Genetic Architecture of Palm Oil Fatty Acid Composition in Cultivated Oil Palm (Elaeis guineensis Jacq.) Compared to Its Wild Relative E. oleifera (H.B.K) Cortés

We searched for quantitative trait loci (QTL) associated with the palm oil fatty acid composition of mature fruits of the oil palm E. guineensis Jacq. in comparison with its wild relative E. oleifera (H.B.K) Cortés. The oil palm cross LM2T x DA10D between two heterozygous parents was considered in our experiment as an intraspecific representative of E. guineensis. Its QTLs were compared to QTLs published for the same traits in an interspecific Elaeis pseudo-backcross used as an indirect representative of E. oleifera. Few correlations were found in E. guineensis between pulp fatty acid proportions and yield traits, allowing for the rather independent selection of both types of traits. Sixteen QTLs affecting palm oil fatty acid proportions and iodine value were identified in oil palm. The phenotypic variation explained by the detected QTLs was low to medium in E. guineensis, ranging between 10% and 36%. The explained cumulative variation was 29% for palmitic acid C16:0 (one QTL), 68% for stearic acid C18:0 (two QTLs), 50% for oleic acid C18:1 (three QTLs), 25% for linoleic acid C18:2 (one QTL), and 40% (two QTLs) for the iodine value. Good marker co-linearity was observed between the intraspecific and interspecific Simple Sequence Repeat (SSR) linkage maps. Specific QTL regions for several traits were found in each mapping population. Our comparative QTL results in both E. guineensis and interspecific materials strongly suggest that, apart from two common QTL zones, there are two specific QTL regions with major effects, which might be one in E. guineensis, the other in E. oleifera, which are independent of each other and harbor QTLs for several traits, indicating either pleiotropic effects or linkage. Using QTL maps connected by highly transferable SSR markers, our study established a good basis to decipher in the future such hypothesis at the Elaeis genus level.     

Oil palm (Elaeis guineensis) protoplasts: isolation, culture and microcallus formation

Procedures are deseribed for the efficient isolation of protoplasts from a variety of oil palm (Elaeis guineensis Jacq.) tissues. Various factors including donor source, composition of enzyme mixture and culture medium affected the yield and viability of the protoplasts Polyembryogenic cultures of oil palm were the most suitable starting material in terms of yield, viability and metabolic competence. Pectolyase Y-23 in association with cellulase and hemicellulase was required for the efficient release of protoplasts from the oil palm tissues. Limited cell division to form microcallus was observed at very low frequency (<0.01%) when glutathione and catalase were incorporated in the culture medium.Abbreviations 2,4-d dichlorophenoxyacetic acid - DTT dithiothreitol - MES 2[N-morpholino] ethanesulphonic acid - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone     

Changes in fatty acid composition of the lipid classes in developing oil palm mesocarp

During fruit development of oil palm (Elaeis guineensis) oil deposition in the mesocarp startedca12–13 weeks after flowering (WAF) and continued until the fruit ripened at 20 WAF. Over the next 1–2 weeks oil continued to be deposited but the fruit became loose and readily detached from the bunch. The lipids extracted at this stage contained over 50 % free fatty acids andca6%, polar lipids. The major fatty acids in the storage triacylglycerols were 16:0,18:1 and 18:2. The fatty acid composition of the neutral lipid classes and polar lipids during oil deposition were similar except that the latter also contained a high proportion of 18:3. Longer chain acids (20:3 and 22:0) were detected in certain lipid classes at 8 and 12 WAF.     

Quantitative trait loci (QTLs) analysis of palm oil fatty acid composition in an interspecific pseudo-backcross from Elaeis oleifera (H.B.K.) Cortés and oil palm (Elaeis guineensis Jacq.)

