Differential gene expression at different stages of mesocarp development in high- and low-yielding oil palm.

BACKGROUND: The oil yield trait of oil palm is expected to involve multiple genes, environmental influences and interactions. Many of the underlying mechanisms that contribute to oil yield are still poorly understood. In this study, we used a microarray approach to study the gene expression profiles of mesocarp tissue at different developmental stages, comparing genetically related high- and low- oil yielding palms to identify genes that contributed to the higher oil-yielding palm and might contribute to the wider genetic improvement of oil palm breeding populations. RESULTS: A total of 3412 (2001 annotated) gene candidates were found to be significantly differentially expressed between high- and low-yielding palms at at least one of the different stages of mesocarp development evaluated. Gene Ontologies (GO) enrichment analysis identified 28 significantly enriched GO terms, including regulation of transcription, fatty acid biosynthesis and metabolic processes. These differentially expressed genes comprise several transcription factors, such as, bHLH, Dof zinc finger proteins and MADS box proteins. Several genes involved in glycolysis, TCA, and fatty acid biosynthesis pathways were also found up-regulated in high-yielding oil palm, among them; pyruvate dehydrogenase E1 component Subunit Beta (PDH), ATP-citrate lyase, ß- ketoacyl-ACP synthases I (KAS I), ß- ketoacyl-ACP synthases III (KAS III) and ketoacyl-ACP reductase (KAR). Sucrose metabolism-related genes such as Invertase, Sucrose Synthase 2 and Sucrose Phosphatase 2 were found to be down-regulated in high-yielding oil palms, compared to the lower yield palms. CONCLUSIONS: Our findings indicate that a higher carbon flux (channeled through down-regulation of the Sucrose Synthase 2 pathway) was being utilized by up-regulated genes involved in glycolysis, TCA and fatty acid biosynthesis leading to enhanced oil production in the high-yielding oil palm. These findings are an important stepping stone to understand the processes that lead to production of high-yielding oil palms and have implications for breeding to maximize oil production.

Enabling chlorophyll photo-response for in-line real-time noninvasive direct probing of the quality of palm-oil during mill process.

During the milling process of palm oil, the degree of palm fruit ripeness is a critical factor that affects the quality and quantity of the oil. As the palm fruit matures, its chlorophyll level decreases, and since chlorophyll in oil has undesirable effects on hydrogenation, bleachability, and oxidative degradation, it's important to monitor the chlorophyll content in palm oil during the milling process. This study investigated the use of light-induced chlorophyll fluorescence (LICF) for non-invasive and real-time monitoring of chlorophyll content in diluted crude palm oil (DCO) located at the dilution and oil classification point in palm oil mill. An LICF probe was installed at the secondary pipe connected to main DCO pipeline, and the system communicates with a computer located in a separate control room via a Wi-Fi connection. Continuous measurements were recorded with an integration time of 500 ms, averaging of 10, and a time interval of 1 min between each recording during the oil mill's operation. All data were stored on the computer and in the cloud. We collected 60 DCO samples and sent them to the laboratory for American Oil Chemists' Society (AOCS) measurement to compare with the LICF signal. The LICF method achieved a correlation coefficient of 0.88 with the AOCS measurements, and it also provided a direct, quantitative, and unbiased assessment of the fruit ripeness in the mill. By incorporating Internet of Things (IoT) sensors and cloud storage, this LICF system enables remote and real-time access to data for chemometrics analysis.

Palm oil supply chain factors impacting chlorinated precursors of 3-MCPD esters.

