UNIVERSITI PUTRA MALAYSIA CARBON FLUX ANALYSIS OF LIPID BIOSYNTHESIS PATHWAYS IN OIL PALM (ELAEIS GUINEENSIS JACQ. TENERA) EMILY QUEK MING POH - PDF

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UNIVERSITI PUTRA MALAYSIA CARBON FLUX ANALYSIS OF LIPID BIOSYNTHESIS PATHWAYS IN OIL PALM (ELAEIS GUINEENSIS JACQ. TENERA) EMILY QUEK MING POH FPSK (M) CARBON FLUX ANALYSIS OF LIPID BIOSYNTHESIS
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UNIVERSITI PUTRA MALAYSIA CARBON FLUX ANALYSIS OF LIPID BIOSYNTHESIS PATHWAYS IN OIL PALM (ELAEIS GUINEENSIS JACQ. TENERA) EMILY QUEK MING POH FPSK (M) CARBON FLUX ANALYSIS OF LIPID BIOSYNTHESIS PATHWAYS IN OIL PALM (ELAEIS GUINEENSIS JACQ. TENERA) By ElVIIL Y QUEK MlNG POH Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Master of Science Ma ch 2002 DEDICATION I dedicate this thesis to my parents and my family. 11 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science CARBON FLUX ANALYSIS OF LIPID BIOSYNTHESIS PATHWAYS IN OIL PALM (ELAEIS GUINEENSIS JACQ. TENERA) By EMILY QUEK MING POH March 2002 Chairman: Associate Professor Ong King Kok, Ph.D. Faculty: Medicine and Health Sciences The aim of this study was to investigate the carbon flux through lipid biosynthesis pathways in the oil palm (Elaeis guineensis Jacq. tenera) using metabolic control analysis (MCA). Three types of tissue from oil palm, namely liquid culture, plastid and mesocarp were used. The results showed mesocarp tissue was the most suitable tissue for carbon flux analysis because it incorporated the radioactive precursors mainly into triacylglycerol (TAG). Further analysis on different stages of fruit development was carried out using mesocarp tissue at 12, 15 and 20 weeks after anthesis (JV AA). It was confirmed that 20-W AA mesocarp tissue was the best stage of fruit development for metabolic flux studies because it reflected biosynthesis of storage lipid. Three modes of radiolabel introduction into the oil palm fruits were investigated, namely injecting the radiolabel into the fruits still attached to the palm, injecting the radiolabel into the loose fruit and injecting the radio label into incubation mixture containing meso carp tissue slices. The level of radioactivity in fruits attached to the palm was lower than the other two modes of radiolabel introduction. Carbon flux of lipid biosynthesis pathways was modulated by temperature and the inhibitor 2-bromooctanoate. Radiolabels [1_ 14 C] III acetate and [U_1-lC] glycerol were used to monitor the carbon flux through the lipid biosynthesis pathways. Temperature caused a constraint in the distribution of radioactivity at the level of diacylglycerol acyltransferase (DAGAT). Therefore, DAGAT may be a regulatory enzyme. 2-Bromooctanoate inhibited the carbon flux of lipid biosynthesis pathways. The overall results of MCA suggested that the control of carbon flux in the oil palm may be distributed over two blocks of the lipid metabolic pathway, namely the fatty acid biosynthesis block and TAG formation block. Acetyl CoA carboxylase (ACCase) plays an important role in fatty acid biosynthesis block while DAGAT plays an important role in the TAG formation block. The molecular structure of ACCase was investigated using immunoblotting with streptavidin and screening of ACCase gene in 15-W AA oil palm meso carp cdna library. Immunoblots with streptavidin showed the presence of large molecular weight (approximately 180 kda) multifunctional ACCase and smaller molecular weight (approximately 58 kda) multisubunit ACCase in oil palm mesocarp. Screening for ACCase gene in 15-W AA oil palm mesocarp cdna library showed several strong signals corresponding to the putative -carboxyl transferase subunit of ACCase. IV Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ij azah Master Sains ANALISIS FLUKS KARBON TAP AK JALAN BIOSINTESIS LIPID DI DALAM POKOK SAWIT (ELAEIS GUINEENSIS JACQ. TENERA) Oleh EMIL Y QUEK MING