Elaeis guineensis: Adenosine phosphates and ...

Elaeis guineensis: Adenosine phosphates and nicotinamides involvement in fatty acid biosynthesis Bee Keat Neoh, Huey Fang Teh, Theresa L. M. Ng, Soon Huat Tiong, Harikrishna Kulaveerasingam & David R. Appleton Journal of Plant Biochemistry and Biotechnology ISSN 0971-7811 J. Plant Biochem. Biotechnol. DOI 10.1007/s13562-013-0234-6

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Author's personal copy J. Plant Biochem. Biotechnol. DOI 10.1007/s13562-013-0234-6

ORIGINAL ARTICLE

Elaeis guineensis: Adenosine phosphates and nicotinamides involvement in fatty acid biosynthesis Bee Keat Neoh & Huey Fang Teh & Theresa L. M. Ng & Soon Huat Tiong & Harikrishna Kulaveerasingam & David R. Appleton

Received: 29 January 2013 / Accepted: 29 July 2013 # Society for Plant Biochemistry and Biotechnology 2013

Abstract Adenosine phosphates (ATP, ADP, AMP) and nicotinamides (NAD, NADH, NADP and NADPH) are nucleotides that are involved in various plant biosynthetic pathways such as photosynthesis, nitrogen uptake, purine metabolism and lipid biosynthesis. In oil palm fruit formation, kernel and mesocarp are separated by a shell and differ mainly by lipid composition. Oil palm mesocarp and kernel tissues were extracted using a modified perchloric acid extraction and separated via HPLC to quantify the accumulation of these seven nucleotides in relation to lipid composition. Principal component analysis on palm mesocarp and kernel samples displayed clustering and indicated that palm mesocarp contained higher ATP and NAD+ than palm kernel. The higher levels of ATP and NADH may be attributable to the higher content of unsaturated and long chain fatty acids in found palm mesocarp. Keywords Adenosine phosphate . Nicotinamide . Oil palm . Mesocarp . Kernel . Fatty acid biosynthesis . Perchloric acid extraction . High performance liquid chromatography Abbreviations ATP ADP AMP NAD NADH

Adenosine triphosphate Adenosine diphosphate Adenosine monophosphate Nicotinamide adenine dinucleotide Reduced nicotinamide adenine dinucleotide

B. K. Neoh : H. F. Teh : T. L. M. Ng : S. H. Tiong : H. Kulaveerasingam : D. R. Appleton (*) Sime Darby Technology Centre Sdn Bhd, 1st Floor, Block B, UPM-MTDC Technology Centre III, Sime Darby Technology Centre, 43400 Serdang, Selangor, Malaysia e-mail: [email protected]

NADP NADPH

Nicotinamide adenine dinucleotide phosphate Reduced nicotinamide adenine dinucleotide phosphate

Introduction Oil palm fruit pericarp comprises of three layers, namely; exocarp (skin), mesocarp (outer pulp containing palm oil), and endocarp (a hard shell enclosing the kernel). Development of a palm fruit starts approximately 2 weeks after pollination (WAP). At 12 WAP, oil deposition in the endosperm starts and is almost complete by 16 WAP. The endosperm and endocarp slowly harden from 12 WAP, and by 16 WAP the endocarp is a hard shell enclosing the kernel. Mesocarp neutral lipids, especially triacylglycerol (TAG) of the Malaysian Tenera variety increase rapidly from 16 WAP onwards, along with the parallel accumulation of total lipids, reaching a maximum at 20 WAP (Kalyana et al. 2003). Fatty acids (FAs) are core components of every membrane in the cell, and are also found extra-cellularly as cuticular lipids (Wanasundara et al. 1999). The FA synthases that are responsible for de novo synthesis of 16- and 18-carbon FAs are localized in the plastid. The FAs formed are then transported to endoplasmic reticulum for FA elongation (Wallis and Browse 2010). FA synthesis has a high energy demand and depends upon the stromal provision of ATP and reducing equivalents. Cofactors such as ATP and NADH are nonprotein chemical compounds that are bound to a protein and are required for the protein’s biological activity. These cofactors are produced in the cellular energy metabolism and are involved in a large number of biosynthetic reactions. De novo FA biosynthesis requires stoichiometric amounts of ATP, NADPH, and NADH for each sequential addition of an acetyl

