Coexpressing Escherichia coli Cyclopropane Synthase with Sterculia foetida Lysophosphatidic Acid Acyltransferase Enhances Cyclopropane Fatty Acid Accumulation

Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation.Modified fatty acids (mFAs; sometimes referred to as unusual fatty acids) obtained from plants play important roles in industrial applications as lubricants, protective coatings, plastics, inks, cosmetics, etc. The hundreds of potential industrial uses of mFAs have led to considerable interest in exploring their production in transgenic crop plants. mFAs are produced by a limited number of species, and the transfer of genes encoding mFA-producing enzymes from source plants to heterologous hosts has generally resulted in only modest accumulation, usually less than 20% of the desired mFA in transgenic seed (Napier, 2007) compared with levels found in the natural source. For example, ricinoleic acid accounts for more than 90% of the fatty acid of castor bean (Ricinus communis) seeds, and tung (Aleuites fordii) seeds accumulate more than 80% α-eleostearic acid (Thelen and Ohlrogge, 2002; Drexler et al., 2003). In order to elevate the content of mFAs in the engineered plants to that found in the native plant, it is necessary to (1) optimize the synthesis of mFA (Mekhedov et al., 2001), (2) minimize its degradation (Eccleston and Ohlrogge, 1998), and (3) optimize its incorporation into triacylglycerol (TAG; Bafor et al., 1990; Bates and Browse, 2011; van Erp et al., 2011).Cyclic fatty acids (CFAs) are desirable for numerous industrial applications. The strained bond angles of the carbocyclic ring contribute to their unique chemistry and physical properties, and hydrogenation of CFAs results in ring opening to produce methyl-branched fatty acids. Branched chain fatty acids are ideally suited for the oleochemical industry as feedstocks for the production of lubricants, plastics, paints, dyes, and coatings (Carlsson et al., 2011). Cyclopropane fatty acids (CPAs) have been found in certain gymnosperms, Malvales, Litchi spp., and other Sapindales species. They accumulate to as much as 40% in seeds of Litchi chinensis (Vickery, 1980; Gaydou et al., 1993). Sterculia foetida accumulates the desaturated CFA (i.e. cyclopropene fatty acid) to more than 60% of its seed oil (Bohannon and Kleiman, 1978; Pasha and Ahmad, 1992). The first step in its synthesis is the formation of the CPA by the cyclopropane synthase (CPS) enzyme, which transfers a methyl group to C9 of the oleoyl-phospholipid followed by cyclization to form the cyclopropane ring (Grogan and Cronan, 1997; Bao et al., 2002, 2003). None of the known natural sources of CPA are suitable for its commercial production. Therefore, it would be desirable to create an oilseed crop plant that accumulates high levels of CPA by heterologously expressing CPS in seeds. However, to date, heterologous expression of plant cyclopropane synthase genes has led to only approximately 1.0% CPA in the transgenic seeds (Yu et al., 2011).Two pathways for the biosynthesis of TAG exist in plants (Bates and Browse, 2012; Fig. 1). The de novo biosynthesis from glycerol-3-phosphate and acyl-CoA occurs via the Kennedy pathway and includes three acyltransferases: glycerol-2-phosphate acyltransferase, acyl-CoA:lysophosphatidic acid acyltransferase (LPAT), and acyl-CoA:diacylglycerol acyltransferase (DGAT; Kennedy, 1961). Alternatively, acyl-CoAs can be redirected