Plant acyl-CoA-binding proteins: An emerging family involved in plant development and stress responses

    Plant acyl-CoA-binding proteins: An emerging family involved in plant development and stress responses Zhi-Yan Du, Tatiana Arias, Wei Meng, Mee-Len Chye PII: DOI: Reference:

S0163-7827(16)30002-9 doi: 10.1016/j.plipres.2016.06.002 JPLR 918

To appear in: Received date: Revised date: Accepted date:

22 January 2016 25 June 2016 26 June 2016

Please cite this article as: Du Zhi-Yan, Arias Tatiana, Meng Wei, Chye Mee-Len, Plant acyl-CoA-binding proteins: An emerging family involved in plant development and stress responses, (2016), doi: 10.1016/j.plipres.2016.06.002

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ACCEPTED MANUSCRIPT Plant acyl-CoA-binding proteins: an emerging family involved in plant

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development and stress responses

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Zhi-Yan Dua,b,c, Tatiana Ariasa,d, Wei Menge, and Mee-Len Chyea*

School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong

e

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College of Life Science, Northeast Forestry University, Harbin, China

Current address:

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing,

Michigan 48824, USA c

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b

Department of Biochemistry and Molecular Biology, Michigan State University, East

Corporacion para Investigaciones Biologicas, Cra. 72 A No 78 B 141, Medellin,

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d

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Lansing, Michigan 48824, USA

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Colombia

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* Corresponding author.

Tel: +852-22990319, Fax: +852-28583477 E-mail address: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT Acyl-CoA-binding protein (ACBP) was first identified in mammals as a neuropeptide,

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and was demonstrated to belong to an important house-keeping protein family that

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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

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AtACBPs are examined to provide an insight on their functions in various plant

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developmental processes, such as embryo and seed development, seed dormancy and germination, seedling development and cuticle formation, as well as their roles under

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various environmental stresses. The significance of the AtACBPs in acyl-CoA/lipid

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metabolism, with focus on their interaction with long to very-long-chain (VLC)

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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

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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.

KEY WORDS acyl-CoA metabolism, lipid transport, long-chain acyl-CoA esters, phylogenetics, stress tolerance, subcellular localization, temporal and spatial expression

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ACCEPTED MANUSCRIPT

Introduction

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Plant acyl-CoA-binding proteins

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Contents

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2.1 Phylogenetic analyses of plant acyl-CoA-binding proteins 2.2 Identification of plant acyl-CoA-binding proteins

3.2

Rice acyl-CoA-binding proteins

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Arabidopsis acyl-CoA-binding proteins

Plant acyl-CoA-binding proteins in development Embryo development

4.2

Seed dormancy, germination and seedling development

4.3

Light regulation in seedlings

4.4

Cuticle formation

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4.1

Plant acyl-CoA-binding proteins participate in stress responses

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3.1

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Temporal and spatial expression of plant acyl-CoA-binding proteins

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5.1

Heavy metal and oxidative stresses

5.2

Drought, hypoxia and cold

5.3

Pathogen defense

5.4

Stress-inducible rice acyl-CoA-binding proteins

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Plant acyl-CoA-binding proteins in lipid metabolism

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Conclusions and perspectives

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Acknowledgements

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References

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ACCEPTED MANUSCRIPT 1. Introduction Lipids form major and critical components in eukaryotic and prokaryotic cells.

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They contribute to the structural basis of cellular membranes, maintaining their

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integrity and composition, and they provide energy for numerous cellular events. In many plants, up to 60% of the total dry weight of seeds consists of lipids while the vegetative tissues contain 5 to 10% lipids, mostly in the membranes [1]. Besides

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delineating the cell and its compartments, these lipid membranes also serve as the key

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sites for essential biological actions, including photosynthesis, signal perception and transduction [1, 2]. Lipids also determine cellular polarity [3], and participate in

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chlorophyll and carotenoid biosynthesis [4]. To enable lipids and their derivatives to

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perform these tasks, they need to be translocated subcellularly [2].

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Various proteins involved in lipid transfer have been identified in Arabidopsis. For example, the lipid-transfer proteins (LTPs) are known to bind and transfer lipids

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between the cell wall and plasma membrane [5, 6]. The ATP-binding cassette (ABC) transporter proteins form a large protein family and have been reported to play important roles in fatty acid and lipid transport [7, 8]. A group of ABC transporters, the trigalactosyldiacylglycerol (TGD) proteins TGD1 to TGD5, mediate the transport of lipids such as phosphatidic acid (PA) from the endoplasmic reticulum (ER) to plastids through the outer and inner chloroplast envelope membranes [2, 6, 9-11]. Some ABC transporters of the A, D and G subfamilies participate in the intercellular transport of lipophilic molecules such as acyl-coenzyme A (acyl-CoA), fatty acids and wax/cutin components [6-8]. Arabidopsis PTEN2a (phosphatase and tensin

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ACCEPTED MANUSCRIPT homologue deleted on chromosome ten) binds PA and participates in lipid signaling in plants [12]. Arabidopsis FAX1 (fatty acid export 1) is localized at the inner

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membrane of chloroplast envelope and mediates free fatty acid export from the

demonstrated

to

bind

various

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chloroplast [6, 10, 13]. Recombinant acyl-CoA-binding proteins (ACBPs) have been acyl-CoAs

and

phospholipids

such

as

phosphatidylcholine (PC), and the ACBPs likely participate in acyl-CoA/phospholipid

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transport in the cytosol and between the plasma membrane and the ER [14-16].

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Plant acyl-CoAs are crucial molecules in lipid metabolism. A fraction of long-chain fatty acids (LCFA, e.g. C16 and C18) generated by de novo fatty acid

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synthesis (FAS) in plants (specifically in the chloroplast stroma) is utilized for lipid

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assembly in the plastids (prokaryotic pathway), whereas the majority of LCFA from

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FAS is first converted into acyl-CoAs by long-chain acyl-CoA synthetase (LACS), and then exported to the ER for further acyl editing and biosynthesis of lipids e.g.

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phospholipids and triacylglycerol (TAG) which is referred to as the eukaryotic pathway [1, 2, 17, 18]. Most likely, cytosolic and ER-associated ACBPs or ER-ABC transporters (e.g. AtABCA9) contribute to the transport of acyl-CoAs/fatty acids from the chloroplasts to the ER [14-16, 19]. In Arabidopsis, AtACBPs particularly ER/plasma membrane-localized AtACBP1, cytosolic AtACBP4 and AtACBP6 and apoplastic AtACBP3 are involved in the transport of VLC acyl-CoAs (C ≥ 20) from the ER to and across the plasma membrane for the biosynthesis of surface lipids such as wax and cutin [6, 20-22]. Plant cytosolic ACBPs likely function in acyl-CoA transport into the peroxisomes for fatty acid -oxidation/lipid degradation, which

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ACCEPTED MANUSCRIPT breaks down fatty acids into acetyl-CoA and provides essential carbon for seed germination and seedling development [6, 16, 18]. In Oryza sativa L. (rice),

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OsACBP6 has been reported in the peroxisomes and demonstrated to participate in

functions will be discussed in the later sections.

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peroxisomal -oxidation [23]. More details on plant acyl-CoA metabolism and ACBP

The first ACBP was identified from rat brain as a neuropeptide that inhibited

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the binding of diazepam to the type A receptor for -aminobutyric acid (GABA); it

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was aptly named the diazepam-binding inhibitor (DBI) or endozepine [24]. Subsequently, 10-kDa cytosolic ACBPs were reported in both eukaryotes and

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prokaryotes [14, 25]. Different methods using fluorescence emission spectra [26],

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isoelectric focusing [27], Lipidex-1000 binding assays [28-30], isothermal titration

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calorimetry (ITC) [21, 31-33] and microscale thermophoresis (MST) analysis [22] have shown that recombinant (r) ACBPs can bind various medium-, long- and VLC

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acyl-CoA esters (C8-C26) with binding affinities (Kd) ranging from nM toM, suggesting the potential of ACBPs in acyl-CoA transport in vivo [31, 34, 35], and in the protection and maintenance of cellular acyl-CoA pools [27, 30, 36-39]. Interestingly, recombinant Arabidopsis and rice ACBPs have been shown to bind phospholipids such as PA and PC by in vitro filter-binding and liposome-binding assays [23, 40-43]. The dissociation constants of the interaction between recombinant ACBP and phospholipids are yet unclear because of technical limitations in conducting the measurements in vitro. However, the presence of di18:2-PC and di18:2-PE (phosphatidylethanolamine) partially replaced the binding of C18:3-CoA

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ACCEPTED MANUSCRIPT and recombinant AtACBP3 by Lipidex competition assays, suggesting high-affinity binding between recombinant ACBP3 and the phospholipids [43]. Besides ACBPs,

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LTPs bind both phospholipids and acyl-CoA esters [44, 45]. However, unlike the

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diverse subcellular distributions of ACBPs, LTPs are apoplastic proteins and participate in the assembly of surface lipids such as wax, suberin and sporopollenin [46-48].

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In yeast, ACBP depletion adversely affected plasma membrane and vacuole

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biogenesis [27, 49]. A recent study revealed that the yeast ACBP is important in the assembly and activity of the Brome Mosaic Virus (BMV) RNA replication complex

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[50]. The depletion of ACBP elevated the expression of genes of fatty acid and

In mammals, ACBPs have been found to modulate the activity of enzymes

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[51].