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unit to the growing FA chain. ATP is required for the carboxylation of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase, whereas the two reductases of the FA synthase complex, namely 3-ketoacyl-ACP reductase and enoyl-ACP reductase, require NADPH and NADH, respectively (Baud and Lepiniec 2010). Since FA regulation is key to oil accumulation in oil palm cells, it is important to understand the involvement of cofactors during lipid biosynthesis occuring in oil palm mesocarp and kernel. Several studies on palm oil biosynthesis-related metabolites solely focussed on mesocarp (Neoh et al. 2013; Teh et al. 2013; Tranbarger et al. 2011), and the comparison of palm kernel and mesocarp fiber fatty acid compositions (Noor Lida et al. 2002; Neoh et al. 2011) have been previously reported. In a study of gene expression in both palm mesocarp and kernel using suppression substracted hybridisation, the myo-inositol-1-phosphate synthase (MIPS) homolog gene was upregulated in the mesocarp. Myo-inositol and its derivatives were reported to play an important role in growth regulation, cell wall biosynthesis and phosphorus storage (Li et al. 2011). In mesocarp, rate limiting KAS II activity impedes the conversion of palmitoyl ACP to stearoyl ACP and thus, resulting in high palmitoyl ACP that is efficiently converted to palmitic acid by a palmitoyl ACP thioesterase. According to Sambanthamurthi et al. (1999), highly active thioesterase will limit the elongation of fatty acids. Thus, palmitic acid is highly accumulated in the palm mesocarp. In the kernel, medium chain thioesterase(s) appears to play a role in accumulating medium chain fatty acids, resulting in high lauric acid accumulation. There have been several scientific publications discussing the importance of cofactors in oil seeds (Byers et al. 1999; Lung and Weselake 2006), but there are no reports discussing the relationship of adenosine phosphates and nicotinamides in oil palm mesocarp and kernel along with their involvement in lipid biosynthesis, or other important pathway regulation. Herein, we describe the analysis of cofactors in both palm mesocarp and kernel and discuss their possible involvement in fatty acid (FA) biosynthesis. It is also our aim to provide a platform to quantify the concentration of adenosine phosphates and nicotinamides in lipid-rich plant tissues. This study adopted a perchloric acid extraction protocol (Hai et al. 2006) and a modification of High Performance Liquid ChromatographyPhoto Diode Array (HPLC-PDA) methods using combinations of phosphate buffers at different concentrations (VecianaNogues et al. 1997; Hai et al. 2006). The modification included the utilisation of single phosphate buffer together with isocratic separation in order to increase reproducibility and reduce technical error. This platform will be useful for future comprehensive metabolomics studies into lipid and other cell metabolism pathways in palm fruit, which may provide useful insights into the key enzymes controlling oil yield and FA species biosynthesis and other traits for this important crop. Alteration of FA

composition of oil crops can broaden the range of end uses (Chopra and Vageeshbabu 1996).

