Characterization of Oil Palm Acyl-CoA-Binding Proteins and Correlation of Their Gene Expression with Oil Synthesis.

Acyl-CoA-binding proteins (ACBPs) are involved in binding and trafficking acyl-CoA esters in eukaryotic cells. ACBPs contain a well-conserved acyl-CoA-binding domain. Their various functions have been characterized in the model plant Arabidopsis and, to a lesser extent, in rice. In this study, genome-wide detection and expression analysis of ACBPs were performed on Elaeis guineensis (oil palm), the most important oil crop in the world. Seven E. guineensis ACBPs were identified and classified into four groups according to their deduced amino acid domain organization. Phylogenetic analysis showed conservation of this family with other higher plants. All seven EgACBPs were expressed in most tissues while their differential expression suggests various functions in specific tissues. For example, EgACBP3 had high expression in inflorescences and stalks while EgACBP1 showed strong expression in leaves. Because of the importance of E. guineensis as an oil crop, expression of EgACBPs was specifically examined during fruit development. EgACBP3 showed high expression throughout mesocarp development, while EgACBP1 had enhanced expression during rapid oil synthesis. In endosperm, both EgACBP1 and EgACBP3 exhibited increased expression during seed development. These results provide important information for further investigations on the biological functions of EgACBPs in various tissues and, in particular, their roles in oil synthesis.

The overexpression of rice ACYL-CoA-BINDING PROTEIN2 increases grain size and bran oil content in transgenic rice.

As Oryza sativa (rice) seeds represent food for over three billion people worldwide, the identification of genes that enhance grain size and composition is much desired. Past reports have indicated that Arabidopsis thaliana acyl-CoA-binding proteins (ACBPs) are important in seed development but did not affect seed size. Herein, rice OsACBP2 was demonstrated not only to play a role in seed development and germination, but also to influence grain size. OsACBP2 mRNA accumulated in embryos and endosperm of germinating seeds in qRT-PCR analysis, while ß-glucuronidase (GUS) assays on OsACBP2pro::GUS rice transformants showed GUS expression in embryos, as well as the scutellum and aleurone layer of germinating seeds. Deletion analysis of the OsACBP2 5'-flanking region revealed five copies of the seed cis-element, Skn-I-like motif (-1486/-1482, -956/-952, -939/-935, -826/-822, and -766/-762), and the removal of any adversely affected expression in seeds, thereby providing a molecular basis for OsACBP2 expression in seeds. When OsACBP2 function was investigated using osacbp2 mutants and transgenic rice overexpressing OsACBP2 (OsACBP2-OE), osacbp2 was retarded in germination, while OsACBP2-OEs performed better than the wild-type and vector-transformed controls, in germination, seedling growth, grain size and grain weight. Transmission electron microscopy of OsACBP2-OE mature seeds revealed an accumulation of oil bodies in the scutellum cells, while confocal laser scanning microscopy indicated oil accumulation in OsACBP2-OE aleurone tissues. Correspondingly, OsACBP2-OE seeds showed gain in triacylglycerols and long-chain fatty acids over the vector-transformed control. As dietary rice bran contains beneficial bioactive components, OsACBP2 appears to be a promising candidate for enriching seed nutritional value.

Kelch-motif containing acyl-CoA binding proteins AtACBP4 and AtACBP5 are differentially expressed and function in floral lipid metabolism.

