Ulf Ståhl

Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis

A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487–6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.

Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids

Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization.

A family of eukaryotic lysophospholipid acyltransferases with broad specificity.

The budding yeast ALE1 gene encodes a lysophospholipid acyltransferase (LPLAT) with broad specificity. We show that yeast LPLAT (ScLPLAT) belongs to a distinct protein family that includes human MBOAT1, MBOAT2, MBOAT4, and several closely related proteins from other eukaryotes. We further show that two plant proteins within this family, the Arabidopsis proteins AtLPLAT1 and AtLPLAT2, possess lysophospholipid acyltransferase activities similar to ScLPLAT. We propose that other members of this protein family, which we refer to as the LPLAT family, also are likely to possess LPLAT activity. Finally, we show that ScLPLAT differs from the specific lysophosphatidic acid acyltransferase that is encoded by SLC1 in that it cannot efficiently use lysophosphatidic acid produced by acylation of glycerol‐3‐phosphate in vitro.

A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine

The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana. Biochemical analysis of T(2) and T(3) A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2(cisDelta9,12) and 18:3(cisDelta9,12,15)) at the expense of very long monounsaturated FA (20:1(cisDelta11)) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from DeltaYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase (AthLPCAT) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro. These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.

Characterization of two Arabidopsis thaliana acyltransferases with preference for lysophosphatidylethanolamine

BackgroundTwo previously uncharacterized Arabidopsis genes that encode proteins with acyltransferase PlsC regions were selected for study based on their sequence similarity to a recently identified lung lysophosphatidylcholine acyltransferase (LPCAT). To identify their substrate specificity and biochemical properties, the two Arabidopsis acyltransferases, designated AtLPEAT1, (At1g80950), and AtLPEAT2 (At2g45670) were expressed in yeast knockout lines ale1 and slc1 that are deficient in microsomal lysophosphatidyl acyltransferase activities.ResultsExpression of AtLPEAT1 in the yeast knockout ale1 background exhibited strong acylation activity of lysophosphatidylethanolamine (LPE) and lysophosphatidate (LPA) with lower activity on lysophosphatidylcholine (LPC) and lysophosphatidylserine (LPS). AtLPEAT2 had specificities in the order of LPE > LPC > LPS and had no or very low activity with LPA. Both acyltransferases preferred 18:1-LPE over 16:0-LPE as acceptor and preferred palmitoyl-CoA as acyl donor in combination with 18:1-LPE. Both acyltransferases showed no or minor responses to Ca2+, despite the presence of a calcium binding EF-hand region in AtLPEAT2. AtLPEAT1 was more active at basic pH while AtLPEAT2 was equally active between pH 6.0 – 9.0.ConclusionThis study represents the first description of plant acyltransferases with a preference for LPE. In conclusion it is suggested that the two AtLPEATs, with their different biochemical and expression properties, have different roles in membrane metabolism/homoeostasis.

Biosynthesis of an Acetylenic Fatty Acid in Microsomal Preparations from Developing Seeds of Crepis Alpina

Over 600 naturally occuring compounds with acetylenic bonds (triple bonds) have been characterized (Bohlmann et al., 1973). Previous in-vivo studies, from mosses accumulating acetylenic fatty acids, indicate that the acetylenic bond is formed by the substraction of two hydrogen atoms from a double bond (Kohn et al., 1994).

Purification and Characterization of a Microsomal Phospholipase A2 from Developing Elm Seeds

The phospholipases A2 (PLA2s) hydrolyse specifically the sn-2-fatty acyl ester bond of phosphoglycerides (Waite, 1987). PLA2s in animal systems are involved in many important processes, such as signal transduction, eicosanoid synthesis and inflammation. The available information about PLA2 from plant tissues is, however, very limited.