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phospholipid biosynthesis, glycolysis and glycerol metabolism, and stress responses

including acyl-CoA:lysophospholipid acyltransferase [52], acyl-CoA:cholesterol [53,

54],

carnitine

palmitoyltransferase

I

[55,

56]

and

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acyltransferase

glycerol-3-phosphate acyltransferase [39, 57, 58]. Cytosolic 10-kDa ACBPs also control gene expression; the overexpression of the 10-kDa ACBP in rat down-regulated the expression of metabolic regulators such as the peroxisome proliferator-activated receptors and sterol regulatory element-binding protein-1 [59]. The rat ACBP interacted with hepatocyte nuclear receptor-4 (HNF-4), and its overexpression enhanced HNF-4-mediated transactivation [60]. Besides the 10-kDa cytosolic ACBPs, many larger forms of ACBPs have been characterized in eukaryotes (plants, Caenorhabditis elegans and mammals) in the past

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ACCEPTED MANUSCRIPT twenty years [15, 16, 61-64]. Each of these large ACBPs contains a conserved acyl-CoA-binding (ACB) domain plus other functional domains such as ankyrin

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repeats, kelch motifs, enoyl-CoA hydratase/isomerase (ECH) domain, Golgi

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dynamics (GOLD) domain and coiled-coil domain [16, 62-64]. Some of these large ACBPs bind long-chain acyl-CoA esters via the ACB domain [28, 61, 65-67] and participate in various biological activities including membrane biogenesis [16, 27, 49],

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vesicular trafficking [61], development [15, 16, 62, 63], biotic and abiotic stress

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responses [15, 16, 68] and lipid biosynthesis [15, 16, 62, 63, 68, 69]. Some recent reviews reported a certain aspect of plant ACBPs: acyl-CoA-binding and lipid-binding

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properties [70], development [71, 72] and function and localization of cytosolic

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ACBPs [73]. In this review, we summarize current reports on plant ACBPs and focus

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on a comparison between the ACBPs from two model organisms representing monocots and dicots, Arabidopsis and rice respectively, in acyl-CoA/lipid metabolism,

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development and stress responses based on their subcelullar distribution and temporal and spatial expression patterns, as well as their interaction with acyl-CoAs, phospholipids and protein partners. This is a thorough review on plant ACBPs with brief coverage on the ACBPs from the other eukaryotes such as mammals and yeast (Supplemental Table 1). Along with progress in the genomic sequencing of various plant species, the identification of plant ACBPs has accelerated. This review investigates the evolution and current discoveries on plant ACBPs and provides new perspectives on future research for this important protein family.

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ACCEPTED MANUSCRIPT 2. Plant acyl-CoA-binding proteins 2.1 Phylogenetic analyses of plant acyl-CoA-binding proteins

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Phylogenetic studies on plant ACBPs conducted within sixteen plant genomes

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[74] revealed the Class I ACBPs as a monophyletic group (BS = 83%). In this study, phylogenetic analysis of ACBPs extracted from thirty plant genomes and using Maximum Likelihood reconstructions with an edited matrix, identified four main

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clades representing the ACBP classes (Fig. 1). Two classes, Class I (78%) and Class

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IV (86%), were strongly supported as monophyletic using bootstrap values. A second moderately supported clade containing Classes II, III and IV (65%) was identified as

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sister to Class I (Fig. 1) consistent with previous results [74]. Three clades appear to

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have higher support values in contrast to earlier studies: (1) Class I (78%), (2) Class

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IV (86%) and (3) a clade containing Classes II, III and IV (65%). Fig. 1 reveals that each acyl-CoA class is monophyletic even though Classes II and III are not

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statistically supported by bootstrap values. Support values within each protein classes of the different plant taxa included here are low, attributed either to the lack of homology or little molecular variation among sequences. The hypothesis of an early-divergent Class I ACBP [74] is strongly supported by phylogeny (Fig. 1). Class I ACBPs are small proteins with only one acyl-CoA-binding (ACB) domain (Fig. 1), and have been widely characterized in eukaryotes and prokaryotes [14, 62, 64, 68]. Fig. 1 not only indicates the diversity and evolution of plant ACBPs but also provides the basis for their functional characterization.

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ACCEPTED MANUSCRIPT 2.2 Identification of plant acyl-CoA-binding proteins The first plant ACBP characterized from Brassica napus L. (oilseed rape) was

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a 10-kDa ACBP homologue (Class I ACBP) strongly expressed in developing seeds,

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flowers and cotyledons [25]. Subsequently, Class I ACBPs were also identified from Arabidopsis thaliana [30, 75], Gossypium hirsutum (cotton) [76], Ricinus communis (castor bean) [77], Digitalis lanata Ehrh. (Woolly Foxglove) [78], rice [74, 79] and

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Vernicia fordii (tung tree) [80]. The B. napus ACBP has been observed to bind

the

activities

of

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long-chain acyl-CoA esters [81, 82], participate in acyl-CoA transport [83], control glycerol-3-phosphate

acyltransferase

(GPAT)

[81],

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lysophosphatidylcholine acyltransferase (LPCAT) [82] and lysophosphatidic acid

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acyltransferase (LPAAT) [84], regulate plastidial glucose 6-phosphate (Glc-6-P) and

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ATP transport [85], and mediate acyl exchange between acyl-CoA and phospholipid pools [82, 86].

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Besides the presence of Class II ACBPs in Arabidopsis [16, 67] and rice [74], a Class II 40-kDa ACBP was reported from Agave americana, which was suggested to be involved in cuticle formation [87]. Class III ACBPs are characterized by the presence of a C-terminal ACB domain and they include Arabidopsis ACBP3 [66], O. sativa ACBP5 [74], V. fordii ACBP3A and ACBP3B [80, 88], and Vitis vinifera (grape) ACBP-1 [89]. Unlike the apoplast-localized AtACBP3 [66], OsACBP5 and VfACBP3A/VfACBP3B were localized to the ER [88, 90] whereas VvACBP-1 was expressed at the cell periphery and cytoskeleton [89], indicating possibility in differential functions for Class III ACBPs. The expression of VvACBP-1 was induced

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ACCEPTED MANUSCRIPT by ER stress, cold and heat shock and its overexpression in Arabidopsis caused retardation in floral transition, changes in morphological development related to the

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thickness of inflorescences and leaves, as well as in pathogen resistance [89],

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suggesting its role in development and stress tolerance. In addition, VfACBP3A and VfACBP3B have been shown to participate in fatty acid metabolism [88]. Four plant Class IV ACBPs have been characterized including AtACBP4 and AtACBP5 [65, 91,

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92], OsACBP6 [74] and VfACBP4 [80, 88]. Depletion of AtACBP4 in Arabidopsis

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resulted in decreases in galactolipids and phospholipids [91], suggesting its role in membrane lipid biosynthesis. AtACBP4 also interacted with an Arabidopsis

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ethylene-responsive element binding protein (AtEBP), also known as RELATED TO

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APETALA2.3 (RAP2.3) [92, 93]. The expression of AtACBP4 was induced by the

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ethylene precursor 1-aminocyclopropane-1-carboxylic acid, methyl jasmonate (JA) and Botrytis cinerea infection, indicating that AtACBP4 may be involved in plant

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defense responses [92]. In contrast, the function of the other plant Class IV ACBPs remains unclear. New ACBPs of Classes I to IV from the Brassicaceae family (B. napus, B. rapa and B. oleracea) share conserved domains with and high amino acid sequence identity to the AtACBPs [94]. Further computational predictions on BnACBP protein structure and experiments in their subcellular localization revealed resemblance to the AtACBPs [95]. Our current knowledge on plant ACBPs with the exceptions of Arabidopsis and rice ACBPs is summarized in Table 1. More detailed information on Arabidopsis ACBPs (Table 2) and rice ACBPs (Table 3) are provided separately.

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3. Temporal and spatial expression of plant acyl-CoA-binding proteins

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The expression of ACBPs has been observed in the majority of plant [42, 81,

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96, 97] and animal [62, 64, 98, 99] organs. Investigations on the 5´-flanking regions of ACBPs indicate that they are housekeeping genes [96, 100-103]. ACBPs exist in eukaryotes in multiple isoforms with diverse distribution and function [14-16, 62, 64].

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For instance, there are multiple isoforms of mammalian Class I ACBPs such as brain

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ACBP (B-ACBP), liver ACBP (L-ACBP) and testis ACBP (T-ACBP) [62, 64]. B-ACBP (also referred to as ACBD7) was expressed in human brain, spleen and

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thymus [62, 64]. Similar B-ACBPs have been identified in frog and duck [104, 105].

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L-ACBP was first isolated from bovine liver [106] whereas T-ACBP was strongly

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expressed in the testes [62, 107-109]. For large ACBPs, the GOLD domain-containing human ACBD3 was

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expressed in organs including the hypothalamus, liver, kidney, testis, ovary, adrenal gland, hippocampus and cortex [110, 111]. In the ornamental plant, A. americana, AaACBP-1 (encoding a Class II ACBP) was found to be expressed in the epidermis of the mature zone in leaves [87]. Two genes encoding Class III ACBPs of the tung tree, VfACBP3A and VfACBP3B, were highly expressed in developing (but not mature) seeds and flowers, while VfACBP3A was predominantly expressed in leaves [88]. Similar to VfACBP3B, VfACBP4, the gene encoding Class IV tung tree ACBP, was highly expressed in developing seeds and flowers but showed very low expression in leaves [80]. In Arabidopsis, AtACBP3 [43, 96, 112], AtACBP4 [28] and AtACBP5 [28]

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ACCEPTED MANUSCRIPT were regulated by light and darkness.

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3.1 Arabidopsis acyl-CoA-binding proteins

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The first Arabidopsis ACBP reported was a Class I 10-kDa ACBP (also designated as AtACBP6 [113]), which binds 16:0- and 18:1-CoA esters and protected 18:1-CoA esters from hydrolysis [30]. AtACBP6 exhibited stronger expression in the

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siliques and developing seeds than roots and leaves [30]. Subsequently, two Class II

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AtACBPs, AtACBP1 and AtACBP2 were identified to be highly expressed in Arabidopsis embryos [67, 103, 114]. The other three AtACBPs were retrieved by a

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search of genes encoding proteins with acyl-CoA-binding domain in the Arabidopsis

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genome (Arabidopsis Genome Initiative, 2000), and were designated as AtACBP3,

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AtACBP4, and AtACBP5 [65], representing the first report of an ACBP family in the Plant Kingdom that included larger members ranging from 37.5 to 73.2 kDa. The

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Classes II and IV ACBPs contain a conserved ACB domain together with other functional domains, i.e. transmembrane domain and ankyrin repeats in Class II and kelch motifs in Class IV (Table 2). To verify whether these large AtACBPs bind acyl-CoA esters, rAtACBPs as well as rAtACBP mutants with single amino acid substitutions were tested in Lipidex-1000 assays [28, 43, 65-67]. The results showed that all six rAtACBPs could bind acyl-CoA esters [28, 43, 65-67] and amino acid substitutions within the ACB domain affected the binding between acyl-CoAs esters and rAtACBP2 [67], rAtACBP3 [66], rAtACBP4 [28, 65] and rAtACBP5 [28, 65]. The acyl-CoA-binding

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ACCEPTED MANUSCRIPT ability of rAtACBPs was further confirmed by ITC or MST, supporting their basic function as acyl-CoA carriers (Table 2). With this characteristic established, their

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temporal and spatial expression patterns were next sought to better understand their

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biological roles in Arabidopsis (Fig. 2).