KEY MESSAGE: We herein demonstrated two of the Arabidopsis acyl-CoA-binding proteins (ACBPs), AtACBP4 and AtACBP5, both function in floral lipid metabolism and they may possibly play complementary roles in Arabidopsis microspore-to-pollen development. Histological analysis on transgenic Arabidopsis expressing ß-glucuronidase driven from the AtACBP4 and AtACBP5 promoters, as well as, qRTPCR analysis revealed that AtACBP4 was expressed at stages 11-14 in the mature pollen, while AtACBP5 was expressed at stages 7-10 in the microspores and tapetal cells. Immunoelectron microscopy using AtACBP4- or AtACBP5-specific antibodies further showed that AtACBP4 and AtACBP5 were localized in the cytoplasm. Chemical analysis of bud wax and cutin using gas chromatographyflame ionization detector and GC-mass spectrometry analyses revealed the accumulation of cuticular waxes and cutin monomers in acbp4, acbp5 and acbp4acbp5 buds in comparison to the wild type (Col-0). Fatty acid profiling demonstrated a decline in stearic acid and an increase in linolenic acid in acbp4 and acbp4acbp5 buds, respectively, over Col-0. Analysis of inflorescences from acbp4 and acbp5 revealed that there was an increase of AtACBP5 expression in acbp4, and an increase of AtACBP4 expression in acbp5. Deletion analysis of the AtACBP4 and AtACBP5 5'-flanking regions indicated the minimal promoter activity for AtACBP4 (-145/+103) and AtACBP5 (-181/+81). Electrophoretic mobility shift assays identified a pollen-specific cis-acting element POLLEN1 (AGAAA) mapped at AtACBP4 (-157/-153) which interacted with nuclear proteins from flower and this was substantiated by DNase I footprinting. In Arabidopsis thaliana, six acyl-CoA-binding proteins (ACBPs), designated as AtACBP1 to AtACBP6, have been identified to function in plant stress and development. AtACBP4 and AtACBP5 represent the two largest proteins in the AtACBP family. Despite having kelch-motifs and sharing a common cytosolic subcellular localization, AtACBP4 and AtACBP5 differ in spatial and temporal expression. Histological analysis on transgenic Arabidopsis expressing ß-glucuronidase driven from the respective AtACBP4 and AtACBP5 promoters, as well as, qRT-PCR analysis revealed that AtACBP4 was expressed at stages 11-14 in mature pollen, while AtACBP5 was expressed at stages 7-10 in the microspores and tapetal cells. Immunoelectron microscopy using AtACBP4- or AtACBP5-specific antibodies further showed that AtACBP4 and AtACBP5 were localized in the cytoplasm. Chemical analysis of bud wax and cutin using gas chromatography-flame ionization detector and GC-mass spectrometry analyses revealed the accumulation of cuticular waxes and cutin monomers in acbp4, acbp5 and acbp4acbp5 buds, in comparison to the wild type. Analysis of inflorescences from acbp4 and acbp5 revealed that there was an increase of AtACBP5 expression in acbp4, and an increase of AtACBP4 expression in acbp5. Deletion analysis of the AtACBP4 and AtACBP5 5'-flanking regions indicated the minimal promoter region for AtACBP4 (-145/+103) and AtACBP5 (-181/+81). Electrophoretic mobility shift assays identified a pollen-specific cis-acting element POLLEN1 (AGAAA) within AtACBP4 (-157/-153) which interacted with nuclear proteins from flower and this was substantiated by DNase I footprinting. These results suggest that AtACBP4 and AtACBP5 both function in floral lipidic metabolism and they may play complementary roles in Arabidopsis microspore-to-pollen development.

Acyl-CoA-Binding Proteins (ACBPs) in Plant Development.

Acyl-CoA-binding proteins (ACBPs) play a pivotal role in fatty acid metabolism because they can transport medium- and long-chain acyl-CoA esters. In eukaryotic cells, ACBPs are involved in intracellular trafficking of acyl-CoA esters and formation of a cytosolic acyl-CoA pool. In addition to these ubiquitous functions, more specific non-redundant roles of plant ACBP subclasses are implicated by the existence of multigene families with variable molecular masses, ligand specificities, functional domains (e.g. protein-protein interaction domains), subcellular locations and gene expression patterns. In this chapter, recent progress in the characterization of ACBPs from the model dicot plant, Arabidopsis thaliana, and the model monocot, Oryza sativa, and their emerging roles in plant growth and development are discussed. The functional significance of respective members of the plant ACBP families in various developmental and physiological processes such as seed development and germination, stem cuticle formation, pollen development, leaf senescence, peroxisomal fatty acid ß-oxidation and phloem-mediated lipid transport is highlighted.

The Arabidopsis cytosolic Acyl-CoA-binding proteins play combinatory roles in pollen development.

In Arabidopsis, six acyl-CoA-binding proteins (ACBPs) have been identified and they have been demonstrated to function in plant stress responses and development. Three of these AtACBPs (AtACBP4-AtACBP6) are cytosolic proteins and all are expressed in floral organs as well as in other tissues. The roles of cytosolic AtACBPs in floral development were addressed in this study. To this end, a T-DNA insertional knockout mutant of acbp5 was characterized before use in crosses with the already available acbp4 and acbp6 T-DNA knockout mutants to examine their independent and combinatory functions in floral development. The single-gene knockout mutations did not cause any significant phenotypic changes, while phenotypic deficiencies affecting siliques and pollen were observed in the double mutants (acbp4acbp6 and acbp5acbp6) and the acbp4acbp5acbp6 triple mutant. Vacuole accumulation in the acbp4acbp6, acbp5acbp6 and acbp4acbp5acbp6 pollen was the most severe abnormality occurring in the double and triple mutants. Furthermore, scanning electron microscopy and transmission electron microscopy revealed exine and oil body defects in the acbp4acbp5acbp6 mutant, which also displayed reduced ability in in vitro pollen germination. Transgenic Arabidopsis expressing ß-glucuronidase (GUS) driven from the various AtACBP promoters indicated that AtACBP6pro::GUS expression overlapped with AtACBP4pro::GUS expression in pollen grains and with AtACBP5pro::GUS expression in the microspores and tapetal cells. Taken together, these results suggest that the three cytosolic AtACBPs play combinatory roles in acyl-lipid metabolism during pollen development.

