance for managing disease risk during influenza outbreaks in horses, and more generally, advance our understanding of the transmission of influenza A viruses under field conditions.     

Implication of climate change for the persistence of raptors in arid savanna

Arid savannas are regarded as one of the ecosystems most likely to be affected by climate change. In these dry conditions, even top predators like raptors are affected by water availability and precipitation. However, few research initiatives have addressed the question of how climate change will affect population dynamics and extinction risk of particular species in arid ecosystems. Here, we use an individual‐oriented modeling approach to conduct experiments on the population dynamics of long lived raptors. We investigate the potential impact of precipitation variation caused by climate change on raptors in arid savanna using the tawny eagle (Aquila rapax) in the southern Kalahari as a case study. We simulated various modifications of precipitation scenarios predicted for climate change, such as lowered annual precipitation mean, increased inter‐annual variation and increased auto‐correlation in precipitation. We found a high impact of these modifications on extinction risk of tawny eagles, with reduced population persistence in most cases. Decreased mean annual precipitation and increased inter‐annual variation both caused dramatic decreases in population persistence. Increased auto‐correlation in precipitation led only to slightly accelerated extinction of simulated populations. Finally, for various patterns of periodically fluctuating precipitation, we found both increased and decreased population persistence. In summary, our results suggest that the impacts on raptor population dynamics and survival caused by climate change in arid savannas will be great. We emphasize that even if under climate change the mean annual precipitation remains constant but the inter‐annual variation increases the persistence of raptor populations in arid savannas will decrease considerably. This suggests a new dimension of climate change driven impacts on population persistence and consequently on biodiversity. However, more investigations on particular species and/or species groups are needed to increase our understanding of how climate change will impact population dynamics and how this will influence species diversity and biodiversity.     

Membrane protein topology of oleosin is constrained by its long hydrophobic domain.

Oleosin proteins from Arabidopsis assume a unique endoplasmic reticulum (ER) topology with a membrane-integrated hydrophobic (H) domain of 72 residues, flanked by two cytosolic hydrophilic domains. We have investigated the targeting and topological determinants present within the oleosin polypeptide sequence using ER-derived canine pancreatic microsomes. Our data indicate that oleosins are integrated into membranes by a cotranslational, translocon-mediated pathway. This is supported by the identification of two independent functional signal sequences in the H domain, and by demonstrating the involvement of the SRP receptor in membrane targeting. Oleosin topology was manipulated by the addition of an N-terminal cleavable signal sequence, resulting in translocation of the N terminus to the microsomal lumen. Surprisingly, the C terminus failed to translocate. Inhibition of C-terminal translocation was not dependent on either the sequence of hydrophobic segments in the H domain, the central proline knot motif or charges flanking the H domain. Therefore, the topological constraint results from the length and/or the hydrophobicity of the H domain, implying a general case that long hydrophobic spans are unable to translocate their C terminus to the ER lumen.