Manipulation of cyanogenesis and starch biosynthesis in cassava

MANIPULATION OF CYANOGENESIS AND STARCH BIOSYNTHESIS IN CASSAVA ROY JOSEPH A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGEMENT I am deeply indebted to my supervisors A/P. Loh Chiang-Shiong and A/P. Yeoh Hock-Hin, Department of Biological Sciences, National University of Singapore for suggesting the field of investigation, skilful guidance, keen interest and constant encouragement throughout my graduate program. I am particularly grateful to them for allowing me to an independent research in this project. I am also thankful to Dr. Sanjay Swarup and A/P. Pan Shen Quan the members of my Ph. D. thesis committee for constant support and meaningful suggestions during our meetings. I express my sincere thanks to Mrs. Ang Swee Eng for her kind help during the whole period of my study. Prof. Norman Brisson, Montreal University, Canada is thankfully acknowledged for kindly providing some antibodies. The support and friendship rendered by my lab mates especially Mr. Yang Maocheng is greatly appreciated. All of my friends in Dr. Sanjay’s and A/P. Pan’s laboratories are acknowledged for their help during this period. Special thanks to Dr. Yu Hao, Mr. Bhinu S Pillai, Mr. Zhang Pingyu, Mr. Srinivasa Rao PS., Mrs. Veena Sujindra Rao and Mr. Gong Haibiao for their help during this study. I thankfully remember the constant encouragement and inspiration rendered by my parents and family members. It would perhaps be superfluous to thank my wife Dr. Tessy Joseph, for the constant use I have made of her thoughts and ideas in this thesis. Finally I thank the National University of Singapore for financial support given to me throughout my studies. ii CONTENTS Table of contents List of Tables List of Figures List of Abbreviations Summary Page No. i v vi viii x Chapter Introduction Chapter Literature review 2.1 Taxonomical aspects 2.1.1 Origin, distribution and nomenclature 2.1.2 Morphological descriptions 2.1.3 Economic importance 2.2 Plant breeding and agronomical aspects 2.2.1 Breeding, cytology and cytogenetics 2.2.2 Pests and diseases of cassava 2.2.3 In vitro studies on cassava 2.2.4 Plant growth, development and storage root yield 2.3 Cyanogenesis in cassava 2.3.1 Distribution of cyanogenic glucosides in higher plants 2.3.2 Linamarin and linamarase 2.3.3 Role of cyanogenesis in cassava 2.3.4 Translocation of cyanogenic glucosides 2.3.5 Nutritional value of storage roots and health problems associated with cassava 2.3.6 Importance of acyanogenic cassava 2.4 Biosynthesis and accumulation of starch 2.4.1 Occurrence of starch in plants 2.4.2 Composition, structure and physico-chemical properties 2.4.3 The pathway of starch synthesis and enzymes 2.4.4 Regulation of starch biosynthesis 2.4.5 Starch degradation 2.5 Starch manipulation and its potential applications Chapter Biosynthesis, accumulation and translocation of cyanogenic glucosides in growing cassava plants 3.1 Introduction 3.2 Materials and methods 3.2.1 Plant materials 3.2.2 Determination of linamarin content of the tissues 3.2.3 In vivo biosynthesis of linamarin in cassava tissues 5 10 11 11 13 13 15 17 17 18 19 20 22 23 24 24 25 29 38 44 45 47 47 49 49 49 50 i 3.2.4 Rate of in vivo biosynthesis of linamarin in various tissues 3.2.5 Micrografting and 14C-labelling 3.3 Results and discussion 3.3.1 Leaf age and linamarin level of field grown plants 3.3.2 Linamarin content and biosynthesis in various tissues 3.3.3 In vivo biosynthesis of linamarin in cassava tissues 3.3.4 Growth characteristics and movement of labelled products in the micrografted plants 3.4 Conclusion P Chapter P Somatic embryogenesis, induced mutations and attempts for genetic transformation in cassava 4.1 