The effects of down-regulating expression of Arabidopsis thaliana membrane-associated acyl-CoA binding protein 2 on acyl-lipid composition

doi:10.1016/j.plantsci.2006.07.009

Plant Science

Volume 172, Issue 1, January 2007, Pages 36-44

https://doi.org/10.1016/j.plantsci.2006.07.009 Get rights and content

Abstract

Multiple classes of acyl-CoA binding proteins are encoded by plant genomes, including a plant-unique class of predicted integral membrane-proteins. Transcript analysis revealed that both of the integral membrane-acyl-CoA binding proteins of Arabidopsis thaliana, ACBP1 and ACBP2, are expressed in all organs. Expression of ACBP2 was highest in developing roots and flowers, and was four-fold greater than expression of ACBP1. Polyclonal antibodies against recombinant ACBP2 specifically recognized a Mr 47k protein that accumulated to similar levels in A. thaliana leaves, flowers, and siliques (0.05–0.07% total protein), but was two-fold more abundant in roots. To study the potential role(s) of ACBP2 in acyl-CoA metabolism, expression was down-regulated using hairpin RNA interference (RNAi). Three RNAi lines with at least 70% reduced levels of the ACBP2 protein were analyzed, but displayed no gross alterations in growth or developmental phenotype. A systematic analysis of lipids from developing leaves by electrospray tandem-mass spectrometry revealed little change in the levels of the eight major lipid classes but significant changes in fatty acid composition were observed, particularly for the phospholipids. These data suggest a specialized rather than general role for ACBP2 in plant acyl-lipid metabolism.

Introduction

De novo fatty acid synthesis in plants occurs exclusively within plastids, after which the preponderance of acyl chains are modified and esterified at the endoplasmic reticulum (ER) [1]. Despite this well-known compartmental separation between fatty acid and complex lipid synthesis, the intracellular transport of fatty acids remains a poorly understood aspect of plant lipid metabolism. The fatty acids released from acyl carrier protein at the completion of de novo synthesis are esterified to CoA by outer envelope-localized acyl-CoA synthetases [2], [3].

Traffic of acyl-CoAs from the plastids to the ER, or within the cytoplasm, is thought to be facilitated by the soluble low molecular weight acyl-CoA binding proteins (ACBP) that are ubiquitous in eukaryotic cells [4]. Examples of ACBP have been identified in plants, fungi, protozoa and higher animals, and are characterized by an acyl-CoA binding motif [4], [5], [6], [7]. Typical plant ACBP are 8–10 kDa and share approximately 60% amino acid identity with the mammalian and yeast orthologs [5], [8]. In developing Brassica napus seeds these proteins accumulate to 65 μM, 10-fold higher than in any other organ examined [8]. The high levels of ACBP in developing oil-rich seeds imply a role in acyl-CoA transit between plastids and ER for triacylglycerol biosynthesis.

A catalog of the lipid biosynthesis genes from Arabidopsis thaliana has been prepared [9], [10]. These analyses revealed a single nuclear gene encoding the ACBP, and three additional classes of putative acyl-CoA binding proteins, each of which was identified by amino acid-similarity to ACBP within the acyl-CoA binding cleft. The first of these classes includes ACBP1 and 2, integral membrane proteins that are 72% identical at the deduced amino acid sequence level [11], [12], [13] and include C-terminal ankyrin repeat motifs [14]. A chimera between ACBP2 and the green fluorescent protein was localized to the plasma membrane [14] where it interacts with an ethylene-responsive element-binding protein through the ankyrin repeat motifs [15]. The other two protein classes include ACBP3, an extracellular protein [16], and ACBP4 and 5, two large cytoplasmic proteins that are 81% identical at the deduced amino acid level [17] and are characterized by C-terminal Kelch domains [18]. Despite the substantial information describing the in vitro characteristics of the recombinant proteins, the in vivo role(s) of the plant acyl-CoA binding proteins remain obscure.

To gain additional insight into the function of the A. thaliana acyl-CoA binding proteins, we have employed RNA interference (RNAi) to down-regulate expression of ACBP2. Although this strategy effectively reduced expression in developing leaves, no gross phenotypic changes were observed. In contrast, however, there were significant changes in acyl-lipid composition, particularly within the phospholipids. To our knowledge, this is the first report describing down-regulation of expression of this class of acyl-CoA binding proteins in any plant.

Section snippets

Plant growth conditions

A. thaliana (ecotype Columbia) plants were grown at 18 °C under low light flux (49 μmol photon m⁻² s⁻¹) with a 16 h photoperiod. Seeds were sown in a 1:1 mixture of vermiculite and peat moss-enriched soil.

Heterologous expression of ACBP2 for antibody preparation

A cDNA encoding A. thaliana ACBP2 was identified from a developing seed library [19] and was sequenced on both strands prior to submission to GenBank (Accession number AF320561). The open reading frame for was amplified using primers which contained NdeI and XhoI restrictions sites for insertion

Analysis of recombinant A. thaliana ACBP2 by mass spectrometry

Under a wide range of experimental conditions, A. thaliana ACBP2 was insoluble when expressed in E. coli. Attempts to solubilize the recombinant protein were unsuccessful; at urea concentrations of less than 1.0 M ACBP2 precipitated. The protein was subsequently purified under denaturing conditions by binding to the Ni-NTA matrix and elution with imidazole. The final preparations were >95% pure as judged by Coomassie blue-staining of SDS-gels (Fig. 1).

