Category Archives: 1999 Special Issue

Characterization and regulation of the oil palm (Elaeis guineensis) stearoyl-ACP Desaturase genes

Two different stearoyl-ACP desaturase genes are expressed in the mesocarp of oil palm (E. guineensis) fruits. Their nucleotide sequences share 93% and 76% homologies within the coding and 3’untranslated regions, respectively. Southern blot analysis showed that there are at least two copies of stearoyl-ACP desaturase genes in the oil palm genome. Northern blot analysis using gene specific probes showed that the two genes from the mesocarp are differentially regulated. One gene is constitutively expressed with a high level of expression in various oil palm tissues. The other gene was found to be developmentally regulated with its expression pattern correlating with the pattern of oil synthesis in both the mesocarp and kernel tissues. Polyclonal anti-bodies were raised against a peptide containing the N-terminal sequence of the mature oil palm stearoyl-ACP desaturase. The antibodies recognized the predicted size protein of 37kDa in total protein fractions of oil palm mesocarp after separation on denaturing polyacrylamide gels. Western blot analysis using the polyclonal antibodies showed the enzyme level is high in the mesocarp at late stage of ripening and remains high in ripe fruits. The leaf form of the enzyme appeared to be about 2kDa larger. High levels of enzyme and gene expression were detected in young mesocarp tissue consistent with the requirement for high levels of unsaturated fatty acids for membrane lipid biosynthesis.

Partial nucleotide sequence of an oleosin cDNA from Elaeis guineensis Jacq.

In an attempt to isolate a full length oleosin cDNA from oil palm kernel, we identified a putative clone by colony hybridization of cDNAs from a 20 weeks after anthesis (WAA) kernel library with the oleosin gene (oleM) from maize (Zea rnays). The recombinant plasmid, pOP-KT115, has an insert of 550 base pair (bp). The nucleotide sequence of this insert showed high similarities with the oleosin genes from maize, barley (Hordeum vulgare) and rice (Oryza sativa).
In a Northern blot analysis with the insert of pOP-KT115 as probe, positive signals were only detected with total RNA from oil palm kernel tissues at 10, 12, and 14 WAA. No hybridization signal was detected with total RNA from mesocarp tissues, leaf, germinating seedlings, and inflorescences at Frond 10 (FIO) and Frond 19 (F19). This result concurs with the findings of previous studies carried out with mesocarp tissues of olive (Olea europea) and avocado (Persea americana).

Biochemical factors that control oil composition in the oil palm

Fatty acid biosynthesis is essentially the same in all plant species. Yet, different plants have unique fatty acid profiles which differentiate one vegetable oil from another. The oil palm produces different oils in the mesocarp and kernel. These two oils have distinct fatty acid profiles. Palm oil is rich in palmitic and oleic acids while palm kernel oil is a rich source of lauric and myristic, i.e. medium chain fatty acids. The role of different biochemical factors in controlling fatty acid composition in the oil palm was investigated. The studies confirmed that fatty acid composition was not controlled by a single enzyme. Instead, a number of enzymnes work in concert to produce the distinct fatty acid composition of palm and palm kernel oil. Beta ketoacyl ACP synthase 11, acyl ACP thioesterases and acyltransferases have an important role in determining the fatty acid profiles of palm and palm kernel oil.

Regulation of triacylglycerol synthesis in oil palm (Elaeis guineensis) and olive (Olea europaea) callus cultures

We have been using callus cultures as convenient model systems to understand any step where regulation of flux control in the triacylglycerol biosynthesis pathway may be important in two major oil-rich fruits, oil palm (Elaeis guineensis) and olive (Olea europaea). Top-down metabolic control analysis (TDCA) has been used to address the question of where possible control step(s) in the lipid biosynthesis pathway are located. With this method, lipid metabolism is conceptually divided into blocks of reactions and these are manipulated to see the effect on a key intermediate. We initially measured changes in the overall flux of carbon from [1-14C]acetate as modified by temperature in oil palm and olive callus cultures. A doubling of lipid synthesis with a 10ºC rise from 20ºC to 30ºC did not, however, cause much change in radioactivity incorporation into the acylthioester pools, acyl-CoAs and acyl-ACPs. This suggested that de novo fatty acid synthesis reactions exerted higher control than complex lipids assembly via Kennedy pathway.

Reactions of the Kennedy pathway were examined in more detail in oil palm callus cultures. By using microsomal fractions pre-pared from such cultures, we showed that radioactivity from [U-14C]glycerol 3-phosphate was effectively incorporated into intermediates of the Kennedy pathway and that the changes in radioactivity caused by temperature manipulation reflected well the endogenous lipid pool levels. Stimulation of triacylglycerol synthesis at 30ºC was accompanied by slight increases in diacylglycerol and phosphatidic acid. This indicates that at higher rates of triacylglycerol synthesis, specific enzymes of the Kennedy pathway may become more limiting.