Chlorinated compounds such as sphingolipid-based organochlorine compounds are precursors for the formation of 3-monochlororopanediol (3-MCPD) esters in palm oil. This study evaluates the effects of several factors within the palm oil supply chain on the levels of sphingolipid-based organochlorine, which in turn may influence the formation of 3-MCPD esters during refining. These factors include application of inorganic chlorinated fertiliser in the oil palm plantation, bruising and degradation of oil palm fruits after harvest, recycling of steriliser condensate as water for dilution of crude oil during oil palm milling, water washing of palm oil and different refining conditions. It was observed that bruised and degraded oil palm fruits showed higher content of sphingolipid-based organochlorine than control. In addition, recycling steriliser condensate during milling resulted in elevated content of sphingolipid-based organochlorine in palm oil. However, the content of sphingolipid-based organochlorine compounds was reduced by neutralisation, degumming and bleaching steps during refining. Although water washing of crude palm oils (CPO) prior to refining did not reduce the content of sphingolipid-based organochlorine, it did reduce the formation of 3-MCPD esters through the removal of water-soluble chlorinated compounds. It was found that the use of inorganic chlorinated fertiliser in plantations did not increase the content of chlorinated compounds in oil palm fruits and extracted oil, and hence chlorinated fertiliser does not seem to play a role in the formation of 3-MCPD esters in palm oil. Overall, this study concluded that lack of freshness and damage to the fruits during transport to mills, combined with water and oil recycling in mills are the major contributors of chlorinated precursor for 3-MCPD esters formation in palm oil.

Diurnal biomarkers reveal key photosynthetic genes associated with increased oil palm yield.

To investigate limiters of photosynthate assimilation in the carbon-source limited crop, oil palm (Elaeis guineensis Jacq.), we measured differential metabolite, gene expression and the gas exchange in leaves in an open field for palms with distinct mesocarp oil content. We observed higher concentrations of glucose 1-phosphate, glucose 6-phosphate, sucrose 6-phosphate, and sucrose in high-oil content palms with the greatest difference being at 11:00 (p-value ≤0.05) immediately after the period of low morning light intensity. Three important photosynthetic genes were identified using differentially expressed gene analysis (DEGs) and were found to be significantly enriched through Gene Ontology (GO) and pathway enrichment: chlorophyll a-b binding protein (CAB-13), photosystem I (PSI), and Ferredoxin-NADP reductase (FNR), particularly for sampling points at non-peak light (11:00 and 19:00), ranging from 3.3-fold (PSI) and 5.6-fold (FNR) to 10.3-fold (CAB-13). Subsequent gas exchange measurements further supported increased carbon assimilation through higher level of internal CO2 concentration (Ci), stomatal conductance (gs) and transpiration rate (E) in high-oil content palms. The selection for higher expression of key photosynthesis genes together with CO2 assimilation under low light is likely to be important for crop improvement, in particular at full maturity and under high density planting regimes where light competition exists between palms.

Femtomol SPR detection of DNA-PNA hybridization with the assistance of DNA-guided polyaniline deposition.

The inability of surface plasmon resonance (SPR) spectroscopy to detect extremely small refractive index changes has hindered its applications in ultrasensitive DNA analysis. In this study we report a signal amplification strategy that uses DNA-templated polyaniline deposition, suitable for DNA hybridization analysis with charge neutral peptide nucleic acid (PNA) being probes. Under acidic conditions, protonated aniline monomers are adsorbed on DNA backbones through electrostatic interaction. The microenvironment provided by the DNA facilitates oxidative aniline polymerization initialized by H(2)O(2) in the presence of horseradish peroxide. Under optimal conditions, the detection limit is lowered from 5nM for conventional SPR detection to 0.1pM. The significant sensitivity improvement is attributed to the in-situ polymer chain growth along DNA strands, which introduces drastic refractive index increases. This signal amplification approach does not involve secondary hybridization processes. The detection sensitivity obtained is much better than that of gold nanoparticle-based amplification involving a secondary hybridization process and labeled DNA detection probes.

Natural Organochlorines as Precursors of 3-Monochloropropanediol Esters in Vegetable Oils.

During high-temperature refining of vegetable oils, 3-monochloropropanediol (3-MCPD) esters, possible carcinogens, are formed from acylglycerol in the presence of a chlorine source. To investigate organochlorine compounds in vegetable oils as possible precursors for 3-MCPD esters, we tested crude palm, soybean, rapeseed, sunflower, corn, coconut, and olive oils for the presence of organochlorine compounds. Having found them in all vegetable oils tested, we focused subsequent study on oil palm products. Analysis of the chlorine isotope mass pattern exhibited in high-resolution mass spectrometry enabled organochlorine compound identification in crude palm oils as constituents of wax esters, fatty acid, diacylglycerols, and sphingolipids, which are produced endogenously in oil palm mesocarp throughout ripening. Analysis of thermal decomposition and changes during refining suggested that these naturally present organochlorine compounds in palm oils and perhaps in other vegetable oils are precursors of 3-MCPD esters. Enrichment and dose-response showed a linear relationship to 3-MCPD ester formation and indicated that the sphingolipid-based organochlorine compounds are the most active precursors of 3-MCPD esters.