Different methodologies have been performed to investigate the expression of AtACBPs

including

Southern-,

Northern-

and

Western-blot

analyses,

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semi-quantitative and quantitative RT-PCR, GUS (-glucuronidase) reporter assays

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and immunolocalization (Fig. 2). As shown in Fig. 2 (right), AtACBPs are widely expressed in various organs throughout Arabidopsis development. AtACBP1 and

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AtACBP2, two highly conserved homologues sharing 82% identity, show similar

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expression patterns in flowers [40, 42, 67, 97, 103, 114, 115], siliques [40, 67, 103,

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114, 115], mature seeds [42, 97, 103, 116, 117], seedlings [42, 97, 116], rosettes [42, 97] and mature plants [21, 40, 42, 67, 97, 114, 115]. However, the results of GUS

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reporter assays revealed that only AtACBP1::GUS was expressed in the trichomes [42] whereas AtACBP2::GUS was detected in the guard cells [97]. These corresponded well with their function in heavy metal and drought stress responses, respectively, as trichomes have been previously reported to accumulate lead [Pb(II)] [118, 119] while the guard cells control water loss. In addition, their high expression in siliques, particularly in developing and mature seeds, indicated potential roles in seed development, dormancy and germination [42, 97, 103, 117]. AtACBP3, a Class III AtACBP, was expressed in flowers [43, 65, 96], siliques [43, 65, 96], seedlings [43, 65, 96], rosettes [43, 65, 96] and mature plants [43, 65, 96].

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ACCEPTED MANUSCRIPT From GUS assays, AtACBP3 was strongly expressed in the phloem tissue of leaves and stems [96]. Considering that rAtACBP3 binds 18:2-, 18:3-, 20:0-, 20:4-, 22:0-

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and 24:0-CoAs [22, 43, 66], as well as PC and PE [43], AtACBP3 could be involved

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in phloem-associated long-distance lipid (acyl-CoA esters) signaling, joining other phloem-mobile proteins related to lipid metabolism [120, 121]. Observations in the expression of AtACBP3 at the stigma further indicate a potential role related to

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reproduction [96].

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AtACBP4, AtACBP5 and AtACBP6, the three cytosolic AtACBPs, were expressed in flowers [28, 65, 92, 113], siliques [28, 30, 65, 92, 113] and mature plants

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[28, 30, 65, 91, 92, 113], while AtACBP4 and AtACBP5 were also expressed in

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seedlings (Fig. 2) [28]. Recent expression profiles by quantitative RT-PCR showed

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that AtACBP4 to AtACBP6 were highly expressed in the inflorescences [122]. Subsequent GUS assays revealed the expression of AtACBP4 in pollen grains,

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AtACBP5 in microspores and tapetal cells, and AtACBP6 in pollen grains, microspores and tapetal cells, suggesting their overlapping roles in pollen development [122]. In addition, AtACBP6 was also expressed in the cotyledons, hypocotyl and cotyledonary-staged embryo in GUS-histochemical assays [32].

3.2 Rice acyl-CoA-binding proteins A 10-kDa rice ACBP designated as RPP10, was first identified as a major phloem sap protein [79]. It belongs to the Class I OsACBPs and is a homologue of AtACBP6 [79, 113]. Subsequently, the complete rice OsACBP family was designated

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ACCEPTED MANUSCRIPT as OsACBP1 to OsACBP6, and RPP10 corresponds to OsACBP1 [74]. Similar to Arabidopsis, six ACBP members co-exist in rice [74]. However, unlike the AtACBP

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family, there are three Class I rice ACBPs (OsACBP1 to OsACBP3) and only one

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member for each of the remaining three classes [74] as shown in Table 3. Similar to rAtACBPs, rOsACBPs bind various acyl-CoA esters with different preferences in the chain-length and degree of saturation (Table 3) [74]. As a more newly-identified

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ACBP family, the expression patterns of OsACBPs are not as well understood as the

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AtACBPs. The expression patterns of OsACBPs are summarized herein (Fig. 3, right) from information of the Rice eFP Browser [123] and past reports [23, 74, 90].

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Using the AtACBPs (Table 2) as a reference, the rice ACBPs have been

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mapped to four classes based on their domain architecture (Table 3). For example,

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OsACBP4, the Class II OsACBP, consists of an ACB domain and ankyrin repeats resembling AtACBP1 and AtACBP2 (Tables 2 and 3). OsACBP4 is also expressed in

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developing and germinating seeds (Figs. 2 and 3). OsACBP4 may be involved in embryogenesis, seed dormancy and germination if it were to resemble AtACBP1 [42] and AtACBP2 [97]. Thus, the comparison between Arabidopsis and rice shown in Figs. 2 and 3 should prompt future investigations on the OsACBP family. Table 3 lists a summary of the subcellular localization of OsACBPs from investigations by computational prediction, transient expression of OsACBP::GFP fusions in tobacco leaf epidermal cells and stable expression of OsACBP::GFP fusions in transgenic Arabidopsis plants [23, 90]. For the three Class I members (OsACBP1 to OsACBP3), they were subcellularly localized to the cytosol, similar to

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ACCEPTED MANUSCRIPT BnACBP, AtACBP6 and VfACBP6 (Table 1). Intriguingly, OsACBP3 was also located at the intracellular irregular membranous structures and randomly distributed

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punctates, which had rarely been encountered with other reported ACBPs [23]. This is

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possibly attributed to the presence of a unique C-terminal 64-amino acid extension that is not evident in the 10-kDa OsACBP1 and OsACBP2 [23]. OsACBP3 not only encompasses an ACB domain but contains a putative acetyl-lysine deacetylase

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domain which may allow it to interact with other proteins [23]. Class II OsACBP4

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was located at both the cisternal and tubular ER, consistent with the ER-targeted Class II AtACBP1 and AtACBP2 (Table 3) [23, 90, 103, 124], thus displaying conservation

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in subcellular localization within Class II in the ER, in addition to similarities in their

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distribution across plant organs (Fig. 3), although it should be noted that AtACBP1

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and AtACBP2 are additionally localized to the plasma membrane [103, 115, 124]. OsACBP5, the Class III OsACBP, was localized to the tubular ER (Table 3) [90],

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which bears resemblance to VfACBP3A/VfACBP3B (Table 1) and AtACBP3 has been detected in the apoplast and periphery of the ER and Golgi [43, 66]. The localization of OsACBP4 and OsACBP5 overlaps at the tubular ER [90]. Given that these rice ACBPs belong to distinct classes, i.e. Class II (OsACBP4) and Class IV (OsACBP5), they may have essential and non-overlapping roles at the ER. Seed storage reserves such as TAG are synthesized at the ER and then stored in the form of lipid droplets and protein bodies [125, 126]. High expression of OsACBP5 during the course of seed formation suggests a potential role of OsACBP5 related to energy storage [74]. It is noteworthy that OsACBP4 and OsACBP5 also co-localized to the

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ACCEPTED MANUSCRIPT membrane of ER bodies and ER-derived spherical structures [90]. ER bodies are ER-derived structures reassociated with protein storage and resistance to insects or

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pathogens in Arabidopsis [127]. Expression of the Arabidopsis ER body membrane

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proteins, MEMBRANE PROTEIN OF ENDOPLASMIC RETICULUM BODY1 (MEB1) and MEB2, in yeast enhanced tolerance to metal stress [128]. These observations indicated that ER bodies are closely related with stress responses. The

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localization of OsACBP4 and OsACBP5 in the membrane of ER bodies suggests that

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they are potentially associated with plant stress responses. This proposition is supported by reports that OsACBP5 is wound- and pathogen-inducible. As OsACBP4

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is drought-inducible and distinctively localized to the cisternal ER, it could be

TE

involved in drought response through protein/lipid translocation [129]. In addition,

CE P

OsACBP4 and OsACBP5 showed different preferences for acyl-CoA esters and phospholipids, suggesting that their functions in lipid trafficking are likely to be

AC

non-redundant (Table 3).

The monocot Class IV OsACBP6 has an unusual localization in the

peroxisomal matrix (Table 3) [23], unlike the other characterized plant ACBPs which are either cytosolic or membranous proteins [15, 16, 23, 68]. The subcellular distribution of other monocot Class IV members await to be elucidated [23]. Inmammalian cells, rat ACBP is directly activated by the peroxisome proliferator-activated receptor γ (PPARγ andPPARα) [130, 131] while in plant cells, the peroxisome is considered to be the prime site of fatty acid β-oxidation [132]. For example, COMATOSE (CTS), also referred to as PEROXISOMAL ABC

18

ACCEPTED MANUSCRIPT TRANSPORTER1 (PXA1) and PEROXISOME DEFICIENT3 (PED3), is responsible for substrate import in peroxisomal β-oxidation of Arabidopsis [133-135]. The

IP

T

thioesterase activity of CTS cleaves CoAs during transport across the peroxisomal

SC R

membrane [136]; CoAs are hypothesized to be cleaved at either the cytosolic side or lumenal side of the peroxisome membrane [136, 137]. In both models, peroxisomes require protein(s) to maintain and regulate the peroxisomal-CoA pool [137].

NU

Interestingly, it has been reported that the disruption of C. elegans ACBP2 resulted in

MA

a reduction in β-oxidation of oleic acid (18:1) and increase in the expression of several genes encoding enzymes of β-oxidation in the peroxisomes and mitochondria

TE

should be further explored.