POH Mac 2002 Pengerusi: Profesor Madya Ong King Kok, Ph.D. Fakulti: Perubatan dan Sains Kesihatan Matlamat kajian ini adalah untuk mengkaji fluks karbon melalui tapak jalan biosintesis lipid di dalam pokok sawit (Elaeis guineensis Jacq. tenera) dengan menggunakan analisis kawalan metabolik (MeA). Tiga jenis tisu daripada pokok sawit iaitu kultur cecair, plastid dan mesokarpa telah digunakan. Hasil menunjukkan bahawa tisu mesokarpa merupakan tisu yang sangat sesuai untuk analisis fluks karbon kerana ia menukarkan prekursor radioaktif kebanyakannya ke dalam triasilgliserol (TAG). Analisis lanjutan ke atas peringkat perkembangan buah yang berlainan telah dilakukan dengan menggunakan tisu mesokarpa pada 12, 15 dan 20 minggu selepas pendebungaan (W AA). Tisu mesokarpa pada 20 W AA telah disahkan sebagai peringkat perkembangan buah yang terbaik untuk kajian fluks metabolik kerana ia mengimbas biosintesis penyimpanan lipid. Tiga cara kemasukan penanda radioaktif ke dalam buah sawit iaitu menyuntik penanda radioaktif ke dalam buah yang masih di pokok, menyuntik penanda radioaktif ke dalam buah yang dipetik dan menyuntik penanda radioaktif ke dalam larutan eraman yang mengandungi hirisan tisu mesokarpa telah dikaji. Aras aktiviti radioaktif dalam buah yang masih di pokok adalah lebih v rendah berbanding dengan dua cara kemasukan penanda radioaktif yang lain. Fluks karbon dalam tapak jalan biosintesis lipid telah dimodulasikan oleh suhu dan perencat 2-bromooktanoat. Penanda radioaktif [1_ 14 C] asetat dan [U_ 1 4 C] gliserol telah digunakan di dalam kajian ini untuk memantau fluks karbon melalui tapak jalan biosintesis lipid. Suhu menyebabkan rencatan dalam penyebaran aktiviti radioaktif pada peringkat diasilgliserol asiltransferase (DAGAT). Oleh itu, DAGAT mungkin merupakan enzim pengawalatur. 2-Bromooktanoat mereneat fluks karbon dalam tapak jalan biosintesis lipid. Hasil keseluruhan MCA mencadangkan kawalan fluks karbon dalam buah sawit disebarkan melalui dua blok dalam tapak jalan metabolik lipid iaitu biok biosintesis asid Iemak dan biok pembentukan TAG. AsetiI-CoA karboksilase (ACCase) memainkan peranan penting dalam biok biosintesis asid Iemak manakaia DAGAT memainkan peranan penting dalam blok pembentukan TAG. Stuktur molekular ACCase telah dikaji dengan menggunakan pembiotan Imuno dengan streptavidin dan penyaringan gen ACCase dalam koleksi edna 15-W AA mesokarpa sawit. Pemblotan imuno dengan streptavidin menunjukkan kehadiran protein berberat molekul besar (kira-kira 180 kda) iaitu ACCase pelbagai-fungsi dan protein berberat molekul keeil (kira-kira 58 kda) iaitu ACCase pelbagai-subunit di dalam mesokarpa sawit. Penyaringan gen ACCase dalam koleksi edna 15-WAA mesokarpa sawit telah menunjukkan beberapa signal yang menyamai subunit p-karboksil transferase ACCase. VI ACKNOWLEDGEMENTS I would like to express my sincere appreciation and million thanks to my supervisors, Associate Professor Ong King Kok, Professor Khor Hun Teik and Dr Ravigadevi Sambanthamurthi for their suggestion, advice, support and guidance throughout my project. My appreciation and gratitude go to my parents and my family for their constant support, love and patient throughout my graduate study. My sincere thanks and gratitude are also extended to all the staff of Metabolics Laboratory especially Ms. Jane Sonia, Mr. Andy Yip, En. Jamil, En. Rahim, Pn. Jabariah and Pn. Siti Hasnah for their help towards the success of this project and also to my friends for their support. Vll I certify that an Examination Committee met on 6t h March 2002 to conduct the final examination of Emily Quek Ming Poh on her Master of Science thesis entitled Carbon Flux Analysis of Lipid Biosynthesis Pathways in Oil Palm (Elaeis guineensis Jacq. tenera) in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as fo llows: Dr Chan Hooi Har, Ph.D. Associate Professor Faculty of Medicine and Heath Sciences, Universiti Putra Malaysia (Chairman) Ong King Kok, Ph.D. Associate Professor Faculty of Medicine and Heath Sciences, Universiti Putra Malaysia (Member) Ravigadevi Sambanthamurthi, Ph.D. Advanced Biotechnology and Breeding Centre, Malaysian Palm Oil Board (Member) Khor Hun Teik, Ph.D. Professor Faculty of Medicine and Heath Sciences, Universiti Putra Malaysia (Member)... SHAMSHER MOHAMAD RAMADILI, Ph.D. ProfessorlDeputy Dean, School of Graduate Studies Universiti Putra Malaysia Date: 5 APR 2002 VUl This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Science. AINI IDERIS, Ph.D. ProfessorlDean, School of Graduate Studies, Universiti Putra Malaysia Date f 3./1,, 2002 IX DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions Date: 4 April OOJ, x TABLE OF CONTENTS DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS Page.. 11 III V Vll Vlll X XIV XV XVlll CHAPTER 2 INTRODUCTION LITERATURE REVIEW 2.1 Oil Palm 2.2 Palm Oil 2.3 Plant Lipids Classification of Lipids Functions of Lipids 2.4 Fatty Acid Biosynthesis Acyl Carrier Protein (ACP) Acetyl-CoA Acetyl-CoA Carboxylase (ACCase) Fatty Acid Synthase (F AS) Desaturase Acyl-ACP Thioesterase 2.5 Triacylglycerol Biosynthesis 2.6 The Kennedy Pathway 2.7 Metabolic Control Analysis (MCA) 2.8 Metabolic Engineering MATERIALS AND METHODS Chemicals and Reagents Tagging of Oil Palm Fruits Experimental Fruits Oil Palm Liquid Cultures Preparation of Meso carp Tissue Slices Isolation of Crude Plastids Bio-Rad Protein Assay Radioactive Incorporation [l Y C] Acetate Incorporation in Liquid Cultures [1_ 14 C] Acetate Incorporation in Plastids [1_ 14 C] Acetate or [U_ 14 C] Glycerol Incorporation in Mesocarp Tissue Slices Xl 3.9 Mode of Radio label Uptake into the Oil Palm Fruits 3.10 Determination of ph Optimum Time Course Experiment of Incorporation of Radio label Optimisation ofmes-naoh Concentration Effect of Temperature Inhibition with 2-Bromooctanoate Preparation of2-bromooctanoate Solution Incubation with 2-Bromooctanoate Analysis of Radioactive Incorporation Products 3.16 Thin Layer Chromatography (TLC) Preparation oftlc Plates 38 3.l6.2 Application of Samples onto TLC Plate Separation of Lipid Classes Detection of Lipids Determination of Radioactivity of Separated Lipids 40 3.l7 Separation of Acyl-ACPs and Acyl-CoAs Liquid Scintillation Counting 41 3.l9 Extraction of Proteins Acetyl-CoA Carboxylase (ACCase) Assay Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) Preparation of SDS-P AGE Gel Electrophoresis Conditions for SDS-PAGE Development of SDS-PAGE Gel vvestern Blotting Preparation for Western Blotting Assembly of the Western Blotting Unit Immunoblotting with Streptavidin Preparation of Detection Solution Immunob lotting 3.24 Preliminary ACCase Gene Isolation Preparation of ACCase Probe Screening of ACCase Gene in 15-W AA Mesocarp cdna Library Storage of Transformed Cells 3.25 Statistical Analysis RESULTS AND DISCUSSION Conditions for Incorporation of Radiolabel in Crude Plastids Determination of Suitable Tissue Mode of Radiolabel Uptake into the Oil Palm Fruits Determination of ph Optimum Time Course Experiment of Incorporation of Radiolabel Optimisation ofivles-naoh Concentration Effect of Temperature on Lipid Biosynthesis in Oil Palm Mesocarp Tissue Effect of Temperature on the Incorporation of Radioactivity into Total Lipids in Oil Palm Mesocarp Tissue 73 XII 4.7.2 Effect of Temperature on the Distribution of Radio label into Lipid Classes in Oil Palm Mesocarp Tissue Effect of Temperature on Acyl-ACP and Acyl-CoA Pools in Oil Palm Mesocarp Tissue Manipulation of Lipid Biosynthesis in Oil Palm Mesocarp Tissue by the Inhibitor 2-Bromooctanoate Effect of2-bromooctanoate on the Incorporation of [1-14 C] Acetate into Total Lipids in Oil Palm Mesocarp Tissue Effect of2-bromooctanoate on the Incorporation of [1_ 14 C] Acetate into Lipid Classes in Oil Palm Mesocarp Tissue Effect of2-bromooctanoate on Acyl-ACP and Acyl-CoA Pools in Oil Palm