Introduction 4.2 Materials and methods 4.2.1 Plant materials 4.2.2 Somatic embryogenesis and regeneration of plantlets in cassava 4.2.3 Agrobacterium-mediated plant transformation 4.2.4 Gamma (γ) irradiation of the somatic embryo explants 4.2.5 Field evaluation and morphological screening for mutants 4.2.6 Determination of amounts of photosynthetic pigments 4.2.7 Linamarin content 4.2.8 Linamarase assay 4.2.9 Protein determination 4.2.10 Starch content 4.2.11 Determination of amylose content in storage roots 4.3 Results and discussion 4.3.1 Somatic embryogenesis and Agrobacterium-mediated plant transformation 4.3.2 Radiation sensitivity of the somatic embryo explants 4.3.3 Regeneration of somatic embryos and screening for mutants 4.3.4 Field evaluation of mutant lines 4.3.5 Cyanogenesis and protein content in mutant lines 4.3.6 Storage root yield, morphology and starch content 4.3.7 Further characterization of the mutant lines 4.4 Conclusion 51 51 52 52 54 57 62 69 70 70 71 71 72 73 74 74 75 77 77 77 78 78 79 79 81 83 85 89 91 95 95 ii Chapter Characterisation of the starch from cassava mutant S9 plants 5.1 Introduction 5.2 Materials and methods 5.2.1 Starch content of storage root at different stages of growth 5.2.2 Amylose content in storage root starch during growth period 5.2.3 Measurement of starch gelatinization properties 5.2.4 Viscosity measurements 5.2.5 Baking properties 5.2.6 Size distribution of starch granules 5.2.7 Scanning electron microscopy 5.2.8 Wide-angle X-ray diffraction analysis of starch 5.2.9 MALDI-TOF MS analysis of starch samples 5.3 Results and discussion 5.3.1 Starch and amylose content during the growth period 5.3.2 Physico-chemical properties of the mutant S9 plant starch 5.3.3 Granule morphology 5.3.4 Starch molecular structure 5.4 Conclusion Chapter Enzyme activities associated with starch biosynthesis in storage roots of mutant cassava S9 plants 6.1 Introduction 6.2 Materials and methods 6.2.1 Plant materials 6.2.2 Enzyme preparation 6.2.3 Sucrose synthase (SuS) assay (EC 2.4.1.13) 6.2.4 UDP-glucose pyrophosphorylase (UGPase) assay (EC 2.7.7.9) 6.2.5 Phosphoglucomutase (PGM) assay (EC 5.4.2.2) 6.2.6 ADP-glucose pyrophosphorylase (AGPase) assay (EC 2.7.7.27) 6.2.7 Soluble starch synthase (SS) assay (EC 2.4.1.2.1) 6.2.8 Granule-bound starch synthase (GBSS) assay (EC 2.4.1.21) 6.2.9 Starch branching enzyme (SBE) assay (EC 2.4.1.18) 6.2.10 Starch phosphorylase (SP) assay (EC 2.4.1.1) 6.3 Results and discussion 6.3.1 Assay of enzymes 6.3.2 Activity of major enzymes that control sucrose metabolism 97 97 99 99 99 99 100 100 101 101 101 102 103 103 108 117 120 131 133 133 135 135 135 137 137 137 138 138 139 140 141 141 141 146 iii 6.3.3 Activity of enzymes that control starch biosynthesis 6.4 Conclusion Chapter Genetic variation and gene expression studies in the cassava mutant S9 plants 7.1 Introduction 7.2 Materials and methods 7.2.1 Isozyme studies 7.2.2 Cytological studies 7.2.3 Analysis for DNA content by flow cytometry 7.2.4 Extraction and SDS-PAGE of leaf and storage root soluble proteins 7.2.5 MALDI-TOF-MS identification of root proteins 7.2.6 Immunoblot analysis for starch phosphorylase enzyme 7.2.7 Nucleic acid isolation 7.2.8 RT-PCR experiments 7.2.9 Cloning and sequencing of PCR products 7.2.10 cDNA gel blot analysis for gene expression studies 7.2.11 RNA differential display analysis 7.3 Results and discussion 7.3.1 Isozyme pattern and cytological observations 7.3.2 SDS-PAGE profiles of leaf and root proteins 7.3.3 7.3.4 7.4 Conclusion Chapter References RT-PCR and cDNA blot studies of AGPaseB, GBSSI and SBE genes Differential expression of GTP binding proteins General conclusion and future perspectives 150 157 158 158 160 160 161 162 163 164 165 