D

[63]. Thus, the potential function of ACBPs such as OsACBP6 in lipid catabolism

CE P

Given that rOsACBP6 binds 18:1- and 18:2-CoAs as well as phospholipids (18:0-PA, 18:1-PA; 18:0-PC, 18:1-PC, 18:2-PC) [23, 74] and ACBPs can regulate

AC

enzymatic activities [68], OsACBP6 could participate in the β-oxidation of fatty acids by the donation of acyl-CoA esters or regulation of enzymes such as CTS. Indeed, evidence demonstrating that acyl-CoA esters (C14-, C18- and C24-CoAs) stimulate the activity of CTS has already emerged [138]. Observations that OsACBP6 overexpression in an Arabidopsis β-oxidation defective mutant pxa1 rescued indole-3-butyric acid sensitivity and post-wounding JA production, further support the role of OsACBP6 in peroxisomal β-oxidation [23]. These findings show that plant ACBPs do not only

protect acyl-CoA esters [30] and facilitate fatty acid

biosynthesis [16, 68], but also participate in fatty acid degradation. Furthermore,

19

ACCEPTED MANUSCRIPT higher diversity appears to occur in Class IV [23, 80, 91, 92]. The unique peroxisomal localization of OsACBP6 implies that diversity in Class IV evolved could have split

IP

T

between monocots and eudicots. Obvious differences between the proteomes of

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Arabidopsis and rice peroxisomes have been reported [139]. Substrate transport for β-oxidation in rice may possibly differ from Arabidopsis. Despite the presence of two orthologues of CTS in rice [139], no reports have emerged on how they participate in

MA

NU

β-oxidation.

4. Plant acyl-CoA-binding proteins in development

D

The expression of ACBPs is closely related to their biological functions in

TE

plant development [42, 96, 97]. In other eukaryotes, yeast (Saccharomyces cerevisiae)

CE P

ACBP is essential in membrane biogenesis and its depletion resulted in a defective plasma membrane and abnormal vacuoles [27, 49]. In C. elegans, loss in activity of

AC

MAA-1 (membrane associated ACBP domain-containing protein-1), a 57-kDa membrane-associated ACBP localized to the intracellular membrane organelles of the secretory and endocytic pathway perturbed endosomal morphology [61]. Besides MAA-1, six other ACBPs are expressed in C. elegans, four of Class I, one of Class II plus an ECH-domain containing ACBP [63]. A quadruple mutation of the four Class I ACBPs (acbp1acbp3acbp4acbp6) adversely affected lipid droplet morphology and caused slight delay in development of C. elegans [63]. In human HeLa, HepG2 and Chang cell lines, the use of an ACBP-specific small interference RNA (siRNA) corresponding to the 10-kDa Class I ACBP to knock down its expression by

20

ACCEPTED MANUSCRIPT transfection culminated in lethality [140]. ACBP1e, a novel human ACBP, was recently detected in the adipose tissue and the hippocampus, suggesting a possible

IP

T

role of ACBP1e in the development of these tissues [141], while several human

SC R

ACBPs were found associated with adipogenesis of the human preadipocyte cell line SGBS [142]. In rodents, the Class I ACBP played critical roles in the development and maintenance of mouse hair and skin tissues [143, 144]. Its disruption in mice

NU

adversely affected the biophysical properties of the stratum corneum [145] and

MA

interfered with metabolic adaptation to weaning leading to growth retardation [146]. In plants, the overexpression of grapevine VvACBP-1 affected floral transition

D

by downregulating proteins of the photoperiodic pathway such as CONSTANS,

TE

FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF

CE P

CONSTANS1 [89]. Changes in plant morphology of the transgenic lines included increases in inflorescence diameter and thickness of rosette leaves which displayed an

AC

accumulation of anthocyanin on their abaxial surface [89]. In Arabidopsis, the overexpression of the Class I B. napus ACBP in seeds affected fatty acid composition with increases in 18:2 and decreases in 20:1, during seed development and maturation [82, 86]. As Arabidopsis ACBPs are currently the best-characterized in the Plant Kingdom [15, 16, 93], the function of AtACBPs in development is further discussed below.

4.1 Embryo development Many studies have shown that acyl-CoA esters are essential in embryo

21

ACCEPTED MANUSCRIPT development and mutations in the genes encoding acyl-CoA metabolism enzymes led to embryo lethality, as exemplified in the mutant of ACC1 (encoding an acetyl-CoA

IP

T

carboxylase) [147], double mutants of ACX3-1 and ACX4-1 (encoding acyl-CoA

SC R

oxidases) [148], double mutants of HAL3a-1 and HALb (encoding CoA biosynthetic enzymes) [149] and the mutant of an Arabidopsis plastidial LPAAT [150], an enzyme which was subject to Class I ACBP regulation in in vitro enzyme assays [84].

NU

Interestingly, studies on mice ACBPs had revealed that depletion of either the 10-kDa

MA

ACBP or ACDB3 (consisting of an ACB domain and a phosphotyrosine-binding domain) resulted in embryonic lethality [151, 152].

D

Investigations on the temporal and spatial expression of AtACBPs suggest

immunogold

labeling

[103],

microarray

analysis

[116]

and

CE P

114],

TE

their roles in development [15, 16]. Based on results of Western-blot analysis [103,

immunohistochemical localization assays [117], AtACBP1 and AtACBP2 are now

AC

known to be highly expressed in the embryo (developing seeds) at the heart, torpedo and cotyledon stages. These results were substantiated in GUS assays using transgenic Arabidopsis expressing AtACBP1pro::GUS [42] and AtACBP2pro::GUS [97]. The role of AtACBP1 and AtACBP2 in embryo development was tested using acbp1 and acbp2 T-DNA insertional mutants [117]. Although no significant morphological changes emerged from the single mutations [117], the acbp1acbp2 double mutant was embryo lethal and defective in callus induction [117]. This result is not surprising because AtACBP1 and AtACBP2 are two highly conserved homologues that share similarities including their subcellular localization to the ER

22

ACCEPTED MANUSCRIPT and plasma membrane [15] and they are believed to possess redundant functions [15]. Further analyses on their expression profiles revealed that the disruption of one gene

IP

T

resulted in compensatory up-regulation of the other [117]. Lipid profiling on the

SC R

acbp1 mutant revealed that depletion of AtACBP1 caused accumulation of MGDG with decreases in phospholipids such as PC, PE, PI and PS [117]. Correspondingly, rAtACBP1 and rAtACBP2 were observed to bind various phospholipids in vitro [117].

NU

Together with an earlier analysis of the lipid contents of AtACBP2 knock-down plants

MA

[153], AtACBP1 and AtACBP2 were thus verified to participate in acyl-CoA-related phospholipid and galactolipid synthesis during embryogenesis in Arabidopsis [117,

D

154]. In addition, given that AtACBP1 and AtACBP2 can potentially bind PC and

TE

acyl-CoA esters at the ER, and that ACBP1 expression in developing seeds coincided

CE P

with stages in lipid deposition [42], their possible role in the formation of lipid

AC

droplets in developing seeds warrants further investigations.

4.2 Seed dormancy, germination and seedling development Following reports demonstrating that plant ACBPs are expressed in seeds

(Table 1) [15], AtACBP1 [42] and AtACBP2 [97] were reported to function in seed dormancy and germination through an abscisic acid (ABA)-mediated pathway. Previous studies have shown that the overexpression of AtACBP1 in transgenic 5-week-old Arabidopsis elevated PA content [41] and rAtACBP1 was observed to bind PA in vitro using dot-blot filter assays [41, 42]. Considering that PA triggers early signal transduction events in response to ABA during seed germination in

23

ACCEPTED MANUSCRIPT Arabidopsis [155], the role of AtACBP1 (as well as AtACBP2) in seed germination was examined in the respective mutant and overexpressing seeds, specifically in the lines

[42]

and

T

mutant/AtACBP1-overexpressing

the

acbp2

IP

acbp1

SC R

mutant/AtACBP2-overexpressing lines [97]. The results confirmed that both AtACBP1 and AtACBP2 can promote ABA signaling in seeds and thereby regulate seed dormancy and germination, as well as seedling development [42, 97]. The acbp1

NU

and acbp2 single mutants showed relatively little difference to the wild type compared

MA

to the overexpressing lines [42, 97], most likely attributed to compensatory expression between ACBP1 and ACBP2 [117]. As the acbp1 acbp2 double mutant is embryo

D

lethal [117], RNAi lines would be more useful in future investigations for defining the

TE

functions of AtACBP1 and AtACBP2 in these processes.

CE P

The three cytosolic AtACBPs from Arabidopsis (AtACBP4 to AtACBP6) have been characterized [65, 113] and information on the function of the kelch

AC

motif-containing cytosolic AtACBPs (AtACBP4 and AtACBP5) is lagging behind AtACBP6 given their relatively low expression, large molecular size and putative redundant functions [16]. The functions of all three cytosolic AtACBPs in the reproductive development were elucidated by using a series of double/triple mutants and GUS-reporter assays to map their spatial expression [32, 122]. Results of Microarray (AtACBP4 to AtACBP6) and GUS stains (AtACBP6) indicated the expression of the cytosolic AtACBPs in seeds, and results from the double/triple mutants revealed that the double mutants (acbp4acbp5, acbp4acbp6 and acbp5acbp6), as well as the triple mutant (acbp4acbp5acbp6), exhibited reduction in seed weight

24

ACCEPTED MANUSCRIPT and hypersensitivity to ABA during seed germination in comparison to the wild type [32]. Intriguingly, AtACBP6 likely plays a more important role in ABA signaling

IP

T

than AtACBP4 and AtACBP5 because acbp4acbp5 was less sensitive to ABA than

SC R

the other mutants [122]. In addition, seed number (per silique) and pollen population (per pollen sac) significantly decreased in acbp4acbp6, acbp5acbp6 and acbp4acbp5acbp6 (but not acbp4acbp5) in comparison to the wild type [122]. Such

NU

differences between AtACBP6 and AtACBP4/AtACBP5 may be attributed to their

MA

variation in acyl-CoA-binding properties from in vitro acyl-CoA-binding assays in ITC; rAtACBP6 showed binding to long-chain acyl-CoA (C16- to C18-CoAs) esters

D

with dissociation constants in the nanomolar range in comparison to rAtACBP4 and

TE

rAtACBP5 which displayed values in the micromolar range (Table 2) [32]. On

CE P

acyl-CoA profiling, the acbp6 knockout mutant demonstrated an accumulation of unsaturated C18-CoAs in developing seeds and seedlings [32] consistent with

AC

previous observations on transgenic Arabidopsis seeds overexpressing the 10-kDa B. napus ACBP [86]. The variation in acyl-CoA composition of the double/triple mutants may ultimately affect seed development and germination. Besides developing seeds, AtACBP4 and AtACBP5, as well as AtACBP6, were also expressed in the anthers in GUS histochemical assays [122]. The acbp4acbp5acbp6 triple mutant showed defects in pollen development such as abnormalities in pollen exine and oil body, and reduction in pollen population and activity, indicating that the cytosolic AtACBPs are involved in acyl-CoA/lipid metabolism during pollen development [122]. Progress on investigations on the cytosolic AtACBPs, including the generation

25

ACCEPTED MANUSCRIPT of double/triple mutants and GUS-expressing lines has provided the necessary tools for future studies on their function in lipid metabolism, development and stress

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IP

T

responses.