An Arabidopsis family of six acyl-CoA-binding proteins has three cytosolic members.

In Arabidopsis thaliana, a gene family of six members encodes acyl-CoA-binding proteins (ACBPs). These Arabidopsis ACBPs (designated ACBP1 to ACBP6) range in size from 10.4kDa to 73.1kDa and display varying affinities for acyl-CoA esters, suggesting that they have different roles in plant lipid metabolism. In contrast, only the 10-kDa ACBPs have been well-characterized from other eukaryote species. Our previous studies have revealed that ACBP1 and ACBP2 are membrane-associated proteins, while ACBP3 is extracellularly-targeted. More recently, we have reported that the remaining three members in this protein family (namely ACBP4, ACBP5 and ACBP6) are subcellularly localized to the cytosol in Arabidopsis. The subcellular localizations of ACBP4, ACBP5 and ACBP6 in the cytosol were demonstrated using a number of different approaches incorporating biochemical fractionation, confocal microscopy of transgenic Arabidopsis expressing autofluorescence-tagged fusions and immunoelectron microscopy using ACBP-specific antibodies. Our results indicate that all three ACBPs in the cytosol are potential candidates for acyl-CoA binding and trafficking in plant cells. In this review, the functional redundancy and differences among the three cytosolic ACBPs are discussed by comparison of their light-regulated expression and substrate affinities to acyl-CoA esters, and from biochemical analyses on their knockout mutants and/or overexpression in transgenic Arabidopsis. The transcriptionally light-induced ACBP4 and ACBP5, which encode the two largest forms of Arabidopsis ACBPs, bind oleoyl-CoA esters and likely transfer oleoyl-CoAs from the plastids (the site of de novo fatty acid biosynthesis) to the endoplasmic reticulum for the biosynthesis of non-plastidial membrane lipids in Arabidopsis.

Downregulation of Solanum americanum genes encoding proteinase inhibitor II causes defective seed development.

Proteinase inhibitor II proteins (PIN2) are serine proteinase inhibitors found in the Solanaceae. Here, we assign functions in seed development to two Solanum americanum genes, SaPIN2a and SaPIN2b, encoding proteinase inhibitor II. Their mRNAs and proteins have been previously localized to the reproductive tissues, including the inner cell layers of ovules in senescent flowers at the beginning of fruit development, suggestive of their endogenous roles in reproductive development. We have employed RNA interference (RNAi)-induced post-transcriptional gene silencing (PTGS) to further investigate the role of SaPIN2a and SaPIN2b during seed development. A SaPIN2a-derived construct that shared 83% nucleotide homology to SaPIN2b was used in PTGS to silence both genes. Northern blot analyses confirmed that the PIN2-RNAi transgenic plants contain small interfering RNAs (siRNAs) and exhibit reduced levels of SaPIN2a and SaPIN2b mRNAs at various stages of floral development. A reduction in seed set due to seed abortion was observed in PIN2-RNAi transgenic lines. Cytological and molecular analyses of these lines showed the lack of SaPIN2a and SaPIN2b mRNAs and proteins at the inner cell layers of the ovules in senescent flowers. Aborted seeds in transgenic fruits had an abnormal endothelium. The anomalous expansion of the endothelium prevented proper development of the endosperm and embryo, leading to seed abortion. Our observations indicate that SaPIN2a and SaPIN2b are essential for seed development and suggest that the endothelium may protect the embryo sac, allowing proper formation of the endosperm and embryo, as a result of its ability to produce proteinase inhibitors.