Induction of full-length ACBP2 (which

Discussion

In plants, de novo fatty acid biosynthesis occurs exclusively in the plastids, though acyl-chains are utilized in every cellular compartment. Additionally, some organs, tissues, and membranes have unique acyl-chain requirements. For instance, epidermal cells require acyl-chains for cuticular wax production [28], and developing seeds require large quantities of fatty acids for storage lipid accumulation (reviewed in ref. [29]). Opposing synthesis are acyl-chain turnover and catabolism, which

Acknowledgements

The authors acknowledge the University of Missouri Proteomics Center for mass spectrometry services, and the Kansas State Lipidomics Center for lipid analyses. The authors acknowledge Dr. Hermann Schmidt of DNA Cloning Service (Hamburg, Germany) for synthesizing the ACBP2 RNAi construct. We also thank Professor John Ohlrogge (Michigan State University) for helpful suggestions and for providing lab resources for a sabbatical leave for Professor Michiyuki Kojima.

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Besides, site-directed mutagenesis demonstrated that some conserved residues of AtACBPs are crucial for binding specific ligands but mutation of the equivalent sites in other isoforms did not produce an adverse effect [35,65,79], suggesting that the structural determinants that define functional specificity are not conserved in all ACBPs, and that the structural and biochemical data attained from an isoform should not be over-interpreted in different classes of plant ACBPs. To provide the first insight into the ligand specificities of various plant ACBPs in vitro, their cDNAs were expressed in Escherichia coli for the production of recombinant (r) proteins, including rAtACBP1 [68,71–73,75,79], rAtACBP2 [65,68–70,73,98], rAtACBP3 [79,80,82], rAtACBP4 [35,61,84,87], rAtACBP5 [35,61,79,84,87], rAtACBP6 [50,51,61], rOsACBP1 to rOsACBP6 [37,38], Class I rBnACBP [7,59] and rVfACBP3s [47]. Subsequent binding tests against a variety of potential interactants by in vitro techniques such as Lipidex-1000, gel- and filter-binding assays indicated substantial difference in ligand preferences of these rACBPs (Table 1).
Acyl-CoA esters are the activated form of fatty acids and play important roles in lipid metabolism and the regulation of cell functions. They are bound and transported by nonenzymic proteins such as the acyl-CoA-binding proteins (ACBPs). Although plant ACBPs were so named by virtue of amino acid homology to existing yeast and mammalian counterparts, recent studies revealed that ligand specificities of plant ACBPs are not restricted to acyl-CoA esters. Arabidopsis and rice ACBPs also interact with phospholipids, and their affinities to different acyl-CoA species and phospholipid classes vary amongst isoforms. Their ligands also include heavy metals. Interactors of plant ACBPs are further diversified due to the evolution of protein–protein interacting domains. This review summarizes our current understanding of plant ACBPs with a focus on their binding versatility. Their broad ligand range is of paramount significance in serving a multitude of functions during development and stress responses as discussed herein. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.
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Correspondingly, rAtACBP1 and rAtACBP2 were observed to bind various phospholipids in vitro [117]. Together with an earlier analysis of the lipid contents of AtACBP2 knock-down plants [153], AtACBP1 and AtACBP2 were thus verified to participate in acyl-CoA-related phospholipid and galactolipid synthesis during embryogenesis in Arabidopsis [117,154]. In addition, given that AtACBP1 and AtACBP2 can potentially bind PC and acyl-CoA esters at the ER, and that ACBP1 expression in developing seeds coincided with stages in lipid deposition [42], their possible role in the formation of lipid droplets in developing seeds warrants further investigations.
Acyl-CoA-binding protein (ACBP) was first identified in mammals as a neuropeptide, and was demonstrated to belong to an important house-keeping protein family that extends across eukaryotes and some prokaryotes. In plants, the Arabidopsis ACBP family consists of six AtACBPs (AtACBP1 to AtACBP6), and has been investigated using gene knock-out mutants and overexpression lines. Herein, recent findings on the AtACBPs are examined to provide an insight on their functions in various plant developmental processes, such as embryo and seed development, seed dormancy and germination, seedling development and cuticle formation, as well as their roles under various environmental stresses. The significance of the AtACBPs in acyl-CoA/lipid metabolism, with focus on their interaction with long to very-long-chain (VLC) acyl-CoA esters and their potential role in the formation of lipid droplets in seeds and vegetative tissues are discussed. In addition, recent findings on the rice ACBP family are presented. The similarities and differences between ACBPs from Arabidopsis and rice, that represent eudicot and monocot model plants, respectively, are analyzed and the evolution of plant ACBPs by phylogenetic analysis reviewed. Finally, we propose potential uses of plant ACBPs in phytoremediation and in agriculture related to the improvement of environmental stress tolerance and seed oil production.
New roles for acyl-CoA-binding proteins (ACBPs) in plant development, stress responses and lipid metabolism
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The expression of ACBPs in wild-type Arabidopsis has provided some knowledge on their biological functions [22–25,33,49,87]. Further, the availability of downregulated Arabidopsis lines including T-DNA insertional mutants and RNAi transgenic plants are now available for all of these Arabidopsis ACBPs and have proven invaluable in elucidating their functions [34,42–44,46,48,87]. Also, transgenic lines overexpressing these AtACBPs have been generated and characterised [32,34,40,42,43,48].
ACBPs are implicated in acyl-CoA trafficking in many eukaryotes and some prokaryotes. Six genes encode proteins designated as AtACBP1–AtACBP6 in the Arabidopsis thaliana ACBP family. These ACBPs are conserved in the acyl-CoA-binding domain, but vary in size from 92 amino acids (10.4 kDa) to 668 amino acids (73.1 kDa), and are subcellularly localised to different compartments in plant cells. Results from in vitro binding assays show that their corresponding recombinant proteins exhibit differential binding affinities to acyl-CoA esters and phospholipids, implying that these ACBPs may have non-redundant biological functions in vivo. By using knockout/downregulated and overexpression lines of Arabidopsis ACBPs, recent investigations have revealed that in addition to their proposed roles in phospholipid metabolism, these ACBPs can influence plant development including early embryogenesis and leaf senescence, as well as plant stress responses including heavy metal resistance, oxidative stress, freezing tolerance and pathogen resistance. In this review, recent progress on the biochemical and functional analyses of Arabidopsis ACBPs, their links to metabolic/signalling pathways, and their potential applications in development of stress tolerance are discussed.
Involvement of low molecular mass soluble acyl-CoA-binding protein in seed oil biosynthesis
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Acyl-CoA-binding protein (ACBP), a low molecular mass (m) (∼10 kDa) soluble protein ubiquitous in eukaryotes, plays an important housekeeping role in lipid metabolism by maintaining the intracellular acyl-CoA pool. ACBP is involved in lipid biosynthesis and transport, gene expression, and membrane biogenesis. In plants, low m ACBP and high m ACBPs participate in response mechanisms to biotic and abiotic factors, acyl-CoA transport in phloem, and biosynthesis of structural and storage lipids. In light of current research on the modification of seed oil, insight into mechanisms of substrate trafficking within lipid biosynthetic pathways is crucial for developing rational strategies for the production of specialty oils with the desired alterations in fatty acid composition. In this review, we summarize our knowledge of plant ACBPs with emphasis on the role of low m ACBP in seed oil biosynthesis, based on in vitro studies and analyses of transgenic plants. Future prospects and possible applications of low m ACBP in seed oil modification are discussed.
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^☆: The cDNA sequence for ACBP2 described herein has been assigned GenBank Accession AF320561.