4.3 Light regulation in seedlings

In Arabidopsis, the cytosolic AtACBPs (AtACBP4 to AtACBP6) and apoplastic

NU

AtACBP3 were regulated by light-dark cycling in Northern-blot analysis and

MA

quantitative RT-PCR [28, 43, 112], and AtACBP4 and AtACBP5 were also shown to be light-dark regulated by Western-blot analysis [156]. AtACBP4 and AtACBP5 were

D

light inducible corresponding well with their proposed role in the protection and

TE

transport of plastid-synthesized acyl-CoAs to the ER for extraplastidial lipid

CE P

biosynthesis [28]. In contrast, AtACBP3 and AtACBP6 were both light-inhibited and dark-induced [28, 43]. To better understand the light-dark regulation of AtACBP3,

AC

transgenic Arabidopsis expressing AtACBP3pro::GUS fusion and its deletion derivatives were subjected to light-dark cycles followed by GUS activity assays [96]. The results confirmed the light-dark regulation of AtACBP3 is consistent with previous observations [28, 43] and two functional motifs (Dof and GT-1) related to light-dark cycles in the 1.7-kb 5´-flanking region of AtACBP3 were identified by electrophoretic mobility shift assays and DNase footprinting assays [96].

4.4 Cuticle formation Differences amongst AtACBPs in subcellular localization, spatial expression,

26

ACCEPTED MANUSCRIPT acyl-CoA-binding, and light regulation (Table 2) indicate that they play various roles in acyl-CoA trafficking in Arabidopsis. In spite of their disparate subcellular

IP

T

localization, AtACBP1, AtACBP3, AtACBP4, and AtACBP6 are required for

SC R

maintaining normal fatty acid/lipid levels and cuticle development [20, 21]. Earlier reports on a membrane-associated A. americana ACBP [87] and on AtACBP1 [21] had suggested the role of ACBPs in cuticle formation. The A. americana ACBP was

NU

shown in Northern-blot analysis to be highly expressed in the epidermis which is

MA

enriched in cuticular waxes, cutin and cutan correlating to function in cuticle formation [87]. In Arabidopsis, AtACBP1 has been reported to play a role in

D

intermembrane lipid transport from the ER to the plasma membrane and its

TE

subsequent immunodetection at the plasma membrane especially at the epidermal

CE P

cells of heart, torpedo and cotyledonary stage embryos, and the cell wall of outer integument cells of the seed coat suggested its potential function in cuticle and cutin

AC

formation [42]. GUS-histochemical assays using AtACBP1pro::GUS-expressing Arabidopsis showed AtACBP1 expression at the stem surface [21]. Furthermore, ITC-binding assays verified that rAtACBP1 binds VLC acyl-CoA esters such as 24:0-, 25:0- and 26:0-CoAs that form the precursors of wax biosynthesis [21]. In contrast to the wild type, the acbp1 knockout mutant showed a reduction of stem cuticular wax and cutin monomers as well as wax crystals, in addition to an irregular stem cuticle layer [21]. Considering that AtACBP1 is localized at the ER, it was proposed that AtACBP1 contributes to stem cuticle formation in Arabidopsis by transferring and donating acyl-CoA esters on the ER side [21]. Another report indicated that cytosolic

27

ACCEPTED MANUSCRIPT AtACBP4 and AtACBP6, as well as extracellularly-targeted AtACBP3 are involved in cuticle development by transporting acyl-CoAs/lipids between the prokaryotic and

IP

T

eukaryotic pathways [21]. Similar to the acbp1 mutant, the acbp3 and acbp4 mutants

SC R

have lower cutin monomer content than the wild type, and the acbp3, acbp4 and acbp6 mutants possess a defective cuticle layer [20]. Taken together, both membrane and soluble AtACBPs appear to be essential in acyl-CoA/lipid trafficking between the

NU

plastid and ER for cuticle development in Arabidopsis, and consequently this leads to

MA

better protection against environmental stresses such as drought and microbial infection [20, 21]. Progress made on AtACBPs in relation to various stresses is

TE

D

discussed next.

CE P

5. Plant acyl-CoA-binding proteins in stress responses The Class I ACBPs have been reported to participate in multiple stress

AC

responses. For example, depletion of the yeast ACBP up-regulated the expression of several stress-responsive genes such as CTT1, HSP26 and DDR2 [51]. In crustaceans, the gene encoding Litopenaeus vannamei LvACBP was more highly expressed in shrimps resistant, rather than those susceptible, to the white spot syndrome virus (WSSV) indicating its role in immunity to viral infection [157]. Similarly, in the shrimp Fenneropenaeus chinensis, FcACBP was induced by WSSV and Vibrio anguillarum infection in the intestine [158]. Besides microbial infections, low or high salinity stresses dramatically increased the expression of PmACBP (shrimp Penaeus monodon) in various tissues such as muscle, gut and gills, suggesting that PmACBP

28

ACCEPTED MANUSCRIPT contributes to salinity tolerance and adaptation [159]. In mice, the expression of ACBP was relatively higher in the epidermis, and its disruption led to alopecia and

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T

scaling of the skin accompanied by a ~50% increase in transepidermal water loss [143,

SC R

145]. Intriguingly, the mouse ACBP was also induced by conditioned emotional stimuli which cause psychological stress [160]. In recent years, much evidence supports the participation of plant ACBPs from Classes I to IV in various stress

MA

NU

responses as shown in Fig. 2 (Arabidopsis) and Fig. 3 (rice).

5.1 Heavy metal and oxidative stresses

D

ACBD1, the human 10-kDa ACBP, also known as human endozepine [161,

TE

162] or DBI [24, 163], was reported to bind Pb(II) in kidney with high affinity (Kd=24

CE P

nM) with a 1:1 binding ratio of ACBD1 to Pb(II) [163]. ACBD1 and thymosin 4, another Pb(II)-binding protein, accounted for greater than 35% of the total bound

AC

Pb(II) in the human kidney cortex tissue [163]. In addition, ACBD1 contains solely an ACB domain (86 residues) [62, 161] and its crystal structure revealed only one Pb(II)and one zinc [Zn (II)]-binding site [164], suggesting an important role for the ACB domain in heavy metal binding. As heavy metals such as Pb(II) and Zn(II) are major pollutants threatening the environment and living creatures, several experiments have been performed to test the function of AtACBPs in heavy metal stress responses [115, 165, 166], and to address whether they can be used for phytoremediation. Initially, the two membrane-associated AtACBPs, AtACBP1 and AtACBP2, were demonstrated to bind heavy metals such as Pb(II) [40, 115, 165]. Resembling human ACBD1,

29

ACCEPTED MANUSCRIPT AtACBP1 binds Pb(II) (Kd=1.6 M) as examined in metal-chelate affinity chromatography and fluorescence binding analysis using dansyl aziridine-labeled

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proteins [165]. Further investigations showed that AtACBP1 was induced by Pb(II)

SC R

treatment and its overexpression conferred Pb(II) tolerance and accumulation in both shoots and roots of transgenic Arabidopsis plants [165]. Using GUS-histochemical assays, AtACBP1pro::GUS was expressed in the trichomes [42] which has been

NU

previously reported to accumulate heavy metals such as Pb(II) in plants [118, 119].

MA

These observations demonstrate the potential application of AtACBP1 for phytoremediation of Pb(II). According to in vitro binding assays, rAtACBP1 and

D

rAtACBP4 bind Pb(II) with the highest affinities amongst the six rAtACBPs [166,

TE

167], while the expression of AtACBP1 and AtACBP4 were induced by Pb(II) in both

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shoots and roots of Arabidopsis [166, 168]. Subsequently, AtACBP1 and AtACBP4 were expressed in Brassica juncea, a promising candidate for heavy metal

AC

phytoremediation, to examine the potential of AtACBPs in Pb(II) accumulation [168]. The resultant transgenic B. juncea plants, confirmed to produce AtACBP1 and AtACBP4, accumulated Pb(II) in the cytosol of root tips and the vascular tissues, but not in the shoots, when grown in Pb(II)-containing media, in contrast to AtACBP1-overexpressing Arabidopsis which translocated Pb(II) from roots to shoots [165, 168]. Nevertheless, this represents a first attempt towards the application of AtACBPs in phytoremediation and the detection of Pb(II)-responsive elements such as Pas and CURE in the AtACBP1 5´-flanking region provides further scope for investigations [168].

