Characterization of two Arabidopsis thaliana acyltransferases with preference for lysophosphatidylethanolamine.

BACKGROUND: Two 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. RESULTS: Expression 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. CONCLUSION: This 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.

Identification of a novel GPCAT activity and a new pathway for phosphatidylcholine biosynthesis in S. cerevisiae.

Turnover of phospholipids in the yeast Saccharomyces cerevisiae generates intracellular glycerophosphocholine (GPC). Here we show that GPC can be reacylated in an acyl-CoA-dependent reaction by yeast microsomal membranes. The lysophosphatidylcholine that is formed in this reaction is efficiently further acylated to phosphatidylcholine (PC) by yeast microsomes, thus providing a new pathway for PC biosynthesis that can either recycle endogenously generated GPC or utilize externally provided GPC. Genetic and biochemical evidence suggests that this new enzymatic activity, which we call GPC acyltransferase (GPCAT), is not mediated by any of the previously known acyltransferases in yeast. The GPCAT activity has an apparent V(max) of 8.7 nmol/min/mg protein and an apparent K(m) of 2.5 mM. It has a neutral pH optimum, similar to yeast glycerol-3-phosphate acyltransferase, but differs from the latter in being more heat stable. The GPCAT activity is sensitive to N-ethylmaleimide, phenanthroline, and Zn(2+) ions. In vivo experiments showed that PC is efficiently labeled when yeast cells are fed with [(3)H]choline-GPC, and that this reaction occurs also in pct1 knockout strains, where de novo synthesis of PC by the CDP-choline pathway is blocked. This suggests that GPCAT can provide an alternative pathway for PC biosynthesis in vivo.

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.

Synthesis of ketocarotenoids in the seed of Arabidopsis thaliana.

A cDNA coding for a gene necessary for synthesis of ketocarotenoids was cloned from the alga Haematococcus pluvialis and expressed in the seed of Arabidopsis thaliana. The expression of the algal beta-carotene-oxygenase gene was directed to the seed by use of the 2S, seed storage protein promoter napA. Extracts from seeds of the transgenic plants were clearly red because of accumulation of ketocarotenoids, and free and esterified forms of ketocarotenoids were found in addition to the normal carotenoid composition in the seed. The major ketocarotenoids in the transgenic plants were: 4-keto-lutein (3,3'-dihydroxy-beta-,epsilon-carotene-4-one), adonirubin (3-hydroxy-beta-,beta'-carotene-4,4'-dione) and canthaxanthin (beta-,beta'-carotene-4,4'-dione). 4-Keto-lutein differs from the more common adonixanthin only in the position of one double bond. To increase the substrate availability for the beta-carotene-oxygenase, these transformants were crossed with transgenic plants overexpressing a construct of an endogenous phytoene synthase gene, also under the control of the napA promoter. The resulting crossings gave rise to seeds with a 4.6-fold relative increase of the total pigment, and the three major ketocarotenoids were increased 13-fold compared to seeds of transgenic plants carrying only the beta-carotene-oxygenase construct.

Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids, chlorophyll, and abscisic acid.

Phytoene synthase catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate to phytoene and has been shown to be rate limiting for the synthesis of carotenoids. To elucidate if the capacity to produce phytoene is limiting also in the seed of Arabidopsis (Wassilewskija), a gene coding for an endogenous phytoene synthase was cloned and coupled to a seed-specific promoter, and the effects of the overexpression were examined. The resulting transgenic plants produced darker seeds, and extracts from the seed of five overexpressing plants had a 43-fold average increase of beta-carotene and a total average amount of beta-carotene of approximately 260 microg g-1 fresh weight. Lutein, violaxanthin, and chlorophyll were significantly increased, whereas the levels of zeaxanthin only increased by a factor 1.1. In addition, substantial levels of lycopene and alpha-carotene were produced in the seeds, whereas only trace amounts were found in the control plants. Seeds from the transgenic plants exhibited delayed germination, and the degree of delay was positively correlated with the increased levels of carotenoids. The abscisic acid levels followed the increase of the carotenoids, and plants having the highest carotenoid levels also had the highest abscisic acid content. Addition of gibberellic acid to the growth medium only partly restored germination of the transgenic seeds.