We chose an Elaeis interspecific pseudo-backcross of first generation (E. oleifera × E. guineensis) × E. guineensis to identify quantitative trait loci (QTLs) for fatty acid composition of palm oil. A dense microsatellite linkage map of 362 loci spanned 1.485 cM, representing the 16 pairs of homologous chromosomes in the Elaeis genus from which we traced segregating alleles from both E. oleifera and E. guineensis grandparents. The relative linear orders of mapped loci suggested the probable absence of chromosome rearrangements between the E. oleifera and E. guineensis genomes. A total of 19 QTL associated to the palm oil fatty acid composition were evidenced. The QTL positions and the species origin as well as the estimated effects of the QTL marker alleles were in coherence with the knowledge of the oil biosynthesis pathway in plants and with the individual phenotypic correlations between the traits. The mapping of chosen Elaeis key genes related to oleic acid C18:1, using intra-gene SNPs, supported several QTLs underlying notably FATA and SAD enzymes. The high number of hyper-variable SSR loci of known relative linear orders and the QTL information make these resources valuable for such mapping study in other Elaeis breeding materials.     

Purification and characterisation of acyl-CoA: glycerol 3-phosphate acyltransferase from oil palm (Elaeis guineensis) tissues

Glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.15) catalyses the first step of the Kennedy pathway for acyl lipid formation. This enzyme was studied using high-speed particulate fractions from oil palm (Elaeis guineensis Jacq.) tissue cultures and mesocarp acetone powders. The fractions were incubated with [¹⁴C]glycerol 3-phosphate and incorporation of radioactivity into Kennedy pathway intermediates studied. Optimal conditions were broadly similar between the two preparations but those from fruit mesocarp clearly contained more active enzymes for the subsequent stages of the Kennedy pathway – as exemplified by the appreciable accumulation of radioactivity in triacylglycerol. Experiments with different acyl-CoA substrates showed that the GPAT in both high-speed particulate preparations had a significant preference for palmitate. Glycerol 3-phosphate acyltransferase was solubilised from both preparations with optimal solubilisation being achieved at 0.5% (w/v) CHAPS concentrations. Solubilised GPATs were purified further using DE52 ion-exchange chromatography and Sephadex G-100 molecular exclusion chromatography. Purifications of up to about 70-fold were achieved. The purified GPATs showed a strong preference for palmitoyl-CoA compared to other acyl-CoA donors, in keeping with the importance of palmitate in palm oil. Received: 22 April 1999 / Accepted: 29 July 1999     

The Metabolism of the Germinating Oil Palm (Elaeis guineensis) Seedling : In Vivo Studies

The metabolism of ¹⁴C-labeled fatty acids and triacylglycerols was followed in intact germinating oil palm seedlings as well as in tissue slices. In the germinating seedling, the shoot contained a normal pattern of membrane fatty acids (mainly C₁₆, C_18:1, C_18:2) but the kernel contained about 68% C₁₂ and C₁₄ fatty acids. Haustorium fatty acids were intermediate between the two. [¹⁴C]Acetate was actively metabolized by shoot and haustorium slices but not so actively by the kernel. Approximately 9% to 17% was converted to water-soluble substances, 4% to 6% to CO₂, and 0.5% to 5.9% to lipids. The fatty acids synthesized in the shoot and haustorium were mainly C₁₆, C₁₈, and C_18:1 fatty acids but in the kernel about 18% to 32% of the ¹⁴C-fatty acids were C₁₂ fatty acids.

[¹⁴C]Lauric acid was absorbed and metabolized by haustorium slices and by the haustorium in intact seedlings; it was partly esterified to triacylglycerols and also converted to water-soluble substances and insoluble tissue material. In contrast, tri-[¹⁴C]laurin was absorbed but not metabolized. The haustorium also absorbed other fatty acids but the longer chain (C₁₆ and C₁₈) fatty acids were not esterified or metabolized further. Preincubation of the haustorium with plant hormones or in the presence of kernel tissue did not alter its inactivity towards tri-[¹⁴C]laurin.

When tri-[¹⁴C]laurin or [¹⁴C]lauric acid were injected into the seed or the shoot, there was no movement or radioactivity to other parts of the seedling. When injected into the shoot, but not into the seed, tri-[¹⁴C] laurin was hydrolyzed and partly metabolized to water-soluble substances.

     

Assessment of nuclear,mitochondrial and chloroplast RFLP markers in oil palm (Elaeis guineensis Jacq.)