30

ACCEPTED MANUSCRIPT AtACBP2 is another Arabidopsis ACBP that is responsive to heavy metal stress. In contrast to rAtACBP1 and rAtACBP4, rAtACBP2 was observed to bind

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several heavy metals including cadmium [Cd(II)], copper [Cu(II)] and Pb(II) but

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showed lower-binding affinity to Pb(II) than rAtACBP1 [115, 165]. Although there are no reports on the accumulation of heavy metals in AtACBP2-overexpressing plants, the overexpression of AtACBP2 did enhance tolerance to Cd(II) and oxidative

to

H2O2

and

Pb(II)

was

observed

in

transgenic

Arabidopsis

MA

tolerance

NU

stress (hydrogen peroxide, H2O2) in transgenic Arabidopsis [115]. Similarly, enhanced

AtACBP1-overexpressors [165, 168]. Pb(II)-tracing assays revealed an enrichment of

D

Pb(II) in the trichomes of the wild type and AtACBP1-overexpressors but not in the

TE

acbp1 mutant [168] when plants were cultured in Pb(II)-containing medium,

CE P

consistent with the expression of AtACBP1pro::GUS in the trichomes [21, 42]. Most likely AtACBP1 sequestrates Pb(II) in the trichomes as a means to detoxification [118,

AC

119]. In

comparison,

the

distribution

and

sequestration

of

Cd(II)

in

AtACBP2-overexpressing Arabidopsisis are less well-understood although AtACBP2 is known to interact with the heavy-metal-binding protein AtFP6 [115] besides lysophospholipase AtLysoPL2 [40]. AtFP6 binds heavy metals similar to AtACBP2, and can thus sequester Cd(II), whereas AtLysoPL2 binds and detoxifies lysoPC generated from Cd(II)-induced oxidative stress. By its ability to interact with AtLysoPL2 and lysoPC, AtACBP2 can facilitate an efficient removal of lysoPC [40, 97]. In addition, AtACBP1 and AtACBP2 are both involved in response to oxidative

31

ACCEPTED MANUSCRIPT stress (H2O2), and their overproduction in transgenic plants reduced lipid hydroperoxide content following H2O2 or heavy-metal treatment [40, 115, 168]. As

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both rAtACBPs can bind 18:2- and 18:3-CoAs, precursors of phospholipid repair

SC R

following lipid peroxidation, AtACBP1 and AtACBP2 promote membrane restoration after oxidative stress [40, 115, 168].

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5.2 Drought, hypoxia and cold

MA

Prompted by the observation of AtACBP2 expression in the guard cells, AtACBP2-overexpressing transgenic Arabidopsis were generated to determine

D

whether AtACBP2 is involved in the drought response [97]. AtACBP2-overexpressing

TE

plants showed improved drought tolerance, whereas the AtACBP2-knockout mutant

CE P

was more sensitive to drought. AtACBP2 overexpression up-regulated the expression of genes encoding two NAD(P)H oxidases AtrbohD and AtrbohF that are essential for

AC

ABA-mediated ROS production, as well as HYPERSENSITIVE TO ABA1 (HAB1), an important negative regulator in ABA signaling [97, 169, 170]. It was proposed that the accumulation of reactive oxygen species (ROS) in the guard cells promoted stomatal closure to reduce water loss [97]. AtACBP3, AtACBP4 and AtACBP6 can contribute to drought tolerance by playing a role in cuticle formation [20]. When leaves of the acbp3, acbp4 and acbp6 mutants were examined by toluidine blue staining and transmission electron microscopy (TEM) they showed increase in permeability and an abnormal morphology at the cuticle layer was seen in comparison to the wild type, consistent with elevation in water loss following drought treatment

32

ACCEPTED MANUSCRIPT [20]. The acbp3 and acbp4 mutants showed higher leaf permeability than acbp6 in toluidine blue stains, which corresponded to significant water loss under drought

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conditions [20]. In addition, acbp3 and acbp4 displayed greater changes in cuticular

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wax and cutin monomer composition than acbp6, in comparison to the wild type, suggesting the importance of these AtACBPs in cuticular lipid metabolism and drought tolerance [20].

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Some AtACBPs possess ankyrin repeats (Class II AtACBP1 and AtACBP2) or

MA

kelch motifs (Class IV AtACBP4 and AtACBP5) that mediate protein-protein interactions [15, 171, 172]. AtACBPs that have been verified to interact with protein

D

partners via these domains include AtACBP2 which interacts with a group VII

TE

ethylene response factor (ERF) protein RAP2.3 [173], although AtACBP2 is localized

CE P

at the plasma membrane and ER while RAP2.3 is a transcription factor targeted to the nucleus. Their interaction observed at the plasma membrane [173] was supported by

AC

the interactions of AtACBP1 and AtACBP2 with RAP2.12, another member of Arabidopsis group VII ERFs in two independent plant-oxygen-sensing studies [174, 175]. Under aerobic conditions, RAP2.12 interacted with AtACBP1 and AtACBP2 at the plasma membrane to each form a membrane-bound complex that limited its access to the nucleus and protected it from degradation by the N-end rule pathway [174, 175]. Upon hypoxia, RAP2.12 dissociated from the plasma membrane and was translocated into the nucleus to activate the expression of hypoxia-responsive genes [174]. When this model was tested using a photoconvertible fluorescent protein, mEos, fused to RAP2.12, the results showed that RAP2.12::mEos was relocated from the plasma

33

ACCEPTED MANUSCRIPT membrane to the nucleus in response to low oxygen conditions, and de novo synthesis of RAP2.12::GFP induced by hypoxia activated the expression of hypoxia-responsive

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genes in the nucleus [176]. The current model supports the translocation of RAP2.12

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under hypoxia but details on how it traverses through the cytoplasm is yet unclear. A recent study has shown that AtACBP3 also participates in response to hypoxia in Arabidopsis, by its interaction with VLC acyl-CoA esters and modulation

NU

of comparable fatty acid/lipid metabolism such as unsaturated VLC ceramides [22].

MA

There was an increase in the degree of unsaturation of VLC ceramides that protects plants against hypoxia-induced damages by regulating ethylene signaling in

D

Arabidopsis [177]. In addition, hypoxia usually occurs with other environmental

TE

stresses such as darkness in flooding/submergence under the natural environment,

CE P

consistent with observations that AtACBP3 contributes to the dark-induced leaf senescence by modulating membrane phospholipids and the stability of an

AC

autophagy-related protein ATG8 [43]. Low temperature stress can cause substantial losses in agriculture and it restricts the geographic distribution of crops. Previous studies have shown that freezing-induced damage on the lamellar membrane systems such as the plasma membrane and chloroplast membranes causes primary injury in plants [178, 179], and strategies that can stabilize plant cell membranes during dehydration and rehydration by remodeling membrane lipid composition represents a viable solution to promote survival [180-182]. In Arabidopsis, AtACBP1 and AtACBP6 have been reported to be engaged in the modulation of membrane lipids (e.g. PC

and

PA)

in

response to

freezing stress

by interaction with

34

ACCEPTED MANUSCRIPT phospholipids/acyl-CoAs and by regulation of freezing-responsive genes such as AtPLD and AtPLD1 encoding two phospholipase D [41, 113]. A major natural

IP

T

cause in agricultural loss comes from cold snaps or frosts during spring ruining

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temperature-sensitive organs such as flowers [183, 184]. A recent study has shown that the overproduction of AtACBP6 in Arabidopsis dramatically improved freezing tolerance

in

flowers;

up

to

30%

more

intact

flowers

survived

in

NU

AtACBP6-overexpressors when compared to the wild type [185]. Subsequent

MA

experiments in lipid analysis suggested that freezing resistance in the flowers of AtACBP6-overexpressors differed from the vegetative tissues [113, 185]. Lipid

D

profiling revealed that AtACBP6-overexpressing flowers accumulated PC especially

TE

diunsaturated species that constitute the bilayer-forming lipids and are important in

CE P

the stabilization of membranes under freezing stress [179, 186], in contrast to a decrease in PC in the vegetative tissues [113]. Sugar and proline, important in the

AC

protection of plant cells under freezing stress [187], accumulated in the leaves but not in the flowers after 3 days of cold acclimation [185]. The differences between vegetative tissues and flowers in response to freezing stress were attributed to a variation in the regulation of lipid metabolism-related and cold tolerance-related genes [113, 185], which was not surprising because disparity in cold regulation amongst plant organs have previously been encountered [185]. This finding suggests that AtACBP6 is a promising candidate for the genetic engineering of cold tolerance.

5.3 Pathogen defense

35

ACCEPTED MANUSCRIPT AtACBPs have been reported to participate in plant defense against infections caused by pathogens, and AtACBP3 is exemplary in this regard [20, 188]. The

IP

T

overexpression of AtACBP3 enhanced resistance to the bacterial pathogen

SC R

Pseudomonas syringae pv tomato DC3000 [188], whereas the acbp3 mutant was more sensitive to this biotrophic pathogen than the wild type [20]. Intriguingly, AtACBP3-overexpressing lines showed greater susceptibility to infection by a

NU

necrotrophic pathogen Botrytis cinerea than the wild type and acbp3 mutant [188].

MA

The different responses in the AtACBP3-overexpressing lines against biotrophic and necrotrophic pathogens may be attributed to differences in plant-pathogen recognition

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as well as the plant-defense pathways mediated by AtACBP3. AtACBP3 was rapidly

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induced by arachidonic acid, an important fungal-derived lipophilic molecule with

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elicitor activity [189], and rAtACBP3 was found to bind to arachidonyl-CoA (20:4-CoA) [66]. It is not known how AtACBP3 interacts with bacterial

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pathogen-secreted lipids or the bacteria, although P. syringae DC3000 infection caused rapid degradation of the AtACBP3::GFP fusion protein [188]. In addition, a previous study has shown that AtACBP3 overexpression promoted the degradation of the autophagy protein ATG8, which belongs to a group of ATGs that are involved in plant innate immunity-associated programmed cell death [190, 191]. Some mutants of these ATGs such as atg5, atg7 and atg18a showed different susceptibilities to B. cinerea and P. syringae DC3000 [192, 193], similar to the AtACBP3-overexpressing lines. Furthermore, pathogen resistance mediated by AtACBP3 was related to the regulation of pathogenesis-related (PR) genes (AtPR1, AtPR2, and AtPR5) and the

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ACCEPTED MANUSCRIPT salicylic acid (SA) signaling pathway [188]. A recent study has shown defects in the cuticle layer of the acbp3 mutant which was impaired in its resistance against fungal

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(B. cinerea and Colletotrichum higginsianum) and bacterial (P. syringae DC3000 and

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avrRpt2) pathogens [20]. A similar phenotype was observed in acbp4 and acbp6 [20], as well as the acbp1 mutant which was subject only to B. cinerea [21]. Further investigations suggested that the knockout mutants of AtACBP3, AtACBP4 or

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AtACBP6 showed diminished capability in the generation of signals such as SA and

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SA glucoside for induction of systemic acquired resistance [20].