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The effects of down-regulating expression of Arabidopsis thaliana membrane-associated acyl-CoA binding protein 2 on acyl-lipid composition☆

Abstract

Introduction

Section snippets

Plant growth conditions

Heterologous expression of ACBP2 for antibody preparation

Analysis of recombinant A. thaliana ACBP2 by mass spectrometry

Discussion

Acknowledgements

Mol. Cell. Proteomics

J. Biol. Chem.

Arch. Biochem. Biophys.

Trends Cell Biol.

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

Prog. Lipid Res.

Metab. Eng.

J. Biol. Chem.

Lipid biosynthesis

Plant Cell

Fatty acid export from the chloroplast. Molecular characterization of a major plastidial acyl-coenzyme A synthetase from Arabidopsis thaliana

Plant Physiol.

Evolution of the acyl-CoA binding protein (ACBP)

Biochem. J.

Molecular cloning of a cDNA from Brassica napus L. for a homologue of acyl-CoA-binding protein

Plant Mol. Biol.

Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae

Mol. Biol. Cell

Toward a functional catalog of the plant genome: a survey of genes for lipid biosynthesis

Plant Physiol.

Arabidopsis thaliana genes involved in acyl lipid metabolism: a 2003 census of the candidates, a study of the distribution of ESTs in tissues and a web-based database

Plant Physiol.

Arabidopsis thaliana cDNA encoding a membrane-associated protein with an acyl-CoA binding domain

Plant Mol. Biol.

Single amino acid substitutions at the acyl-CoA-binding domain interrupt 14[C] palmitoyl-CoA binding of ACBP2, an Arabidopsis thaliana acyl-CoA-binding protein with ankyrin repeats

Plant Mol. Biol.

Membrane localization of Arabidopsis thaliana acyl-CoA binding protein ACBP2

Plant Mol. Biol.

Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally

Proteins

Arabidopsis Acyl-CoA-binding protein ACBP2 interacts with an ethylene-responsive element-binding protein, AtEBP, via its ankyrin repeats

Plant Mol. Biol.

Arabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding protein

Planta

ACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA

Plant Mol. Biol.