Protein analysis of shoot apical meristem of oil palm during transition from vegetative meristem to an inflorescence meristem

Oil palm shoots were grown in vitro on high cytokinin media. Terminal inflorescences were observed after four to five months of growth. Two-dimensional polyacrylamide gel electrophoresis was carried out to identify unique proteins in shoot apices producing terminal infloreseence. Three proteins were identified, the absence of which could be associated with the conversion of a vegetative meristem to an inflorescence meristem. Histological studies of the inflorescence revealed that it had no flowers. This showed that in vitro, under high cytokinin growth condition, the vegetative meristem could be induced to transform into an inflorescence meristem. However, the inflorescence meristems produced are not competent to produce flowers.

Glycerol-3-phosphate acyltransferase reactions and edible oil in synthesis in oil palm (Elaeis guineensis) tissue

Acyltransferase enzymes are used in three of the four steps of the Kennedy pathway for storage lipid formation. Their specifities, especially those of the first two reactions involving glycerol-3-phosphate acyltransferase (GPAT) and 1-acylglycerol-3-phosphate acyltransferase (LPAAT), determine the acyl quality of triacylglycerol (TAG) to a significant extent. Therefore, we determined the characteristics of the acyltransferases in oil palm (Elaeis guineensis), one of the world’s most important agricultural species and the most productive oil crop. Two tissue sources were used. Calli were established and used for in situmanipulation and labelling studies as well as a source of microsomal fractions for enzyme measurements. In addition, acetone powder was prepared from oil palm fruits (14-18 weeks after pollination) for enzyme purification. High speed particulate fractions isolated from mesocarp acetone powder or calli were incubated with [14C]glycerol 3-phosphate and the formation of Kennedy pathway intermediates followed. Conditions were optimized with regard to substrate concentrations, etc. and the overall rate manipulated using temperature. GPAT was solubilized from particulate fractions of the acetone powder and calli. Optimal solubilization of GPAT activity using CHAPS treatment was achieved at 0.5% (wlv) concen-tration. Details of the purification procedure and properties of the solubilized enzyme are discussed.

Analysis of the inheritance of AFLP markers in an interspecific cross of oil palm using the pseudo-testcross strategy

The newly developed technique of amplified fragment length polymorphism (AFLP) was used to study the polymorphism and segregation of AFLP markers in an interspecific cross of oil palm. The technique revealed a high level of polymorphism between the Colombian oleifera (female parent) and Nigerian guineensis (male parent) used to generate the cross. The segregation of AFLP markers was studied in a population of 77 F, individuals and analysed as a pseudo-testcross. Using only 10 primer pair combinations, we were able to amplify 674 bands out of which 91 show segregation. All markers detected were scored as dominant, and the segregation ratios indicated that majority of these markers (80%) were inherited in a Mendelian manner. The results also showed that the male parent (palm T128, a Nigerian guineensis) was more heterozygous than the female parent (UP1026, a Colombian oleifera). The AFLP technique was also found to be suitable for detecting contaminants and would therefore be useful for assessing the fidelity of controlled crosses.

Application of genomic In situ hybridization (GISH) on Elaeis hybrids

The introgressed parental genomes of Elaeis oleifera x Elaeis guineensis(OxG) hybrids were investigated using the genomic in situ hybridization technique. This technique was able to show clear differentiation between the E. oleifera and E. guineensis genome. It was observed that there are 16E. oleifera and 16 E. guineensis chromosomes in the OxG hybrids. The interphase nuclei of the OxG hybrids had groups of chromosomes from both parental genomes in discrete, non-intermixed domains.

Isolation of cDNA clone encoding an acyl-acyl carrier protein thioesterase from the mesocarp of oil palm (Elaeis guineensis)

A 1450 base pair (bp) cDNA encoding an acyl-ACP thioesterase was isolated from a developing mesocarp cDNA library. The cDNA sequence corresponding to the mature protein is 66% identical to Cuphea hookeriana Ch FatB1, and 35% identical to Garcinia mangostana Garm FatA1, indicating that this clone belongs to the FatB type of acyl-ACP thioesterases. Although the sequence showed the cDNA to be incomplete, lacking the 5′ untranslated region and most of the transit peptide, the mature protein is intact and will be expressed in Escherichia coli to investigate the substrate specificity of the acyl-ACP thioesterase.

Isolation and sequencing of cDNA clones coding for oil palm (Elaeis guineensis) acyl carrier protein (ACP)

Five cDNA clones encoding the oil palm acyl carrier protein (ACP) were IV isolated and characterized. Four of the clones, pACP1, pACP3, pACP4 and pACP9, were completely sequenced and the sizes of inserts shown to be 529, 701, 505 and 370bp, respectively. The clones could be divided into two classes, which probably represent two different isoforms of ACP. pACPI, which was classed separately from the others, is about 92% identical to the other three clones. The clones also had a high similarity to ACPs in other plants – 73%, 72% and 70% identical to barley ACPI, castor bean ACP and barley ACPIII, respectively. The similarity of the transit peptide was lower at about 45% to 52%. Based on the comparison, we predict that pACP3 contains the full length insert of oil palm ACP with an open reading frame of 432bp which starts at nucleotide 47 from the 5′ end and ends at nucleotide 478. This comprises 144 amino acid residues including 65 residues of a transit peptide.