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5.4 Stress-inducible rice acyl-CoA-binding proteins

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In contrast to the extensive studies on AtACBPs from the eudicot model plant,

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Arabidopsis, the study on the stress responsiveness of OsACBPs from the monocot model, O. sativa, is at its infancy [74]. Current knowledge on OsACBPs in stress

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responses has focused on the responses of OsACBPs upon environmental stimuli such as drought and cold as analyzed by quantitative RT-PCR (Fig. 3) [74]. However, acyl-CoA-binding assays using ITC and Lipidex-1000 as well as nitrocellulose membrane-bound phospholipid-binding assays have revealed that rOsACBPs interact with various species of acyl-CoA esters and phospholipids (mainly PA and PC), suggesting that the basic roles of OsACBPs in lipid metabolism resemble that of the AtACBPs (Table 3) [23, 74]. With reference to the data on AtACBPs (Fig. 2) and OsACBPs (Fig. 3), the proposed biological functions of OsACBPs can be inferred from findings on the AtACBPs. Subsequently OsACBP functions can be verified

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ACCEPTED MANUSCRIPT using knockout/knockdown mutants and OsACBP-overexpressing transgenic rice plants. Given that stable transgenic rice is more difficult and time-consuming to

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obtain, the function of OsACBPs can be initially investigated in transgenic

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Arabidopsis. As a case in point the OsACBP6-overexpression in the Arabidopsis pxa1 mutant was found to restore the wound-induction of JA [23]. Furthermore, OsACBP::GFP-overexpressing Arabidopsis lines that were originally generated for

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studies on subcellular localization assays [23] are available to address OsACBP

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function before the rice lines are available. Genetic complementation analysis can be

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carried out by expressing OsACBPs in the background of Arabidopsis acbp mutants.

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6. Plant acyl-CoA-binding proteins in lipid metabolism

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Previous studies on AtACBPs in lipid metabolism were mainly focused on the polar glycerol lipids such as phospholipids and galactolipids, which have

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demonstrated that variations in the expression of AtACBPs (no data for AtACBP5 to date) lead to changes in the abundance and composition of polar glycerol lipids in the development and stress response (Table 2) [70]. However, some recent studies have shown that plant ACBPs are also involved in the biosynthesis of neutral lipids such as TAG in Arabidopsis seeds [32, 68, 82, 86]. In mammals, ACBPs have been shown to affect TAG accumulation [39, 144, 194]. In C. elegans, mutations in different CeACBPs (CeACBP1 to CeACBP3 and CeACBP5) perturbed lipid droplet formation [63]. For example, the C. elegans acbp1 mutant contained approximately 30% of lipid droplets when compared to the wild type and most of these droplets were significantly

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ACCEPTED MANUSCRIPT bigger, on average ~3.8 times the diameter of the wild type. Using Nile Red staining, a CeACBP1 RNAi mutant was found to display a reduction in storage lipids [195] that

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is consistent with observation of 25% decline in TAG content in the C. elegans acbp1

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mutant [63]. Although lipid droplets, typically TAG, are fundamental in lipid metabolism and form the major reservoir of storage lipids, their de novo biogenesis remains unclear [196, 197]. Currently, a model based on ER-derived lipid droplets

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suggests that there are two populations of basic lipid droplets: small lipid

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droplets/buds derived from the extracellular (outer) side of the ER which packs TAG generated by diacylglycerol acyltransferase DGAT1 that synthesizes TAG by the

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acylation of a diacylglycerol (DAG) and an acyl-CoA, and cytosolic lipid droplets

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which expand with TAG synthesized by DGAT2 [196, 197]. The sources of the

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acyl-CoAs and phospholipids need to be addressed because each lipid droplet consists of a monolayer organelle that relies on phospholipids such as PC to form the

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monolayer surface during lipid droplet expansion [196, 198]. As ACBPs are known to be associated with both acyl-CoAs and PCs, they are viable candidates for this role (Tables 2 and 3) and the putative function of ACBPs is supported by observations in irregular lipid droplets in the C. elegans acbp1 and acbp1acbp6acbp4acbp3 quadruple (four Class I ACBPs) mutants, as well as by the dramatic decrease in TAG in the C. elegans acbp1 mutant [63]. In plants, the overexpression of the 10-kDa B. napus ACBP affected fatty acid composition in Arabidopsis seeds [82, 86], wherein ~ 60% of the total dry weight are lipid (mainly TAGs) derived [1]. In Arabidopsis ACBPs, a double mutation in AtACBP1 and AtACBP2, the two ER

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ACCEPTED MANUSCRIPT membrane-localized AtACBPs, proved lethal during seed development [117]. Considering that rAtACBP1 and rAtACBP2 interacted with various acyl-CoAs and

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PCs (Table 2) [21, 42, 115], a plausible reason for the lethality of the double mutation

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may be attributed to failure in regular lipid droplet formation arising from the loss in acyl-CoA and PC donors at the ER membranes (Fig. 4). This hypothesis can be

acbp2 or acbp1 mutant, respectively.

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further tested using the AtACBP1 or AtACBP2 RNAi knockdown mutation in the

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The three cytosolic AtACBPs that also interacted with acyl-CoAs and PCs (Table 2) were reported to function in germination, pollen development, and seed

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development in investigations using double/triple mutants because the single mutants

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lacked phenotypic changes given their redundant roles [32, 122]. Thus these

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double/triple mutants can now be utilized further to examine their potential role in lipid droplet expansion in the cytosol (Fig. 4). Furthermore, it does not seem like an

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ordinary coincidence that all the AtACBPs are highly expressed in developing seeds [32, 42, 97, 117] with the exception of the apoplast-targeted AtACBP3 [43, 96]. Besides its significance in reservation of metabolic energy and lipids in seeds, lipid droplet (TAG) accumulation has been reported in plant vegetative tissues upon environmental stresses such as ozone fumigation, freezing and desiccation that all lead to the degradation of cellular membranes and remodeling of membrane lipids [180, 199-201]. Stress-induced membrane degradation will generate toxic compounds such as free fatty acids and phytols, which can be removed and utilized for TAG synthesis [202]. Thus, TAG profiling on the AtACBP-overexpressing transgenic lines

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ACCEPTED MANUSCRIPT may reveal whether their role in stress tolerance is by any way related to the TAG

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accumulation and lipid recycling.

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7. Conclusions and Perspectives

Ever since the identification of the very first ACBP in rat [24] and the first plant ACBP in oilseed rape [25], knowledge on the ACBP family has expanded

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rapidly, especially from investigations in Arabidopsis the past decade. Taking

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advantage of new technologies in lipid profiling, the function of AtACBPs in various aspects of the life cycle of the plant in development and during stress responses has

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become clearer and a model for future studies based on our results on the subcellular

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localization and temporal/spatial expression of AtACBPs (Fig. 2). This model should

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guide further exploration on the diverse function of highly conserved proteins such as AtACBP1 (trichomes and stem surface) and AtACBP2 (guard cells) in development

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and stress responses [21, 97, 168, 203]. Various T-DNA knockout and RNAi knockdown mutants of AtACBPs have been generated and characterized especially the double/triple mutants of cytosolic AtACBPs that led to recent findings on their contribution in reproductive tissues [32, 122]. The very basic functions of acyl-CoA/lipid-binding proteins in the interaction with various acyl-CoA esters and phospholipids and the modulation of the acyl-CoA and membrane lipid composition are being deciphered (Table 2). Findings on the AtACBPs also provide the foundation for research on other organisms such as rice which represents an important crop (Fig. 3; Table 3). Along with the information on AtACBPs, including the analysis of

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ACCEPTED MANUSCRIPT 5´-flanking regions of several AtACBPs (AtACBP1, AtACBP3 and AtACBP6) [32, 96, 168] and stress tolerance acquired in the overexpression or in knockout AtACBP lines

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(Fig. 2; Table 2), these findings can now be extended beyond Arabidopsis for

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engineering better crops.

Furthermore, the phylogenetic analyses on plant ACBPs have revealed a common existence of larger ACBPs besides the Class I 10-kDa ACBPs in plants and

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data on the evolution within them (Fig. 1) [74]. Besides the acyl-CoA-binding domain,

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many larger ACBPs also possess multiple functional domains, e.g. protein interactingankyrin repeats and kelch motifs [171, 172]. Protein-protein interactions are essential

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for numerous biological events. From studies on the interactors of AtACBPs such as

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RAP2.12, PLD1, AtFP6 and lysoPL2, novel roles of AtACBPs in plant development

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and stress response have emerged in the last decade [93]. However, this only marks the beginning of studies on the interactions between ACBPs and their putative

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partners. For example, a very recent study on Arabidopsis RAP2.12, RAP2.2 and RAP2.3 revealed their functions in anoxia, oxidative (H2O2), osmotic (mannitol) and ABA-mediated (root elongation) stress responses [204]. It is not surprising that the AtACBP interactors are involved in the same processes as the AtACBPs themselves, and these findings especially on RAP2.3 enlighten us on the importance of interactions between AtACBP2/AtACBP4 and RAP2.3 that had been quite a mystery previously because of their differences in their predicted subcellular localizations [92, 173]. In addition, the initial screens of AtACBP interactors have demonstrated that many putative protein partners are yet to be characterized [92, 115, 205]. Using the

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ACCEPTED MANUSCRIPT Arabidopsis acbp mutants in analysis, future studies on AtACBPs and their protein partners could eventually lead to a fuller understanding on the roles of AtACBPs in

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cell-to-cell and intercellular acyl-CoA/lipid trafficking, as well as in acyl-CoA/lipid

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biosynthesis and turnover in plant development and stress responses.

