Michael S. Greer

Biology and Biochemistry of Plant Phospholipases

Phospholipases are a complex group of enzymes that hydrolyze phospholipids. The plant phospholipase family is composed of multiple members with varying positional specificity, and each type is represented by multiple isoforms distinguishable by their structural, catalytic, and physiological characteristics. A large number of phospholipase genes and gene families have been identified and the biochemical properties of several members have been characterized, revealing considerable molecular and catalytic diversity. Forward and reverse genetics has further revealed that phospholipases are widely involved in physiological processes including lipid metabolism, cell signaling, and responses to biotic and abiotic stresses. Such studies have highlighted the potential biotechnological value of phospholipases as targets for improving stress tolerance. The catalytic diversity of various phospholipase isoforms is also of increasing interest for industrial biocatalysis. This review focuses on recently acquired information on biochemical, molecular and functional aspects of plant phospholipases.

Ammonium nitrate improves direct somatic embryogenesis and biolistic transformation of Triticum aestivum.

Triticum aestivum is of major importance both nutritionally and economically. Introduction of new genes has been difficult to apply to elite wheat varieties mainly as a result of their recalcitrance to prerequisite tissue culture. We attempted to improve the frequency of wheat transformation by exposing plants to high level of ammonium nitrate. Our experiments showed that modification of the ammonium nitrate content in the direct somatic embryogenesis induction medium can increase the number of primary embryos produced over twofold in the elite hard red wheat cultivar Superb. The number of primary embryos that were capable of transitioning into shoot development also increased twofold. Biolistic transformation efficiency improved as much as sevenfold when targeted scutellar tissue was exposed to elevated ammonium nitrate levels. This simple approach could become extremely useful for increasing transformation efficiency in wheat.

In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase

Background: Plant polyunsaturated fatty acids (PUFAs) are mainly synthesized on phosphatidylcholine (PC). Results: Diacylglycerol acyltransferase (DGAT) produced higher amount of PUFA-containing TAG in the presence of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT). Conclusion: The LPCAT-catalyzed reverse reaction can be coupled to the DGAT reaction for PUFA accumulation. Significance: A mechanism for enhancing the transfer of PUFAs from PC into TAG has been confirmed. Seed oils of flax (Linum usitatissimum L.) and many other plant species contain substantial amounts of polyunsaturated fatty acids (PUFAs). Phosphatidylcholine (PC) is the major site for PUFA synthesis. The exact mechanisms of how these PUFAs are channeled from PC into triacylglycerol (TAG) needs to be further explored. By using in vivo and in vitro approaches, we demonstrated that the PC deacylation reaction catalyzed by the reverse action of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) can transfer PUFAs on PC directly into the acyl-CoA pool, making these PUFAs available for the diacylglycerol acyltransferase (DGAT)-catalyzed reaction for TAG production. Two types of yeast mutants were generated for in vivo and in vitro experiments, respectively. Both mutants provide a null background with no endogenous TAG forming capacity and an extremely low LPCAT activity. In vivo experiments showed that co-expressing flax DGAT1-1 and LPCAT1 in the yeast quintuple mutant significantly increased 18-carbon PUFAs in TAG with a concomitant decrease of 18-carbon PUFAs in phospholipid. We further showed that after incubation of sn-2-[14C]acyl-PC, formation of [14C]TAG was only possible with yeast microsomes containing both LPCAT1 and DGAT1-1. Moreover, the specific activity of overall LPCAT1 and DGAT1-1 coupling process exhibited a preference for transferring 14C-labeled linoleoyl or linolenoyl than oleoyl moieties from the sn-2 position of PC to TAG. Together, our data support the hypothesis of biochemical coupling of the LPCAT1-catalyzed reverse reaction with the DGAT1-1-catalyzed reaction for incorporating PUFAs into TAG. This process represents a potential route for enriching TAG in PUFA content during seed development in flax.

Identification and characterization of an LCAT-like Arabidopsis thaliana gene encoding a novel phospholipase A

A previously uncharacterized Arabidopsis lecithin:cholesterol acyltransferase (LCAT) family gene (At4g19860) was functionally expressed in yeast, where it was demonstrated to encode a novel cytosolic and calcium‐independent phospholipase A with preferences for the sn‐2 position. This enzyme shows optimal activity at pH 5.0, exhibits a headgroup specificity for phosphatidylcholine > phosphatidic acid > phosphatidylethanolamine > phosphatidylglycerol > phosphatidylserine and has an acyl chain specificity for oleoyl > linoleoyl > ricinoleoyl. The expression of AtLCAT‐PLA inhibited yeast cell growth and fatty acid accumulation. AtLCAT‐PLA transcript in Arabidopsis was detected at high levels in roots and siliques.