A variety of DNA probes was used to screen a diverse set of oil palm accessions in order to identify markers with a utility in genotype discrimination. This survey included samples of the commercial oil palm native to Africa (Elaeis guineensis Jacq.), the closely-related South American species [E.oleifera (HBK) Cortes] and inter-specific hybrids of the two. Of 106 major chloroplast bands none showed differences between E. guineensis and E. Oleifera. Mitochondrial and ribosomal probes were more informative inter-specifically (the former allowing identification of the maternal inheritance of mitochondria) and may be useful in hybrid breeding programmes; however, they were unable to identify polymorphism within E. guineensis. In contrast, low-copy nuclear genomic clones were able to identify intra-specific variation, though in most cases they revealed a relatively small number of allelic variants. One DNA probe showed a much larger number of band variants, revealing ten patterns amongst 13 E. guineensis accessions, and should prove useful in genetic fingerprinting and evaluation of oil-palm germplasm collections.     

Lipid Composition and the Role of the Haustorium in the Young Seedling of the West African Oil Palm, Elaeis guineensis Jacq.

The lipid and fatty acid composition of the haustorium of thedeveloping seedling of the West African oil palm, Elaeis guineensisJacq. has been studied using the combined techniques of thin-layerand gas-liquid chromatography. In addition to triglycerides,which represented over 75 per cent of the total lipids, therewere present small quantities of free fatty acids, diglyceridesand polar lipids. The two glycolipids, monogalactosyl and digalactosyldiglycerides, formed the bulk of the polar lipids with smallamounts of phosphatidyl choline and phosphatidyl inositol. In general, the fatty acid pattern of the haustorium was intermediatebetween that of the palm kernel oil and the palm fruit mesocarp,and resembled to a great extent the fatty acids of the kerneltesta. It is suggested, from the presence of the biological membranelipids and lipolytic enzymes, that the main function of thehaustorium is that of food mobilization and transport for theyoung plant.     

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.     

Chilling-induced Lipid Hydrolysis in the Oil Palm (Elaeis guineensis) Mesocarp

Oil palm fruits exposed to temperatures of 15 °C and belowshowed a significant increase in free fatty acid (FFA) contentin the mesocarp. This effect was most pronounced in fruits exposedto 5 °C when FFA levels exceeding 70% of the total oil wereobserved. The increase in FFA was accompanied by an increasein lipid-soluble phosphorus levels and a decrease in carotenecontent. Chilling did not have an effect on palm kernel oil.The results suggest the activation of a lipase in the mesocarpby low temperature stress. Key words: Lipase, oil palm, free fatty acid     

Transfer of fatty acids and proteins between cytoplasmic membranes and mesosomes of Bacillus subtilis var. niger

A transfer of labelled branched-chain fatty acids or proteins between mesosomes, periplasmic space and protoplasts, is suggested in vivo and demonstrated in vitro.In the sole mesosomal fraction, [¹⁴C]valine or [¹⁴C]isoleucine are, always, more incorporated in fatty acids than in proteins.Mesosomal fatty acids can be transferred to the protoplasts, but protoplasts seems to give essentially amino acids to mesosomes.     

Differential Gene Expression and Characterization of Tissue-specific cDNA Clones in Oil Palm using mRNA Differential Display

The mRNA differential display method was utilized to study the differential expression and regulation of genes in two species of oil palm, the commercially grown variety Elaeis guineensis, var. tenera and the South American species, Elaeis oleifera. We demonstrated the differential expression of genes in the mesocarp and kernel at the week of active oil synthesis (15 week after anthesis) during fruit development as compare to the roots and leaves and the isolation of tissue-specific and species-specific cDNA clones. A total of eight specific cDNA clones were isolated and their specificities were confirmed by Northern hybridization and classified into three groups. Group one contains four clones (KT3, KT4, KT5 and KT6) that are kernel-specific for E. guineensis, tenera and E. oleifera. The second group represents clone FST1, which is mesocarp and kernel-specific for E. guineensis, tenera and E. oleifera. The third group represents clones MLT1, MLT2 and MLO1 that are mesocarp and leaf-specific. Northern analysis showed that their expressions were developmentally regulated. Nucleotide sequencing and homology search in GenBank data revealed that clones KT3 and KT4 encode for the same maturation protein PM3. While clones MLT1 and MLT2 encode for S-ribonuclease binding protein and fibrillin, respectively. The other clones (KT5, KT6, FST1 and MLO1) did not display any significant homology to any known protein.     