8. Acknowledgements

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This work was supported by the Research Grants Council of the Hong Kong Special

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Administrative Region, China (projects no. HKU765813M and HKU17105615M) and the Wilson and Amelia Wong Endowment Fund. We thank Ziwei Ye and Terry S.C.

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Lung for critical comments on the manuscript.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Fig. 1. Phylogeny analysis of acyl-CoA-binding proteins (ACBPs) using Maximum

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Likelihood methods. Phylogenies were inferred from a protein alignment with ACBP

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sequences obtained by BLAST searches using Arabidopsis ACBPs in PLAZA v. 2.5 and

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Phytozome v. 9.1, and 83 sequences were included in the final alignment using Homo sapiens ACBD5 as the outgroup. The program automatically determines the number of

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bootstrap runs necessary to reach completion. The gamma model of heterogeneity was

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used for protein sequences and the amino acid substitution model was BLOSUM 62. Methods for the identification, alignment construction and phylogenetic analysis of

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plant ACBPs are described in Supplemental Methods. Plant species and gene

TE

identifier or accession numbers of selected ACBPs are listed in Supplemental Table 2.

CE P

A neighbor-joining (Supplemental Fig. S1) tree was built by complete protein

AC

alignments (Supplemental Fig. S2) using H. sapiens ACBD5 as the outgroup.

Fig. 2. A model showing the temporal and spatial expression of Arabidopsis acyl-CoA-binding proteins at various stages of plant development and potential roles in environmental stress responses. Pb(II), lead; Cd(II), cadmium; H2O2, hydrogen peroxide; ABA, abscisic acid; PM, plasma membrane; ER, endoplasmic reticulum.

Fig. 3. A summary of recent findings on the subcellular localization of rice acyl-CoA-binding proteins and their temporal/spatial expression during development and stress. IBA, indole-3-butyric acid; ER, endoplasmic reticulum.

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Fig. 4. Hypothetical model of Arabidopsis acyl-CoA-binding proteins in lipid droplet

IP

T

formation via diacylglycerol acyltransferase (DGAT) at the endoplasmic reticulum (ER),

SC R

or in the cytosol. CW, cell wall; PM, plasma membrane; ER, endoplasmic reticulum;

AC

CE P

TE

D

MA

NU

LD, lipid droplet; PC, phosphatidylcholine; TAG, triacylglycerol; DAG, diacylglycerol.

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Vernicia fordii (tung tree)

Vitis vinifera (grape)

16:0, 18:1, 18:2, 18:3

GhACBP

10.0

-

RcACBP

10.1

-

ACBP9.9 ACBP10.0

9.9 10.0

16:0 16:0

ACBP-1

40

-

VfACBP3A

20.2

18:1, 20:4

VfACBP3B

26.2

VfACBP4

-

-

VfACBP6

10

-

VvACBP-1

31

20:4

-

IP

References

activity regulation of GPAT, LPCAT and LPAAT; regulation of Glc-6-P and ATP transport; acyl exchange between acyl pools; acyl-CoA transport

[14, 19, 56-61, 69, 70]

-

-

[51]

-

-

[52]

epidermis of mature zone of leaves ER membranes; seeds, flowers, leaves ER membranes; seeds, flowers, leaves seeds, flowers, leaves cytosol; seeds, flowers, leaves

-

[53]

putative function in cuticle formation

[62]

fatty acid metabolism

[55, 63]

fatty acid metabolism

[55, 63]

fatty acid metabolism

[55, 63]

fatty acid metabolism

[55, 63]

ER stress; cold and heat shock stresses; photoperiodic pathway-mediated floral transition; morphological development; pathogen resistance

[64]

CR

10.2

Function

seeds (embryo), flowers, cotyledons, leaves, roots

US

BnACBP

Expression

MA N

(castor bean) Digitalis lanata Ehrh. (Woolly Foxglove) Agave americana L. (maguey)

Acyl-CoA binding

TE D

Gossypium hirsutum (cotton) Ricinus communis

Size (kDa)

CE P

Brassica napus L. (oilseed rape)

Proteins

AC

Species

T

Table 1 Plant ACBPs (except Arabidopsis and rice ACBPs).

cell periphery; cytoskeleton

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AC

CE P

TE D

MA N

US

CR

IP

T

Abbreviations: LPAAT, lysophosphatidic acid acyltransferase; LPCAT, lysophosphatidylcholine acyltransferase; Glc-6-P, plastidial glucose 6-phosphate; GPAT, glycerol-3-phosphate acyl-transferase; ER, endoplasmic reticulum.

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References

AtACBP1

AtACBP2

+(3-87) 10.4 nM range Kd 14:0, 16:0, 18:0, 18:1, 18:2, 18:3

+(11-31) +(94-180) +(251-314) 37.5 M range Kd 18:1, 18:2, 18:3, 24:0, 25:0, 26:0

18:1, 18:2

-

PC

Class III

Class IV

AtACBP3

AtACBP4

AtACBP5

+(10-31) +(104-191) +(266-329) 38.5

+(1-26) +(7-26) +(231-311) 39.3

+(33-99) +(184-225) 73.2

+(31-101) +(184-225/295-340) 71

Unpublished

M range Kd 18:2, 20:0, 22:0, 24:0

M range Kd 18:0, 16:0, 14:0, 18:2, 18:1, 18:3

M range Kd 18:0, 16:0, 14:0, 18:2, 18:1, 18:3

-

16:0, 18:0, 18:3

-

-

PA, PC

PC, lysoPC

PC, PE

PC

PC

PA, PC, PS

PC, PA, DGDG, MGDG, PE, PG, PI, PS

freezing, pathogen, drought cuticle, pollen development, seed development and germination

AREB1, RAP2.12, PLD1 Pb2+, freezing, H2O2, hypoxia embryogenesis, seed dormancy, germination, seedling development stem cuticle formation

DGDG, MGDG, PG, PC, PE, PI RAP2.3, AtFP6, lysoPL2 Cd2+, H2O2, drought, hypoxia

DGDG, MGDG,PE, PG, PC, PI, PS, PA darkness, pathogen, drought, hypoxia

DGDG, MGDG, PE, PG, PC, PI RAP2.3 2+ Pb , pathogen, drought cuticle, pollen development, seed development and germination

[17, 19, 21, 91, 103, 139,

[72, 79, 92, 96, 136, 137, 147, 150,

TE D

MA N

US

CR

AtACBP6

IP

Class II

CE P

Signal peptide Transmembrane ACB domain Ankyrin repeats Kelch motifs Size (kDa) acyl-CoA binding (ITC or MST) acyl-CoA content Phospholipid binding Phospholipid content Interactors Stress responses Development

Class I

AC

Proteins

T

Table 2 Characterization of Arabidopsis ACBPs.

embryogenesis, seed dormancy, germination, seedling development

senescence, cuticle

[46,73, 93, 94, 96, 106, 155,

[44, 71, 89, 100, 139, 168]

[17, 21, 45, 66, 67, 103, 139,

pollen development, seed development and germination [17, 21, 45, 66, 103]

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165]

156, 183]

156, 183]

150]

IP

T

Abbreviations: ITC, isothermal titration calorimetry; MST, microscale thermophoresis; nM, nanomolar; Kd, dissociation constant; M, micromolar; dissociation

AC

CE P

TE D

MA N

US

CR

constant; PC, phosphatidylcholine; PA, phosphatidic acid; PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; AREB1, ABARESPONSIVE ELEMENT BINDING PROTEIN1; RAP, RELATED TO APETALA; PLDα1, phospholipase Dα1; AtFP6, Arabidopsis FARNESYLATED PROTEIN6; lysoPL2, LYSOPHOSPHOLIPASE2; Pb2+, lead; Cd2+, cadmium; H2O2, hydrogen peroxide.

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T

Table 3 Rice ACBPs.

+(3-87) 10.2

+(16-87) 10.3

+(3-87) 17.7

Acyl-CoA binding

16:0, 18:1, 18:2, 18:3

18:3

MA N TE D

PA (18:0/18:1) PC (18:0/18:1/18:2)

18:3

CE P

PSORT Transient expression

PA (18:0/18:1) PC (18:0/18:1/18:2)

Class III OsACBP5

Class IV OsACBP6

+(1-30) +(11-31) +(109-177) +(223-320) 36

+(1-30) +(7-31) +(436-504) 61.2

+(15-32,462-479) +(37-95) +(182-223,394-437) 71.4

16:0, 18:2, 18:3

16:0, 18:3

18:2, 18:3

PA (18:0/18:1) PC (18:0/18:1/18:2)

PA (16:0/18:0/18:1) PC (18:0/18:1/18:2)

PA (18:0/18:1) PC (18:0/18:1/18:2)

PA (18:0/18:1) PC (18:0/18:1/18:2)

cytoplasm

cytoplasm

cytoplasm

outside

ER

chloroplast stroma

cytosol

cytosol

cytosol

ER

ER

putative organelle

ER

ER

peroxisomes

[49, 65, 104, 105]

[49, 65, 104, 105]

[49, 104, 105]

Stable expression

cytosol

References

[49, 54, 104, 105]

AC

Phospholipid binding

Class II OsACBP4

IP

Signal peptide Transmembrane ACB domain Ankyrin repeats Kelch motifs Size (kDa)

CR

OsACBP3

US

OsACBP1

Class I OsACBP2

Proteins

cytosol

[49, 104, 105]

cytosol, irregular membranous structures and punctates [49, 104, 105]

Abbreviations: PA, phosphatidic acid; PC, phosphatidylcholine; ER, endoplasmic reticulum.

69

AC

CE P

TE D

MA N

US

CR

IP

T

ACCEPTED MANUSCRIPT

Fig. 1

70

AC

CE P

TE D

MA N

US

CR

IP

T

ACCEPTED MANUSCRIPT

Fig. 2

71

AC

CE P

TE D

MA N

US

CR

IP

T

ACCEPTED MANUSCRIPT

Fig. 3

72

AC

CE P

TE D

MA N

US

CR

IP

T

ACCEPTED MANUSCRIPT

Fig. 4

73