Purification and properties of recombinant Brassica napus diacylglycerol acyltransferase 1

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in the acyl‐CoA‐dependent triacylglycerol biosynthesis. Although the first DGAT1 gene was identified many years ago and the encoded enzyme catalyzes a key step in lipid biosynthesis, no detailed structure–function information is available on the enzyme due to difficulties associated with its purification. This study describes the purification of recombinant Brassica napus DGAT1 (BnaC.DGAT1.a) in active form through solubilization in n‐dodecyl‐β‐d‐maltopyranoside, cobalt affinity chromatography, and size‐exclusion chromatography. Different BnaC.DGAT1.a oligomers in detergent micelles were resolved during the size‐exclusion process. BnaC.DGAT1.a was purified 126‐fold over the solubilized fraction and exhibited a specific activity of 26 nmol TAG/min/mg protein. The purified enzyme exhibited substrate preference for α‐linolenoyl‐CoA > oleoyl‐CoA = palmitoyl‐CoA > linoleoyl‐CoA > stearoyl‐CoA.

Plant phospholipase A: advances in molecular biology, biochemistry, and cellular function.

Abstract Plant phospholipase As (PLAs) are a complex group of enzymes that catalyze the release of free fatty acids from phospholipids. Plant PLAs can be grouped into three families, PLA1, PLA2, and patatin-like PLA, that catalyze the hydrolysis of acyl groups from the sn-1 and/or sn-2 position. Each family is composed of multiple isoforms of phospholipases that differ in structural, catalytic, and physiological characteristics. In this review, recently acquired information on molecular, biochemical, and functional aspects of plant PLAs will be discussed.

A novel assay of DGAT activity based on high temperature GC/MS of triacylglycerol.

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG), a high-energy compound composed of three fatty acids esterified to a glycerol backbone. In vitro DGAT assays, which are usually conducted with radiolabeled substrate using microsomal fractions, have been useful in identifying compounds and genetic modifications that affect DGAT activity. Here, we describe a high-temperature gas chromatography (GC)/mass spectrometry (MS)-based method for monitoring molecular species of TAG produced by the catalytic action of microsomal DGAT. This method circumvents the need for radiolabeled or modified substrates, and only requires a simple lipid extraction prior to GC. The utility of the method is demonstrated using a recombinant type-1 Brassica napus DGAT produced in a strain of Saccharomyces cerevisae that is deficient in TAG synthesis. The GC/MS-based assay of DGAT activity was strongly correlated with the typical in vitro assay of the enzyme using [1-14C] acyl-CoA as an acyl donor. In addition to determining DGAT activity, the method is also useful for determining substrate specificity and selectivity properties of the enzyme.

Genetic Engineering of Lipid Biosynthesis in Seeds

The demand for vegetable oils, including those derived from crucifer (Brassicaceae) species, has been increasing rapidly over recent years for use in both food and industrial applications. In order to meet these demands, biotechnological approaches will almost certainly be a necessity to generate crops with improved lipid traits. In addition to the clear need to increase the seed oil content of crucifer species, there has also been growing interest in generating transgenic lines that display improved compositions of fatty acids and non-acyl lipids, including carotenoids and tocochromanols, for enhanced nutritional or industrial applicability. Fortunately, knowledge concerning oilseed metabolism and lipid biosynthesis are accumulating at a rapid pace, which is enabling attempts to genetically engineer crucifer species with enhanced oil content and quality. This chapter outlines the various attempts and successes in this field to date.

Two Clades of Type-1 Brassica napus Diacylglycerol Acyltransferase Exhibit Differences in Acyl-CoA Preference

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to produce triacylglycerol, which is the main component of the seed oil of Brassica oilseed species. Phylogenetic analysis of the amino acid sequences encoded by four transcriptionally active DGAT1 genes from Brassica napus suggests that the gene forms diverged over time into two clades (I and II), with representative members in each genome (A and C). The majority of the amino acid sequence differences in these forms of DGAT1, however, reside outside of motifs suggested to be involved in catalysis. Despite this, the clade II enzymes displayed a significantly enhanced preference for linoleoyl-CoA when assessed using in-vitro enzyme assays with yeast microsomes containing recombinant enzyme forms. These findings contribute to our understanding of triacylglycerol biosynthesis in B. napus, and may advance our ability to engineer DGAT1s with desired substrate selectivity properties.