Materials and methods Chemicals and reagents High purity ATP, ADP, AMP, NAD, NADH, NADP, NADPH, uric acid, and all other reagents were purchased from Sigma Chemical Co. All standards used were used as is and dissolved in deionized water, and then filtered through 0.20 μm Nylon membrane filters. Plant materials Three biological replicates of palm mesocarp and kernel of Elaeis guineensis were harvested from Sime Darby Plantation in Carey Island, Selangor, Malaysia, and were preserved in liquid nitrogen during transportation. All samples were pulverized using a mortar and pestle and liquid nitrogen. The powdered samples were freeze-dried and kept at −80 °C until use. Extraction of co-factors from palm tissue Cofactors were extracted from powdered samples of mesocarp or kernel (600 mg) with 0.6 M perchloric acid (2.7 mL) and water (0.2 mL), being a slight modification of the method reported by Hai et al. (2006). Uric acid (100 μL) was added as an internal standard. Extraction was performed in an ice bath before vortexing for 30 s. The extraction mixture was then centrifuged for 40 min at 9,000 × g at 4 °C, after which 2 mL of the supernatant was collected and quickly neutralised to pH 6.5– 6.8 with 1 M potasium hydroxide solution. The neutralised supernatant was then allowed to stand for 30 min in an ice bath to precipitate most of the potassium perchlorate, which was subsequently removed by 0.2 μm nylon filtration. The filtrate (PCA extract) was freeze dried and kept at −80 °C until further use. Prior to analysis the extract was dissolved in water (200 μL).

Table 1 Quantification of cofactors (nmol) in palm mesocarp and kernel via perchloric acid extraction

ATP ADP AMP NAD NADH NADP

Palm mesocarp (nmol)

Palm kernel (nmol)

603±3.93 4.56±0.30 10.1±2.70 149±5.50 57.2±5.52 29.0±2.58

183±4.34 76.2±1.38 16.0±0.09 106±7.27 10.7±1.46 43.4±1.67

Author's personal copy J. Plant Biochem. Biotechnol. Fig. 1 HPLC chromatograms of (a) standard solution of seven co-factors using uric acid as internal standard; (b) cofactors extracted from the palm mesocarp; (c) kernel tissue

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Extraction of lipid from palm tissue Freeze dried palm tissue (200 mg) was extracted with hexane (10 mL) and incubated at 50 °C for 6 h with constant shaking at 250 rpm. The mixture was then centrifuged and the supernatant was collected. The solvent was removed using nitrogen (oxygen free). The lipid extracts were weighed and then subjected to fatty acid composition analysis (FAC). HPLC apparatus The high performance liquid chromatography (HPLC) system consisted of a pump (Waters 2695 Separation Module), photodiode array detector (PDA,Waters 2996) and reversed phase column. The columns used in this study were LiChroCART® 250-4, LiChrospher®100 RP-18 endcapped (5 μm) with guard column LiChroCART® 250-4, LiChrospher®100 RP-18e (5 μm) (Merck, Malaysia), and set to 40 °C during analysis. Empower® software was used for peak analysis and quantification. UV chromatograms were obtained at a wavelength of 254 nm. HPLC method The mobile phase consisted of phosphate buffer (65 mM) composed of 39 mM dipotassium hydrogen phosphate and

Fig. 2 a Principal component analysis (PCA) of palm mesocarp and kernel; b Bioplot of overlaying samples’ PCA and cofactor’s loading plot. Palm mesocarp (black up-pointing triangle), palm kernel (black diamond suit), co-factors (black circle)

26 mM potassium dihydrogen phosphate, adjusted to pH 6 (21.5–22.5 °C) with orthophosphoric acid (modification of method previously described by Hai et al. (2006)). The buffer was prepared in deionized water and filtered through a 0.25 μm filter before use. The column was equilibrated with 10 column volumes of buffer at a flow rate of 1 mL/min. The retention times were stabilised with five injections of spiked standards prior to analytical separation. The runtime for the elution of relevant compounds was 30 min. Upon completion of ten sample injections, the system was re-calibrated using standards. Blank injections were performed before and after the re- calibration. Concentrations were calculated by comparison of sample compound peak areas with calibration curves for each standard.

GCMS apparatus One microliter aliquot of samples were injected at a 1:100 split ratio into a gas chromatography mass spectrometry system consisting of an Agilent 6890N Gas Chromatograph (GC) coupled with an Agilent 5973i Mass Detector and 7683 series auto sampler. Chromatography was performed using a 30 cm×0.25 mm, 0.25 μm film thickness HP-5MS column (Agilent, Malaysia). Mass spectra were recorded at 2.48 scans/s with a mass scanning range of 50 to 650m/z.