Mesocarp Tissue Effect of2-bromooctanoate on the Incorporation of [U } 4 C] Glycerol into Total Lipids in Oil Palm Mesocarp Tissue Effect of2-bromooctanoate on the Distribution of [U )4 C] Glycerol into Lipid Classes in Oil Palm Mesocarp Tissue Application of Metabolic Control Analysis (MCA) on the Lipid Biosynthesis Pathway in Oil Palm Mesocarp Tissue Acetyl-CoA Carboxylase (ACCase) Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) Immunoblotting with Streptavidin Preliminary ACCase Gene Isolation Future Studies CONCLUSION 105 REFERENCES 107 APPENDIX VITAE 119 Xlll LIST OF TABLES Table Page 2.1 Lipid classification (King, 1996) The percentage (%) incorporation of radioactivity into total lipids in the plastid without and with cofactors, ACP, ATP, NADH and NADPH The percentage (%) incorporation of [1_ 14 C] acetate into total lipids in the plastid, liquid culture and mesocarp tissue system The percentage (%) distribution of [1 }4 C] acetate into lipid classes in the plastid, liquid culture and mesocarp tissue system The percentage (%) distribution of [1_ 14 C] acetate into lipid classes in 12-W AA, 16-W AA and 20-W AA oil palm mesocarp tissue The percentage (%) incorporation of radioactivity into total lipids in three different modes of radiolabel uptake into 20-W AA oil palm fruits The percentage (%) distribution of [1_ 14 C] acetate into lipid classes in three different modes of radio label uptake into 20-W AA oil palm fruits The calculation of elasticity coefficients and flux control coefficients of the lipid biosynthesis pathway of the oil palm mesocarp tissue affected by temperature perturbation (see Appendix 1). 94 XIV LIST OF FIGURES Figure 2. 1 Oil palm fruit. 2.2 Fatty acid biosynthesis pathway in plants. 2.3 The Kennedy pathway in plants. 2.4 Top-down metabolic control analysis (TDCA). 2.5 Bottom-up metabolic control analysis (BUCA). 3.1 A 25-ml conical flask containing the CO2 trap and the radioactive incubation mixture. Page Assembly of the Western blotting unit The separation of radio labelled lipids by TLC. 1: Radiolabelled lipids extracted from mesocarp tissue. 2: Radiolabelled lipids extracted from plastid. 3: Radiolabelled lipids extracted from liquid cultures. 4: Lipid standards (Sigma) Optimisation of ph for the incorporation of radioactivity The percentage (%) incorporation of radioactivity measured at one-hour intervals The percentage (%) incorporation of radioactivity for vanous concentrations ofwies-naoh buffer. 4.5 The percentage (%) incorporation of radioactivity into total lipids at various temperatures. 4.6 The percentage (%) incorporation of radioactivity into lipid classes at various temperatures. 4.7 The separation of the acyl-acps and acyl-coas through the SEP-PAK C 1 8 column (Waters). 4.S Effect of various temperatures on the incorporation of radioactivity into acyl-acps in 20-WAA oil palm mesocarp tissue SO Sl 4.9 Effect of various temperatures on the incorporation of radioactivity into acyl-coas in 20-WAA oil palm mesocarp tissue. 81 xv 4.10 Effect of vanous concentrations of 2-bromooctanoate on the incorporation of [1_ 14 C] acetate into total lipids in 20-W AA oil palm mesocarp tissue Effect of various concentrations of 2-bromooctanoate on the distribution of [1_ 1 4 C] acetate into lipid classes in 20-WAA oil palm mesocarp tissue Effect of vanous concentrations of 2-bromooctanoate on the incorporation of radioactivity into acyl-acps in 20-W AA oil palm meso carp tissue Effect of vanous concentrations of 2-bromooctanoate on the incorporation of radioactivity into acyl-coas in 20-W AA oil palm meso carp tissue Effect of vanous concentrations of 2-bromooctanoate on the incorporation of [U_ 1 4 C] glycerol into total lipids in 20-WAA oil palm mesocarp tissue Effect of various concentrations of 2-bromooctanoate on distribution of [U_ 1 4 C] glycerol into lipid classes in 20-WAA oil palm mesocarp tissue Top-dovvn approach ofmca on