166 166 168 169 170 171 171 176 184 192 195 196 199 iv List of Tables Table 3.1 Table 3.2 Leaf position and linamarin content in leaf tissues of different varieties of cassava Linamarin content in various tissues of field grown plants of cassava variety PRC 60a. Table 3.3 Radioactivity (nCi/gFW) of all 14C-incorporated compounds and linamarin in micrografted plants of cassava variety PRC 60a. Table 4.1 The minimum descriptor list to explain the morphological features of different accessions of cassava accepted by the International Cassava Genetic Resources Network Morphological parameters of the 10-month-old field grown mutant plants Details of leaf morphology of the mutant lines at 10-months of growth Cyanogenesis and total protein content in the mutant lines. Storage root yield and starch content of wild-type and mutant cassava plants DSC gelatinization temperatures and enthalpy changes of the starch samples Biochemical activity of the major enzymes of sucrose metabolism in storage roots of cassava Biochemical activity of starch-biosynthetic enzymes in the storage roots of cassava Table 4.2 Table 4.3 Table 4.4 Table 4.5 Table 5.1 Table 6.1 Table 6.2 14 53 55 C- 65 76 86 87 90 92 112 147 152 v List of Figures Fig. 2.1 Fig. 2.2 Fig. 3.1 Morphological details of mature cassava plant. Possible fates of sucrose in the cells of developing, nonphotosynthetic storage organs Biosynthesis of linamarin in various tissues of field grown cassava plants (variety PRC 60a) after incubation with 14C valine Micrografted plant of cassava (variety PRC 60a) after 12-days of growth Various developmental stages of somatic embryogenesis and induction of mutation in cassava variety PRC 60a. Morphological mutants obtained through γ-irradiation in cassava variety PRC 60a. Starch content in the cassava storage roots harvested at each month of growth Amylose content in the starch samples isolated from different cassava plants at various stages of growth Iodine affinity of cassava starch samples collected from wild-type as well as mutant S9 plants at various stages of growth Effects of concentration of urea on gelatinization of starch granules from the cassava plants Effect of M urea on the swelling of starch granules of wild-type and mutant S9 plants Changes in viscosity of the starch samples Starch granule size distribution in 10-month-old cassava plants Scanning electron microscopic images of starch grains isolated from both wild-type and mutant S9 plants at 3-, 6- and 10-months of growth X-ray diffraction pattern of the starch samples from different plants of cassava MALDI-TOF-MS spectrum of starch samples from 3-month-old PRC 60a and mutant S9 plants MALDI-TOF-MS spectrum of starch samples from 5-month-old PRC 60a and mutant S9 plants MALDI-TOF-MS spectrum of starch samples from 7-month-old PRC 60a and mutant S9 plants MALDI-TOF-MS spectrum of total starch samples from 10-monthold PRC 60a and mutant S9 plants Rate of reaction in assays of enzymes with respect to volume of extract used Rate of reaction in the assays of enzymes with respect to amount of starch used Rate of reaction in the assays of enzymes with respect to time Rate of reaction in the assays of enzymes with respect to time Isozyme pattern of the cassava leaf soluble proteins P Fig. 3.2 Fig. 4.1 Fig. 4.2 Fig. 5.1 Fig. 5.2 Fig. 5.3 Fig. 5.4 Fig. 5.5 Fig. 5.6 Fig. 5.7 Fig. 5.8 Fig. 5.9 Fig. 5.10 Fig. 5.11 Fig. 5.12 Fig. 5.13 Fig. 6.1 Fig. 6.2 Fig. 6.3 Fig. 6.4 Fig. 7.1 P 7, 30 58 63 80 88 104 105 107 109 110 115 118 119 121 125 126 127 128 142 143 144 145 172 vi Fig. 7.2 Fig. 7.3 Fig. 7.4 Fig. 7.5 Fig. 7.6 Fig. 7.7 Fig. 7.8 Fig. 7.9 Fig. 7.10 Fig. 7.11 Photomicrographs of somatic chromosomes of cassava Flow cytogram of nuclei from PRC 60a and