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

Cytosolic 10-kDa acyl-CoA-binding proteins (ACBPs) function in the storage and intracellular transport of acyl-CoA esters in eukaryotes. Fatty acids synthesized de novo in plant chloroplasts are exported as oleoyl-CoA and palmitoyl-CoA esters. In Arabidopsis, other than the 10-kDa ACBP, there exists five larger ACBPs (ACBP1 to ACBP5) of which homologues have not been characterized in other organisms. To investigate the significance of this gene family, we have attempted to subcellularly localize them and compare their acyl-CoA-binding affinities. We have previously shown that Arabidopsis ACBP1 and ACBP2 are membrane-associated proteins while ACBP4 and ACBP5 contain kelch motifs. Here, to localize ACBP3, we have expressed ACBP3-red fluorescent protein (DsRed2) from the CaMV 35S promoter. ACBP3-DsRed was localized extracellularly in transiently expressed tobacco BY-2 cells and onion epidermal cells. The function of the acyl-CoA-binding domain in ACBP3 was investigated by in vitro binding assays using (His)(6)-ACBP3, which was observed to bind [(14)C]arachidonyl-CoA with high affinity in comparison to [(14)C]palmitoyl-CoA and [(14)C]oleoyl-CoA. To identify the residues functional in binding, five mutants with single amino acid substitutions in the acyl-CoA-binding domain of (His)(6)-ACBP3 and (His)(6)-ACBP1 (which also binds [(14)C]arachidonyl-CoA) were generated by site-directed mutagenesis. Binding assays with arachidonyl-CoA revealed that replacement of a conserved R residue (R150A in ACBP1 and R284A in ACBP3), disrupted binding. In contrast, other substitutions in ACBP1 (Y126A, K130A, K152A and Y171A) and in ACBP3 (F260A, K264A, K286A and Y305A) did not affect arachidonyl-CoA binding, unlike their equivalents in (His)(6)-ACBP2, (His)(6)-ACBP4 and (His)(6)-ACBP5, which had altered binding to palmitoyl-CoA or oleoyl-CoA.

Brassica juncea HMG-CoA synthase: localization of mRNA and protein.

3-Hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) synthase (HMGS; EC 2.3.3.10) synthesizes HMG-CoA, a substrate for mevalonate biosynthesis in the isoprenoid pathway. It catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA and HS-CoA. In Brassica juncea (Indian mustard), HMGS is encoded by four isogenes (BjHMGS1-BjHMGS4). We have already enzymatically characterized recombinant BjHMGS1 expressed in Escherichia coli, and have identified its residues that are significant in catalysis. To further study HMGS mRNA expression that is developmentally regulated in flowers and seedlings, we have examined its mRNA distribution by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). We observed predominant localization of HMGS mRNA in the stigmas and ovules of flower buds and in the piths of seedling hypocotyls. RT-PCR analysis revealed that BjHMGS1 and BjHMGS2 but not BjHMGS3 and BjHMGS4were expressed in floral buds. To investigate the subcellular localization of BjHMGS1, we fused BjHMGS1 translationally in-frame either to the N- or C-terminus of green fluorescent protein (GFP). BjHMGS1-GFP and GFP-BjHMGS1 fusions were used in particle gun bombardment of onion epidermal cells and tobacco BY-2 cells. The GFP-BjHMGS1 construct was also used in agroinfiltration of tobacco leaves. Both GFP-fusion proteins were observed transiently expressed in the cytosol on confocal microscopy of onion epidermal cells, tobacco BY-2 cells, and agroinfiltrated tobacco leaves. Further, subcellular fractionation of total proteins from transgenic plants expressing GFP-BjHMGS1 derived from Agrobacterium-mediated transformation confirmed that BjHMGS1 is a cytosolic enzyme. We suggest that the presence of BjHMGS isoforms is likely related to the specialization of each in different cellular and metabolic processes rather than to a different intracellular compartmentation of the enzyme.

An agglutinating chitinase with two chitin-binding domains confers fungal protection in transgenic potato.

Brassica juncea BjCHI1 is a unique chitinase with two chitin-binding domains. Here, we show that, unlike other chitinases, potato-expressed BjCHI1 shows hemagglutination ability. BjCHI1 expression in B. juncea seedlings is induced by Rhizoctonia solani infection, suggesting its protective role against this fungus. To verify this, transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing BjCHI1 generated by Agrobacterium-mediated transformation were challenged with R. solani. We also transformed potato with a cDNA encoding Hevea brasiliensis beta-1,3-glucanase, designated HbGLU, and a pBI121-derivative that contains cDNAs encoding both BjCHI1 and HbGLU. In vitro fungal bioassays using Trichoderma viride showed that extracts from transgenic potato lines co-expressing BjCHI1 and HbGLU inhibited fungal growth better than extracts from transgenic potato expressing either BjCHI1 or HbGLU, suggesting a synergistic effect. Consistently, in vivo fungal bioassays with soil-borne R. solani on young transgenic potato plants indicated that the co-expressing plants showed healthier root development than untransformed plants or those that expressed either BjCHI1 or HbGLU. Light microscopy and transmission electron microscopy revealed abundant intact R. solani hyphae and monilioid cells in untransformed roots and disintegrated fungus in the BjCHI1-expressing and the BjCHI1 and HbGLU co-expressing plants. Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.