lipid biosynthesis pathway (a) SDS -P AGE using standard gel to separate the crude proteins extracted from 15-WAA mesocarp. The standard gel consisted of a separating gel (12%) and a stacking gel (4%). Well 1: crude proteins extracted from 15-W AA mesocarp, and well 2: high range protein molecular weight markers (Bio-Rad). (b) Graph of 10glO molecular weight against distance of migration SDS-PAGE using gradient gel to separate the crude proteins extracted from 15-WAA mesocarp. The gradient gel consisted of a separating gel (5-20%) and a stacking gel (4%). Well 1 and 2: crude proteins extracted from ls-waa mesocarp, and well 3: high range biotin-labelled calibration proteins (Boehringer Mannheim) Immunoblotting of separated proteins with streptavidin. The highlighted bands indicate the biotin-containing proteins. These proteins were detected using chemiluminescence blotting kit (Boehringer Mannheim). Well 1: high range biotin-labelled calibration proteins (Boehringer Mannheim), and well 2: crude proteins extracted from ls-waa mesocarp. 101 XVl 4.20 Gel electrophoresis of restriction enzyme digestion products on 1.2% agarose. Well 1: marker 100 bp DNA ladder, well 2: uncut plasmid DNA, well 3: plasmid DNA digested with Nco I, well 4: uncut plasmid DNA, well 5: plasmid DNA digested with Hind III and Bg II, well 6: uncut plasmid DNA, well 7: plasmid DNA digested with Hind III and B g II, and well 8: marker 1 kbp DNA ladder Screening of ACCase gene in 15-WAA mesocarp cdna library. Several strong signals corresponding to putative -ct subunit of ACCase were obtained. 103 XVll LIST OF ABBREVIATIONS ACCase ACP ADP A1vlP ANOVA ATP BC BCCP BCS BSA BUCA C12:0 C16:0 C18:0 C18:1 C18:2 C18:3 CI6:0-ACP CI8:0-ACP CI8: l-acp cpm CPO CT DAG DAGAT Acetyl-CoA carboxylase Acyl carrier protein Adenosine biphosphate Adenosine monophosphate Analysis of variance Adenosine triphosphate Biotin carboxylase Biotin carboxyl carrier protein Biodegradable counting scintillant Bovine serum albumin Bottom-up metabolic control analysis Lauric acid Palmitic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Palmitoyl-ACP Stearoyl-ACP Oleoyl-ACP 16 carbons 18 carbons Curie Coenzyme A Carbon dioxide Counts per min Crude palm oil Carboxyl transferase Diacylglycerol Diacylglycerol acyltransferase XVIll DNA EDTA FAS FFA HCl HC03- HEPES H2S04 IPTG KAS KCl kda KHC03 KOH LB LPC :tvlag MgClz MgS04 l\1nclz MCA MES NaCl NADH NADPH NaH l4 C03 NaOH PAGE pfu ph PKO Deoxyribonucleic acid Ethylenediaminetetraacetic acid Fatty acid synthase Free fatty acid Hydrochloric acid Ion bicarbonate N-[2-Hydroxylethyl]piperazine-N' -2-ethanesulphonic acid Sulphuric acid Isopropylthio-p-D-galactoside p-ketoacyl-acp synthetase Potassium chloride Kilo Dalton Potassium bicarbonate Potassium hydroxide Luria-Bertani Lysophosphatidylcholine Monoacylglycerol Magnesium chloride Magnesium sulphate Manganous chloride Metabolic control analysis 2-[N-morpholino ]ethanesulphonic acid Sodium chloride Nicotinamide adenine dinucleotide reduced form Nicotinamide adenine dinucleotide phosphate reduced fonn Sodium e 4 C] bicarbonate Sodium hydroxide Polyacrylamide gel electrophoresis Plaque fonning unit Hydrogen potential Palm kernel oil XIX PL POD PI PPI ppm PVDF SD SDS sn SOC SPSS SSC TAG TBS TBST TCA TDCA TLC V var. v/v WAA w/v X-gal Phospholipid Peroxidase Inorganic phosphate Pyrophosphate Part per million Polyvinylidene difluoride Standard deviation Sodium dodecyl sulphate Stereospecific number Sodium citrate Statistical package fo r social sciences Sodium-citrate saline Triacylglycerol Tris-buffered saline TBS-Tween 20 Tricarboxylic acid Top-down metabolic control analysis Thin layer chromatography Volt Variety Volume/volume Weeks after anthesis Weight/volume 5-Bromo-4-chloro-3 -indolyl- -D-galactoside xx CHAPTER 1 INTRODUCTION Recent advances in biotechnology, such as in genomics, proteomi
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