from phosphatidylcholine (PC) via the action of a phospholipase C, choline phosphotransferase, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT; Hu et al., 2012; Lu et al., 2009), or phospholipid:diacylglycerol acyltransferase (PDAT; Dahlqvist et al., 2000). An acyl group can be released from PC to generate lysophosphatidylcholine (LPC) by the back reaction of acyl-CoA:LPC acyltransferase (Stymne and Stobart, 1984; Wang et al., 2012) or a phospholipase A/acyl-CoA synthase (Chen et al., 2011).Open in a separate windowFigure 1.Schematic representation of the plant TAG biosynthesis network. Acyl editing can provide PC-modified fatty acids for de novo diacylglycerol/TAG synthesis. ACS, acyl-CoA synthase; CPT, CDP-choline:diacylglycerol choline phosphotransferase; G3P, glycerol-3-phosphate; GPAT, acyl-CoA:glycerol-3-phosphate acyltransferase; LPC acyltransferase, acyl-CoA:LPC acyltransferase; mFAS, modified fatty acid synthase (in this work, mFAS is CPS); PAP, phosphatidic acid phosphatase; PLA, phospholipase A; PLC, phospholipase C.LPAT is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid (LPA) into different phosphatidic acids (PAs) in diverse tissues. Membrane-associated LPAT activities, identified in bacteria, yeast, plant, and animal cells, catalyze the transfer of acyl groups from acyl-CoA to LPA to synthesize PA. In plants and other organisms, LPAT activities have been identified in the endoplasmic reticulum (Kim et al., 2005), plasma membrane (Bursten et al., 1991), and mitochondria (Zborowski and Wojtczak, 1969). In higher plants, endoplasmic reticulum-localized LPAT plays an essential role transferring fatty acid from CoA esters to the sn-2 position of LPA in the synthesis of PA, a key intermediate in the biosynthesis of membrane phospholipids and storage lipids in developing seeds (Maisonneuve et al., 2010). LPAT from developing seeds of flax (Linum usitatissimum), rape (Brassica napus), and castor bean preferentially incorporate oleoyl-CoA, weakly incorporate cyclopropane acyl-CoA, and were unable to incorporate methyl-branched acyl-CoA when presented with an equimolar mix of these potential substrates (Nlandu Mputu et al., 2009). Thus, LPAT activity from agronomic plants constitutes a potential bottleneck for the incorporation of branched chain acyl-CoA into PA. In this work, we investigate the utility of an LPAT from a cyclopropanoid-syntheizing plant, S. foetida, with respect to its ability to enhance CPA accumulation. In our efforts to enhance CPA accumulation in transgenic plants, we screened CPS genes from diverse sources and identified Escherichia coli CPS (EcCPS) as an effective enzyme for the production of CPA in plants. However, EcCPS is reported to prefer the sn-2 position of E. coli phospholipid (Hildebrand and Law, 1964), and the data presented here show that its expression primarily leads to the accumulation of CPA at the stereospecific numbering (sn)-1 position. Moreover, coexpression of S. foetida lysophosphatidic acid acyltransferase (SfLPAT) results in the incorporation of CPA at the sn-2 position of LPA. Thus, coexpression of EcCPS and SfLPAT enables a cycle that enriches the accumulation of CPA at both sn-1 and sn-2 positions of PC and increases the accumulation of CPA. This work underscores the utility of coexpressing an acyltransferase from mFA-accumulating species with mFA-synthesizing enzymes to help mitigate bottlenecks in mFA TAG synthesis.     