In vitro developmental study of oil palm (Elaeis guineensis Jacq.) polyembryoids from cell suspension using scanning electron microscopy

In the present study, we report the in vitro development of polyembryoids with identification of a definite stage that can be used for subsequent uniform plantlet regeneration in oil palm (Elaeis guineensis Jacq.). Induction and maturation of polyembryoids was accomplished when cell suspension culture was transferred in MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) semisolid medium consisting of 30 g L^?1 sucrose and 3.5 g L^?1 gelrite^® devoid of any plant growth regulator. Growth and development of cell suspension culture into polyembryoids were assessed by stereo and scanning electron microscopy (SEM) to identify the sequential events as well as the differentiation that occur during each stage. Observations on the differentiation symptoms showed that the embryos pass through distinct morphological characteristics indicating distinctively varied stages. SEM observations indicated the development of extracellular network at an early stage of differentiation and acts as the structural marker of differentiation leading to the development of polyembryoids via formation of globular proembryo and haustorium. Eventually, a specific developmental stage comprising haustorium and torpedo-shaped structure was identified, for conservation, regeneration or multiplication, based on the embryogenic competence.     

Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

Background

Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants.

Methodology/Principal Findings

We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation.

Conclusions/Significance

We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.     

Microsatellite-based high density linkage map in oil palm (Elaeis guineensis Jacq.).

A microsatellite-based high-density linkage map for oil palm (Elaeis guinensis Jacq.) was constructed from a cross between two heterozygous parents, a tenera palm from the La Mé population (LM2T) and a dura palm from the Deli population (DA10D). A set of 390 simple sequence repeat (SSR) markers was developed in oil palm from microsatellite-enriched libraries and evaluated for polymorphism along with 21 coconut SSRs. A dense and genome-wide microsatellite framework as well as saturating amplified fragments length polymorphisms (AFLPs) allowed the construction of a linkage map consisting of 255 microsatellites, 688 AFLPs and the locus of the Sh gene, which controls the presence or absence of a shell in the oil palm fruit. An AFLP marker E-Agg/M-CAA132 was mapped at 4.7 cM from the Sh locus. The 944 genetic markers were distributed on 16 linkage groups (LGs) and covered 1,743 cM. Our linkage map is the first in oil palm to have 16 independent linkage groups corresponding to the plants 16 homologous chromosome pairs. It is also the only high-density linkage map with as many microsatellite markers in an Arecaceae species and represents an important step towards quantitative trait loci analysis and physical mapping in the E. guineensis species.     

Biochemical characterisation during seed development of oil palm (Elaeis guineensis)

Developmental biochemical information is a vital base for the elucidation of seed physiology and metabolism. However, no data regarding the biochemical profile of oil palm (Elaeis guineensis Jacq.) seed development has been reported thus far. In this study, the biochemical changes in the developing oil palm seed were investigated to study their developmental pattern. The biochemical composition found in the seed differed significantly among the developmental stages. During early seed development, the water, hexose (glucose and fructose), calcium and manganese contents were present in significantly high levels compared to the late developmental stage. Remarkable changes in the biochemical composition were observed at 10 weeks after anthesis (WAA): the dry weight and sucrose content increased significantly, whereas the water content and hexose content declined. The switch from a high to low hexose/sucrose ratio could be used to identify the onset of the maturation phase. At the late stage, dramatic water loss occurred, whereas the content of storage reserves increased progressively. Lauric acid was the most abundant fatty acid found in oil palm seed starting from 10 WAA.     

Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants

Photosynthetically active chloroplasts retaining high rates of fatty acid synthesis from [1-¹⁴C]acetate were purified from leaves of both 16:3 (Solanum nodiflorum, Chenopodium album) and 18:3 plants (Amaranthus lividus, Pisum sativum). A comparison of lipids into which newly synthesized fatty acids were incorporated revealed that, in 18:3 chloroplasts, enzymic activities catalyzing the conversion of phosphatidate to diacylglycerol and of diacylglycerol to monogalactosyl diacylglycerol (MGD) were significantly less active than in 16:3 chloroplasts. In contrast, labeling rates of MGD from UDP-[¹⁴C]gal were similar for both types of chloroplasts.