mutant S9 plants SDS-PAGE protein profile of soluble extracts from cassava leaves SDS-PAGE profile of soluble proteins from cassava storage roots Mass spectral data of two proteins identified by MALDI-TOF-MS, which were differentially present in the wild-type (PRC 60a plants) total storage root soluble proteins and absent or weekly present in mutant S9 plants Immunoblot analysis for starch phosphorylase enzyme RT-PCR experiments for 1. ADP-glucose pyrophosphorylase B (AGPase B), 2. granule-bound starch synthase I (GBSSI) and 3. starch branching enzyme (SBE) genes Electrophoresis and cDNA blot analysis for cassava starchbiosynthetic genes of PRC 60a and mutant S9 plants Differentially expressing gene of Ran like GTP binding proteins in cassava storage roots Partial DNA sequence of Ran-like GTP binding protein identified from cassava 174 175 177 178 179 181 185 187 193 194 vii LIST OF ABBREVIATIONS 2,4-D ADP AGPase amf AMP ATP BA BSA CRBC CTAB DAPI DEPC DP DSC DTT dUTP EDTA EMS FW GBSS GTP HCN HEPES HNLyase HPAEC-PAD Hsp IBA IgG MALDI-TOF MS MES MS NAD NADP NBT-BCIP PBS PGM PI 2,4-Dichlorophenoxyacetic acid Adenosine diphosphate ADP-glucose pyrophosphorylase Amylose-free Adenosine monophosphate Adenosine triphosphate 6-Benzyl aminopurine Bovine serum albumin Chicken red blood cells Hexa decyl trimethyl ammonium bromide 4',6-Diamidino-2-phenylindole Diethylpyrocarbonate Degree of polymorphism Differential scanning calorimetry Dithiothreitol 2'-Deoxyuridine 5'-triphosphate Ethylenediamine tetra-acetic acid Ethyl methane sulfonate Fresh weight Granule-bound starch synthase Guanosine triphosphate Hydrogen cyanide N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid Hydroxynitrile lyase High-performance anion-exchange chromatography with pulsed amperometric detection Heat-shock protein Indole-3-butyric acid Immuno globulin G Matrix assisted laser desorption ionisation time-offlight mass spectrometry 2-(4-Morpholino)-ethane sulfonic acid Murashig and Skoog Nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide phosphate Nitro blue tetrazolium chloride- 5-bromo-4-chloro-3indolyl phosphate Phosphate buffer saline Phosphoglucomutase Propidium iodide viii Sambrook J, Fritsch EF and Maniatis T. 1987. 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New York, pp: 23-44 Zobel HF and Stephen AM. 1995. Starch: structure, analysis and application. In: Food Polysaccharides and Their Applications, Stephen AM. (Ed.), Marcel Dekker, New York, p: 19 Zrenner R, Salanoubat M, Willmitzer L and Sonnewald U. 1995. Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). The Plant Journal, 7: 97-107 Zrenner R, Schuler K and Sonnewald U. 1996. Soluble acid invertase determines the hexose-tosucrose ratio in cold-stored potato tubers. Planta, 198: 246-252 254 [...]... relatively rich in vitamin C and calcium but poor in proteins and other vitamins or minerals In addition to the low protein content, the protein has also been found to be deficient in the essential amino acids (Splittstoesser and Martin, 1975; Yeoh and Truong, 1996) The main amino acids found in cassava flour by Close et al (1953) were glutamic acid, ornithine, alanine, aspartic acid, lysine and arginine while... First, starch is a major component in harvested parts of many crops and an understanding of regulation of its biosynthesis will aid to make directed changes in composition/quality Second, increase in human population and shrinking land and water resources are threatening the right to food in many regions of the world This demands working strategies to produce a range of cultivars with starches of different... manipulate cyanogenesis and starch biosynthesis in cassava Biosynthesis, accumulation and translocation of cyanogenic glucoside