Author's personal copy J. Plant Biochem. Biotechnol. Fig. 3 Irreversible reaction committed step in fatty acid synthesis using ATP for acyl-CoA elongation

FAC analysis Esterification of lipid extract was performed using a modified method based on Malaysian Palm Oil Board (MPOB) test methodology (Ainie 2005). Derivatised methyl esters were injected at 280 °C, interface 280 °C. Separations were achieved using the following temperature program: 3 min isothermal heating at 80 °C, followed by a 5 °C/min oven ramp to 315 °C, and a final isothermal heating at 315 °C for 12 min. Fatty acid identifications were determined using GC-MS spectral database matching against the current National Institute of Standards and Technology library (NIST05).

Statistical analysis Principle component analysis (PCA), biplot and variation plot analysis using Simca-P version 12 (Umetrics) were used to monitor the clustering of different tissues according to concentration of cofactors.

Results and discussion In this study, 600 mg of fresh palm mesocarp and kernel tissue was sufficient to determine the concentrations of ATP, ADP, AMP, NAD, NADH and NADP. Additional steps of freeze drying pre- and post- extraction enhanced the extraction rate and concentration of targeted compounds without requiring pre-concentration steps such as solid phase micro-extraction. Due to the high activities of ATPases in fresh samples, changes in the adenine nucleotide level occurs very quickly when fruit are harvested. Snap-freezing of tissues in liquid nitrogen followed by freeze drying was practised to stop any enzymatic activities and minimize any degradation of ATP (zur Nedden et al. 2009). The HPLC method using a single phosphate buffer and isocratic separation was applied to determine the concentrations of ATP, ADP, AMP, NAD, NADH, NADP and NADPH of three replicates of palm mesocarps and kernels. Six cofactors (ATP, ADP, AMP, NAD, NADH & NADP) were successfully detected and quantified (Table 1) in palm mesocarp. NADPH was detected using the standards mixture (Fig. 1a) but co-eluted with an unknown peak, RT 2.44–2.58 in

mesocarp (Fig. 1b) and kernel (Fig. 1c) using these chromatographic parameters. Concentration data of cofactors in palm mesocarp and kernel were subjected to multivariate analysis. Principal component analysis (Fig. 2a) indicated that clear clustering can be observed between palm mesocarp and kernel samples. Palm mesocarp samples clustered closer together compared to palm kernel samples in this study, giving a correlation coefficient of 0.99 compared to 0.92, respectively. The bi-plot of samples and cofactors (Fig. 2b) indicated that palm mesocarp contained higher concentrations of ATP, NAD and NADH, while ADP and NADP were relatively more concentrated in palm kernel. The lower amount of AMP detected in palm kernel 3 resulted in the deviation from other two replicates (Fig. 2b). Palm mesocarp and kernel are sink organs and only well separated physically at 16 WAP. Cell development and lipid accumulation are contributed to by the deposition of phosphates before and during lipid biosynthesis (Li et al. 2011). Compared to Arabidopsis seeds, oil palm ATP-dependent enzymes are reported to show a preferential up-regulation over pyrophosphate-dependent steps of sucrose breakdown and early glycolysis, suggesting that ATP availability might be an important factor related to higher levels of oil (Bourgis et al. 2011). Comparison of ATP levels between high and low yielding palms further established its importance by showing significantly lower concentration in high yielding oil palms throughout fruit developmental, but most markedly during the period of lipid biosynthesis (16–20 WAP). The low concentration of ATP is likely a result of increased energy demand

Table 2 Fatty acids composition of palm oil and kernel lipid extract Fatty acid composition, % Mesocarp lipid extract Lauric acid (12:0) Myristic acid (14:0) Palmitic acid (16:0) Stearic acid (18:0) Oleic acid (18:1) Linoleic acid (18:2) Linoleneic acid (18:3) Arachidinic acid (20:0)