The composition and positional distribution of labeled fatty acids within the glycerides synthesized by isolated 16:3 and 18:3 chloroplasts were similar and in each case only a C18/C16 diacylglycerol backbone was synthesized. In nodiflorum chloroplasts, C18:1/C16:0 MGD assembled de novo was completely desaturated to the C18:3/C16:3 stage.

Whereas newly synthesized C18/C18 MGD could not be detected in any of these chloroplasts if incubated with [¹⁴C]acetate after isolation, chloroplasts isolated from acetate-labeled leaves contained MGD with labeled C18 fatty acids at both sn-1 and sn-2 positions. Taken together, these results provide further evidence on an organellar level for the operation of pro- and eucaryotic pathways in the biosynthesis of MGD in different groups of plants.

     

The histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.)

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the somatic embryogenesis (SE) mechanisms. In the present study, the histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) was investigated. Zygotic embryos were inoculated on SE induction medium, and at 14 days the first divisions of the procambial and perivascular cells were observed. This region progressed to the formation of meristematic masses at 21 days, indicating their procambial and perivascular origin. Primary calli emerged at 45 days of culture, followed by progression to embryogenic calli at 90 days. The formation of proembryos (PE) from the meristematic cells occurred at 135 days of cultivation. The PE were isolated from the tissue of origin by the slight thickening of the cell wall, indicating their unicellular origin. When transferred to the maturation phase, differentiation of the somatic embryos at different developmental stages (globular and torpedo) was observed. The differentiated somatic embryos presented protoderm, procambial strands and plumules. Afterwards, they were transferred to culture medium without growth regulators in which conversion of the somatic embryos from torpedo stage into plants was observed. These results enable a greater understanding of the SE process and plantlet formation in E. guineensis.     

Insights from the GC content analysis of 76genome survey sequences (GSS) from Elaeisoleifera

South American oil-palm (Elaeis oleifera) is not cultivated in tropical countries like Malaysia on large scale due to low yield of palm oil derived from its fruit mesocarp. However, its fruit mesocarp oil contains about 68.6 % oleic acid (C_18:1) which is more than double in comparison to commercially cultivated oilpalm, E. guineensis Jacq Tenera (hybrid of Dura (♀) x Pisifera (♂)). It is also known that E. oleifera is a good source of tocotrienols and carotenoids. Therefore, it is of interest to know the genome sequence of E. oleifera. The objective of this study is to generate genome survey sequences (GSS) to get GC content insight in the E. oleifera genome. The nuclear genomic DNA isolated from young leaf‐tissues was digested with EcoRI and NdeI/DraI restriction enzymes; and three genomic DNA libraries were constructed using Lambda ZAP‐II, pGEM®‐T Easy, and pDONR 222™ as cloning vectors. Generated 76 GSSs were analyzed by using Bioinformatics tools. The analysis result indicates that the adenine, cytosine, guanine and thymine content in generated GSSs are 30%, 20%, 20%, and 30% respectively. In conclusion, based on the precise GC content analysis of the randomly isolated 76 GSSs by using Bioinformatics tools we hypothesize that GC content in E. oleifera genome is 40%. The hypothesized 40% GC content in E. oleifera genome is expected to remain close to the GC content based on the whole genome analysis.^ψThe nucleotide sequence data reported in this paper have been submitted to dbGSS division of the international DNA database (GenBank/DDBJ/EMBL) under accession numbers: {"type":"entrez-nucleotide-range","attrs":{"text":"DX575945- DX575972","start_term":"DX575945","end_term":"DX575972","start_term_id":"94323534","end_term_id":"94323561"}}DX575945- DX575972 and {"type":"entrez-nucleotide-range","attrs":{"text":"EI798032-EI798079","start_term":"EI798032","end_term":"EI798079","start_term_id":"146199019","end_term_id":"146199066"}}EI798032-EI798079.

Abbreviations

gDNA - Nuclear genomic DNA, GSSs - Genome survey sequences K12, SAOP - South American oil‐palm Db1