Amino Acid and Secondary Metabolite Production in Embryogenic and Non-Embryogenic Callus of Fingerroot Ginger (Boesenbergia rotunda).

Interest in the medicinal properties of secondary metabolites of Boesenbergia rotunda (fingerroot ginger) has led to investigations into tissue culture of this plant. In this study, we profiled its primary and secondary metabolites, as well as hormones of embryogenic and non-embryogenic (dry and watery) callus and shoot base, Ultra Performance Liquid Chromatography-Mass Spectrometry together with histological characterization. Metabolite profiling showed relatively higher levels of glutamine, arginine and lysine in embryogenic callus than in dry and watery calli, while shoot base tissue showed an intermediate level of primary metabolites. For the five secondary metabolites analyzed (ie. panduratin, pinocembrin, pinostrobin, cardamonin and alpinetin), shoot base had the highest concentrations, followed by watery, dry and embryogenic calli. Furthermore, intracellular auxin levels were found to decrease from dry to watery calli, followed by shoot base and finally embryogenic calli. Our morphological observations showed the presence of fibrils on the cell surface of embryogenic callus while diphenylboric acid 2-aminoethylester staining indicated the presence of flavonoids in both dry and embryogenic calli. Periodic acid-Schiff staining showed that shoot base and dry and embryogenic calli contained starch reserves while none were found in watery callus. This study identified several primary metabolites that could be used as markers of embryogenic cells in B. rotunda, while secondary metabolite analysis indicated that biosynthesis pathways of these important metabolites may not be active in callus and embryogenic tissue.

Hormones, polyamines, and cell wall metabolism during oil palm fruit mesocarp development and ripening.

Oil palm is one of the most productive oil-producing crops and can store up to 90% oil in its fruit mesocarp. Oil palm fruit is a sessile drupe consisting of a fleshy mesocarp from which palm oil is extracted. Biochemical changes in the mesocarp cell walls, polyamines, and hormones at different ripening stages of oil palm fruits were studied, and the relationship between the structural and the biochemical metabolism of oil palm fruits during ripening is discussed. Time-course analysis of the changes in expression of polyamines, hormones, and cell-wall-related genes and metabolites provided insights into the complex processes and interactions involved in fruit development. Overall, a strong reduction in auxin-responsive gene expression was observed from 18 to 22 weeks after pollination. High polyamine concentrations coincided with fruit enlargement during lipid accumulation and latter stages of maturation. The trend of abscisic acid (ABA) concentration was concordant with GA4 but opposite to the GA3 profile such that as ABA levels increase the resulting elevated ABA/GA3 ratio clearly coincides with maturation. Polygalacturonase, expansin, and actin gene expressions were also observed to increase during fruit maturation. The identification of the master regulators of these coordinated processes may allow screening for oil palm variants with altered ripening profiles.

Profiling of metabolites in oil palm mesocarp at different stages of oil biosynthesis.

Oil palm is one of the most productive oil producing crops and can store up to 90% oil in its fruit mesocarp. However, the biosynthetic regulation and drivers of palm mesocarp development are still not well understood. Multiplatform metabolomics technology was used to profile palm metabolites during six critical stages of fruit development in order to better understand lipid biosynthesis. Significantly higher amino acid levels were observed in palm mesocarp preceding lipid biosynthesis. Nucleosides were found to be in high concentration during lipid biosynthesis, whereas levels of metabolites involved in the tricarboxylic acid cycle were more concentrated during early fruit development. Apart from insights into the regulation of metabolites during fruit development in oil palm, these results provide potentially useful metabolite yield markers and genes of interest for use in breeding programs.