0.72±0.11 38.14±2.17 5.42±0.93 42.88±5.39 11.9±1.04 0.53±0.049 0.4±0.075

Kernel lipid extract 50.12±4.39 18.24±1.26 10.2±0.55 2.5±0.02 16.15±1.22 2.79±0.25

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required to produce the observed differences in lipid production levels in the high yielding oil palms (Teh et al. 2013). ATP in palm mesocarp was found in higher concentration compared to palm kernel, while palm kernel contained higher ADP. In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acylcoenzyme A (CoA) before their elongation by acyl-CoA synthase (ACS), and that the ATP dependence of fatty acid elongation is evidence of its involvement (Hlousek-Radojcic et al. 1998; Ranjan and Randhir 1996; Rawsthorne 2002; Lung and Weselake 2006). Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA (Harwood 1988; Salas et al. 2000; Berg et al. 2002) (Fig. 3) Hlousek-Radojcic et al. showed that stearic acid ([1-14C]18:0)-CoA was synthesized from [1-14C]18:0 in the presence of CoA-free malonylCoA or acetyl-CoA, and that [1-14C]18:0-CoA synthesis under these conditions was ATP dependent. In Brassica napus seeds, both microsomes and oil body fractions have acyl-CoA elongation ATP-dependent activity. While ATP is essential, ADP may limit FA biosynthesis, presumably because of participation in the shuttle mechanism for regeneration of ATP. This may explain why kernel oil is more concentrated with shorter chain FAs compared to mesocarp oil (Table 2). Mesocarp contained more than three times the ATP and NADH than kernel, at the same time as producing almost three times amount of C18 FA. This is possibly related to FA synthesis requiring at least seven ATP and 14 NADPH to assemble an 18-carbon FA (Ohlrogge and Jaworski 1997). NAD+ content in palm mesocarp was found to be higher than in palm kernel (Table 1). In fatty acid biosynthesis, unsaturated FAs are produced from the saturated FAs and the surplus hydrogen is transferred onto NAD+. This finding is correlated to the level of unsaturation of FA in palm mesocarp oil being higher compared to palm kernel oil, where the iodine values are 52.80 and 18.10, respectively (Basiron et al. 2000). Unsaturation of fatty acids was also found to be higher in corn exocarp compared kernel at all stages of corn kernel development (Saoussem et al. 2009). According to Harwood (1996), experiments carried out on safflower, corn and castor bean have proven that NADH acts as an electron source to enhance the desaturation of oleate to linoleate. The desaturation of oleate to linoleate was reduced by 93 % when the safflower NADH-dependent cytochrome b5 was inhibited by antibodies raised against Brassica oleracea cytochrome b5. NAD+ may also be related to total lipid content. Total lipid determination indicated that palm mesocarp produced 28.2±0.2 % (oil extracted/fresh fruitlet) of palm oil, which was higher compared to the oil content in palm kernel (18.6±0.2 %). In transgenic Brassica napus, it is reported that sn-glycerol-3phosphate dehydrogenase catalyzes the conversion of dihydroacetone phosphate using NADH to glycerol-3phosphate (G3P) and NAD+. The G3P content of the maturing seed increased three- to four-fold, and the mature seed

displayed up to a 40 % relative increase in lipid content (Weselake et al. 2009). This also suggests that five-fold increase NADH content in palm mesocarp relative to palm kernel may correlate with the higher lipid content in palm mesocarp. ATP and NAD+, which are two cofactors that play important roles in FA synthesis were highly concentrated in palm mesocarp. This is possibly one of the factors contributing to the higher proportion of longer FA chains and greater degree of unsaturation in palm mesocarp compared to kernel. Further analysis of palm mesocarp and kernel at other stages of development, together with the metabolites involved in TAG and FA synthesis may reveal the key biosynthetic processes responsible for oil yield increases in palm. Acknowledgments This work was financially supported by Sime Darby Plantation. We are grateful for the co-operation of Sime Darby Research Centre, Carey Island, Malaysia for providing sample materials and Dr. Mohamad Sanusi Jangi for proof reading.

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