The role of glycosphingolipids in HIV signaling, entry and pathogenesis

Although HIV uses CD4 and coreceptors (CCR5 and CXCR4) for productive infection of T cells, glycosphingolipids (GSL) may play ancillary roles in lymphoid and non-lymphoid cells. Interactions of the HIV Envelope Glycoprotein (Env) with GSL may help HIV in various steps of its pathogenesis. Physical-chemical aspects of the interactions between HIV Env and GSL leading to CD4-dependent entry into lymphocytes, the role of GSL in HIV transcytosis, and CD4-independent entry into non-lymphoid cells are reviewed. An overview of signaling properties of HIV receptors is provided with some speculation on how GSL may play a role in these events by virtue of being in membrane rafts. Finally, we summarize how interactions between HIV and coreceptors leading to signaling and/or fusion can be analyzed by the use of various tyrosine kinase and cytoskeletal inhibitors. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Alpha-crystallin and ATP facilitate the in vitro renaturation of xylanase: enhancement of refolding by metal ions

Alpha-crystallin is a multimeric protein that functions as a molecular chaperone and shares extensive structural homology to small heat shock proteins. For the functional in vitro analysis of alpha-crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus was used as a model system. The mechanism of chaperone action of alpha-crystallin is less investigated. Here we studied the refolding of Gdn HCl-denatured Xyl II in the presence and absence of alpha-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1-naphthalene sulfonate binding studies, suggest that alpha-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of Xyl II. The alpha-crystallin.Xyl II complex exhibited no functional activity. Addition of ATP to the complex initiated the renaturation of Xyl II with 30%-35% recovery of activity. The nonhydrolyzable analog 5'-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active Xyl II to a lesser extent than ATP. Although the presence of Ca(2+) was not required for the in vitro refolding of Xyl II, the renaturation yield was enhanced in its presence. Experimental evidence indicated that the binding of ATP to the alpha-crystallin.Xyl II complex brought about conformational changes in alpha-crystallin facilitating the dissociation of xylanase molecules. This is the first report of the enhancement of alpha-crystallin chaperone functions by metal ions.     

Changes in ecosystem functioning after replacement of forest by Lantana shrubland in Kumaun Himalaya

Abstract. Lantana camara shrubland is compared with the adjacent Quercus leucotrichophora and Pinus roxburghii forests to understand changes occurring in net primary productivity and nutrient cycling, as a consequence of degradation of these forests. The total net primary productivity of Lantana camara shrubland was 17 t ha^-1 yr^-1, which is similar to the values reported for forests: 16 - 21 t ha^-1 yr^-1. Total nutrient content (N, P) in the soil in the L. camara shrubland: 2932 kg ha^-1 N and 111 kg ha^-1 P, was lower than that of the forest soils.     

Non-timber forest products (NTFPs): a viable option for biodiversity conservation and livelihood enhancement in central Himalaya

The present study aims to document detail information of some of the selected wild edible having enormous potential for livelihood enhancement and socio-economic development by making a variety of value added products. To this end, some of the wild edibles of central Himalaya were selected and prioritized for harnessing their economic potential along with their detail information in terms of distribution, ethnobiology, phenophases and appropriate time of harvesting so as to make communities well aware about the resource availability and their harvesting period round the year. The cost-benefit analysis of each value added product prepared from selected wild edibles was worked out in detail and these analyses revealed that total monetary output, as well as the net return, is very high for all value added products prepared. Since wild edible fruits or other edible parts can be collected from wild free of cost except labour is involved in collection of these wild edibles bio-resources. In addition, information on a participatory action research framework & approaches for promoting participatory conservation of these wild edible species were also highlighted for appropriate management of these resources. The present attempt provides a practical example of sustainable utilization of wild edibles, their potential in livelihood improvement of local people, distribution and phenophases and availability in natural conditions, participatory conservation of these wild edibles may help policy planners at the regional and national levels to link livelihood/socio-economic development with conservation.     

A diploid wheat TILLING resource for wheat functional genomics

Background

Cultivated peanut (Arachis hypogaea L.) is an important crop worldwide, valued for its edible oil and digestible protein. It has a very narrow genetic base that may well derive from a relatively recent single polyploidization event. Accordingly molecular markers have low levels of polymorphism and the number of polymorphic molecular markers available for cultivated peanut is still limiting.

Results

Here, we report a large set of BAC-end sequences (BES), use them for developing SSR (BES-SSR) markers, and apply them in genetic linkage mapping. The majority of BESs had no detectable homology to known genes (49.5%) followed by sequences with similarity to known genes (44.3%), and miscellaneous sequences (6.2%) such as transposable element, retroelement, and organelle sequences. A total of 1,424 SSRs were identified from 36,435 BESs. Among these identified SSRs, dinucleotide (47.4%) and trinucleotide (37.1%) SSRs were predominant. The new set of 1,152 SSRs as well as about 4,000 published or unpublished SSRs were screened against two parents of a mapping population, generating 385 polymorphic loci. A genetic linkage map was constructed, consisting of 318 loci onto 21 linkage groups and covering a total of 1,674.4 cM, with an average distance of 5.3 cM between adjacent loci. Two markers related to resistance gene homologs (RGH) were mapped to two different groups, thus anchoring 1 RGH-BAC contig and 1 singleton.

Conclusions

The SSRs mined from BESs will be of use in further molecular analysis of the peanut genome, providing a novel set of markers, genetically anchoring BAC clones, and incorporating gene sequences into a linkage map. This will aid in the identification of markers linked to genes of interest and map-based cloning.