linamarin in various tissues of cassava plants were studied It was found that leaf, petiole, stem and root tissues could synthesize linamarin and it accumulated to different levels in these organs Radiolabelling studies failed to detect any translocation of linamarin into the... contrast to germinating cassava seedlings there is less evidence to support the operation of a linustatin pathway in mature cassava plants There are several contradictory reports on the presence of linustatin in mature cassava tissues Linustatin has been detected in very low quantities from mature cassava tissues by Selmar (1994) However, several other investigators failed to detect linustatin in cassava plant... germinating seeds indicated that the biosynthesis and degradation of cyanogenic glucosides occurred initially in the roots Enzyme systems from microsomes, which catalysed the synthesis of the aglycone of linamarin and lotaustralin have been studied from etiolated cassava seedlings (Bokanga et al., 1994; Koch et al., 1992) Distributions of linamarin and its metabolizing enzymes linamarase, rhodanase and. .. significance and implications of scope of manipulating starch biosynthesis or cyanogenesis are discussed The work presented here demonstrates the use of somatic embryos for mutation studies The scope of cassava mutant S9 plants as a genetic resource for further research on starch biosynthesis has also been discussed These findings would enhance our understanding on cyanogenesis and starch biosynthesis in cassava. .. countries In Africa, Latin America and Asia, about 70, 35-40 and 40% of the cassava produced, respectively, is used for human consumption Cassava is a cheap source of calories and often supplements where there are insufficient rice supplies According to Food and Agricultural Organization (FAO) reports, cassava production and utilization is increasing and the estimate of world cassava production in 2001... composition and quality in storage roots of cassava To achieve these goals this study explored the scope of gene transformation and induced mutation techniques in order to obtain variations in the cassava germplasm Furthermore, the biochemical and molecular regulation of starch biosynthesis in cassava was attempted in general and compared with a mutant cassava which has less or no storage root formation in. .. βcyanoalanine synthase in various tissues and different cultivars with high and low cyanide have been studied by Nambisan and Sundaresan (1994) Even if the HCN content of the inner part of the root is less than that in leaves and bark of the tuberous roots, the 17 protein content of M esculenta leaves is much higher than the inner part of the storage roots (Yeoh and Paul, 1989) 2.3.2 Linamarin and linamarase... potential (Bellotti and van Schoonhoven, 1978) 2.3.4 Translocation of cyanogenic glucosides The physiological basis of the cultivar-dependent differences in root linamarin content remains one of the controversial aspects of cyanogenesis in cassava Several researchers believe that linamarin is synthesized in leaves and transported to roots Stemgirdling experiments have indicated that linamarin could be transported . 3.2.2 Determination of linamarin content of the tissues 49 3.2.3 In vivo biosynthesis of linamarin in cassava tissues 50 i ii 3.2.4 Rate of in vivo biosynthesis of linamarin in various. certain knowledge of the way in which properties of starch are determined during its biosynthesis. Therefore, an understanding of the biochemistry, physiology and genetics of starch biosynthesis. Cyanogenesis in cassava 17 2.3.1 Distribution of cyanogenic glucosides in higher plants 17 2.3.2 Linamarin and linamarase 18 2.3.3 Role of cyanogenesis in cassava 19 2.3.4 Translocation of