Expression of proteinase inhibitor II proteins during floral development in Solanum americanum.

The heterologous expression of serine proteinase inhibitor II (PIN2) proteins confers insect resistance in transgenic plants, but little is known of their endogenous roles. We have cloned two cDNAs encoding Solanum americanum PIN2 proteins, SaPIN2a and SaPIN2b. SaPIN2a is highly expressed in stem, particularly in the phloem, suggesting it could possibly regulate proteolysis in the sieve elements. When SaPIN2a was expressed in transgenic lettuce, we observed an inhibition of endogenous trypsin- and chymotrypsin-like activities. Here, we demonstrate that both SaPIN2a and SaPIN2b are expressed in floral tissues that are destined to undergo developmental programmed cell death (PCD), suggesting possible endogenous roles in inhibiting trypsin- and chymotrypsin-like activities during flower development. Northern and western blot analyses revealed that SaPIN2a and SaPIN2b mRNAs and proteins show highest expression early in floral development. In situ hybridization analysis and immunolocalization on floral sections, localized SaPIN2a and SaPIN2b mRNAs and their proteins to tissues that would apparently undergo PCD: the ovules, the stylar transmitting tissue, the stigma and the vascular bundles. Detection of PCD in floral sections was achieved using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. Examination of the mid-style before, and 1 day after, pollination revealed that high expression of SaPIN2a and SaPIN2b in the style was inversely correlated with PCD.

Membrane localization of Arabidopsis acyl-CoA binding protein ACBP2.

Cytosolic acyl-CoA binding proteins bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and pool formers. Recently, we have characterized Arabidopsis thaliana cDNAs encoding novel forms of ACBP, designated ACBP1 and ACBP2, that contain a hydrophobic domain at the N-terminus and show conservation at the acyl-CoA binding domain to cytosolic ACBPs. We have previously demonstrated that ACBP1 is membrane-associated in Arabidopsis. Here, western blot analysis of anti-ACBP2 antibodies on A. thaliana protein showed that ACBP2 is located in the microsome-containing membrane fraction and in the subcellular fraction containing large particles (mitochondria, chloroplasts and peroxisomes), resembling the subcellular localization of ACBP1. To further investigate the subcellular localization of ACBP2, we fused ACBP2 translationally in-frame to GFP. By means of particle gene bombardment, ACBP2-GFP and ACBP1-GFP fusion proteins were observed transiently expressed at the plasma membrane and at the endoplasmic reticulum in onion epidermal cells. GFP fusions with deletion derivatives of ACBPI or ACBP2 lacking the transmembrane domain were impaired in membrane targeting. Our investigations also showed that when the transmembrane domain of ACBP1 or that of ACBP2 was fused with GFP, the fusion protein was targeted to the plasma membrane, thereby establishing their role in membrane targeting. The localization of ACBP1-GFP is consistent with our previous observations using immunoelectron microscopy whereby ACBPI was localized to the plasma membrane and vesicles. We conclude that ACBP2, like ACBP1, is a membrane protein that likely functions in membrane-associated acyl-CoA transfer/metabolism.

G-box binding coincides with increased Solanum melongena cysteine proteinase expression in senescent fruits and circadian-regulated leaves.

We have previously shown that SmCP, the gene encoding Solanum melongena cysteine proteinase, is expressed during developmental events associated with programmed cell death (PCD) suggesting its involvement in protein degradation during these events (Xu and Chye, Plant Journal 17 (1999) 321-327). Here, we investigated the regulation of SmCP expression and showed that it is ethylene-inducible and is under circadian control. This circadian rhythm is entrained by light/dark (LD) cycling with peak expression in the late light period, as opposed to that in early light for rbcS, suggesting that protein degradation and photosynthesis are temporally separated by circadian control. Northern blot analysis shows that the pattern of ethylene induction of SmCP is consistent with our previous observation of its significantly increased expression at leaf senescence and fruit ripening when endogenous ethylene is abundant. To further understand SmCP regulation, we have cloned the SmCP promoter and identified a G-box (CACGTG) at -85/-80 by DNase I footprinting analysis of the -221/+17 region. Its specific interaction with nuclear proteins in S. melongena leaves and fruits was confirmed by competitive electrophoretic mobility shift assays using oligonucleotides containing the G-box and mutant derivatives. G-box binding activity was stronger in senescent than young fruits. In circadian-regulated leaves, stronger binding activity coincided with peak circadian expression of SmCP. This correlation between binding activity and expression suggests that G-box binding factors enhance SmCP transcription and that the G-box likely plays a role in circadian regulation of genes affected by LD cycling.