Differential metabolite profiles during fruit development in high-yielding oil palm mesocarp.

To better understand lipid biosynthesis in oil palm mesocarp, in particular the differences in gene regulation leading to and including de novo fatty acid biosynthesis, a multi-platform metabolomics technology was used to profile mesocarp metabolites during six critical stages of fruit development in comparatively high- and low-yielding oil palm populations. Significantly higher amino acid levels preceding lipid biosynthesis and nucleosides during lipid biosynthesis were observed in a higher yielding commercial palm population. Levels of metabolites involved in glycolysis revealed interesting divergence of flux towards glycerol-3-phosphate, while carbon utilization differences in the TCA cycle were proven by an increase in malic acid/citric acid ratio. Apart from insights into the regulation of enhanced lipid production in oil palm, these results provide potentially useful metabolite yield markers and genes of interest for use in breeding programmes.

Enzyme-based colorimetric detection of nucleic acids using peptide nucleic acid-immobilized microwell plates.

The development of label-free or nonlabeling assays for nucleic acids is important in basic biological research and biomedical diagnosis. In this study, we have developed an enzyme-based colorimetric assay for nucleic acids, which combines the robustness of nonlabeling of DNA and RNA samples and the adequate sensitivity of enzymatic reactions. The core of this assay is the use of neutral peptide nucleic acid (PNA) as capture probe and the electrostatic adsorption of horseradish peroxidase (HRP) on hybridized, negatively charged nucleic acids to report the hybridization events, through HRP-catalyzed color reactions of 3,3',5,5'-tetramethylbenzidine and H(2)O(2). The proposed assay has been validated with fully complementary and single base-mismatched DNAs of different chain lengths. The proposed assay has also been validated with total RNA samples extracted from two human cancer cell lines (A 549 lung cancer cell and HeLa cell) for microRNA detection in real samples. Through extensive optimizations of HRP adsorption and nucleic acid hybridization conditions, detection limits of 0.1-0.2 nM for DNA (depending on chain length) and approximately 2 microg of total RNA have been achieved. Surface plasmon resonance spectroscopy has been used to elucidate the HRP adsorption and PNA-nucleic acid hybridizations through real-time measurements and to provide guidance for the development of the colorimetric assay.

Characterization of protein--DNA interactions using surface plasmon resonance spectroscopy with various assay schemes.

Specific protein-DNA interactions play a central role in transcription and other biological processes. A comprehensive characterization of protein-DNA interactions should include information about binding affinity, kinetics, sequence specificity, and binding stoichiometry. In this study, we have used surface plasmon resonance spectroscopy (SPR) to study the interactions between human estrogen receptors (ER, alpha and beta subtypes) and estrogen response elements (ERE), with four assay schemes. First, we determined the sequence-dependent receptors' binding capacity by monitoring the binding of ER to various ERE sequences immobilized on a sensor surface (assay format denoted as the direct assay). Second, we screened the relative affinity of ER for various ERE sequences using a competition assay, in which the receptors bind to an ERE-immobilized surface in the presence of competitor ERE sequences. Third, we monitored the assembly of ER-ERE complexes on a SPR surface and thereafter the removal and/or dissociation of the ER (assay scheme denoted as the dissociation assay) to determine the binding stoichiometry. Last, a sandwich assay (ER binding to ERE followed by anti-ER recognition of a specific ER subtype) was performed in an effort to understand how ERalpha and ERbeta may associate and compete when binding to the DNA. With these assay schemes, we reaffirmed that (1) ERalpha is more sensitive than ERbeta to base pair change(s) in the consensus ERE, (2) ERalpha and ERbeta form a heterodimer when they bind to the consensus ERE, and (3) the binding stoichiometry of both ERalpha- and ERbeta-ERE complexes is dependent on salt concentration. With this study, we demonstrate the versatility of the SPR analysis. With the involvement of various assay arrangements, the SPR analysis can be further extended to more than kinetics and affinity study.

Understanding ligand binding effects on the conformation of estrogen receptor alpha-DNA complexes: a combinational quartz crystal microbalance with dissipation and surface plasmon resonance study.

Estrogen receptors are ligand-activated transcription factors that regulate gene expression by binding to specific DNA sequences. To date, the effect of ligands on the conformation of estrogen receptor alpha (ERalpha)-DNA complex remains a poorly understood issue. In our study, we are introducing the quartz crystal microbalance with dissipation monitoring (QCM-D) as a new alternative to study the conformational differences in protein-DNA complexes. Specifically, we have used QCM-D, in combination with surface plasmon resonance (SPR) spectroscopy, to monitor the binding of ERalpha to a specific DNA (estrogen response element, ERE) and a nonspecific DNA in the presence of either the agonist ligand, 17b-estradiol, the partial antagonist ligand, 4-hydroxytamoxifen, or vehicle alone. Both with presence and absence of ligand, the specific ERalpha-ERE complexes are observed to adopt a more compact conformation compared to nonspecific complexes. This observation is well correlated to the biophysical changes occurring during protein-DNA interaction shown by past structural and mechanism studies. Notably, pretreatment of ERalpha with E2 and 4OHT affects not only the viscoelasticity and conformation of the protein-DNA complex but also ERalpha binding capacity to immobilized ERE. These results affirm that ligands have remarkable effects on ERalpha-DNA complexes. Understanding these effects will provide insight into how ligand binding promotes subsequent events required for gene transcription.

Combinational application of surface plasmon resonance spectroscopy and quartz crystal microbalance for studying nuclear hormone receptor-response element interactions.

Conventional methodologies for studying protein-DNA complexes, such as electrophoretic mobility shift assays (EMSAs), lack the real-time sensitivity and precision to accurately characterize the complex dynamics of interactions between transcription factors and their binding sites. To better understand the interactions between estrogen receptor (ER) subtypes and the estrogen response elements (EREs), we employed surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance with dissipation measurement (QCM-D) and made the following observations: (1) base substitutions in ERE half-sites reduced binding affinity for both ERalpha and ERbeta, (2) ERalpha has a higher sequence specificity than ERbeta or there were more nonspecific interactions between ERbeta and control DNA, and (3) ERalpha bound ERE as dimers and ERbeta bound as tetramers. These findings highlight intrinsic differences in DNA-binding properties between receptor subtypes, which are not apparent based on the high degree of conservation (96% identity) in their DNA-binding domains and results from EMSA studies. With this study, we demonstrate the potential of utilizing SPR and QCM in combination for a comprehensive characterization of ER-DNA interactions, including sequence-dependent binding mechanisms and structural differences in ERalpha-DNA and ERbeta-DNA complexes.

Multiplatform genome-wide identification and modeling of functional human estrogen receptor binding sites.

BACKGROUND: Transcription factor binding sites (TFBS) impart specificity to cellular transcriptional responses and have largely been defined by consensus motifs derived from a handful of validated sites. The low specificity of the computational predictions of TFBSs has been attributed to ubiquity of the motifs and the relaxed sequence requirements for binding. We posited that the inadequacy is due to limited input of empirically verified sites, and demonstrated a multiplatform approach to constructing a robust model. RESULTS: Using the TFBS for the estrogen receptor (ER)alpha (estrogen response element [ERE]) as a model system, we extracted EREs from multiple molecular and genomic platforms whose binding to ERalpha has been experimentally confirmed or rejected. In silico analyses revealed significant sequence information flanking the standard binding consensus, discriminating ERE-like sequences that bind ERalpha from those that are nonbinders. We extended the ERE consensus by three bases, bearing a terminal G at the third position 3' and an initiator C at the third position 5', which were further validated using surface plasmon resonance spectroscopy. Our functional human ERE prediction algorithm (h-ERE) outperformed existing predictive algorithms and produced fewer than 5% false negatives upon experimental validation. CONCLUSION: Building upon a larger experimentally validated ERE set, the h-ERE algorithm is able to demarcate better the universe of ERE-like sequences that are potential ER binders. Only 14% of the predicted optimal binding sites were utilized under the experimental conditions employed, pointing to other selective criteria not related to EREs. Other factors, in addition to primary nucleotide sequence, will ultimately determine binding site selection.