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1 Title Isolation, characterization, and expression analysis of genes encodingstarch synthesizing enzymes from grain amaranth Author(s) Lu, Bei.; 呂蓓. Citation Issue Date 2006 URL Rights The author retains all proprietary rights, (such as patent rights) and the right to use in future works.

2 ISOLATION, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF GENES ENCODING STARCH SYNTHESIZING ENZYMES FROM GRAIN AMARANTH by Lu Bei B.Sc. Graduate School of Chinese Academy of Agricultural Sciences; M.A. China Agricultural University A thesis submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy at The University of Hong Kong January 2006

3 Abstract of the thesis entitled ISOLATION, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF GENES ENCODING STARCH SYNTHESIZING ENZYMES FROM GRAIN AMARANTH Submitted by Lu Bei for the degree of Doctor of Philosophy at The University of Hong Kong in January 2006 Amaranth species (Amaranthus) are C4 photosynthetic plants that possess drought resistance and can grow well in saline, alkaline, acidic or poor soil. Grain amaranth (A. cruentus, A. hypochondriacus, or A. caudatus) is a pseudo-cereal which contains a high proportion of starch in the grain, similar in concentration to that in cereal grains. Storage starch is present as highly organized, water insoluble granules, which vary in size in different plants from 1 to 100 µm. Amaranth starch has the smallest granules found in nature and typically has very low amylose concentration (7.8%), with some genotypes having almost no amylose (0.2%). The physical properties of the starch suggest

4 that amaranth amylopectin contains a high proportion of short chains, as compared to other plant starches. Therefore, grain amaranth is an ideal system for studying starch biosynthesis, to determine the activity and regulation of key biosynthetic genes that confer unique properties on its starch. In this project, granule-bound starch synthase (GBSS), starch soluble synthase (SS) and starch branching enzyme (SBE) were identified by immunoassay in A. cruentus. To isolate starch synthesizing genes, a cdna library was constructed from developing seeds of A. cruentus. The genes (full-length cdnas) encoding two major starch synthesizing enzymes, A. cruentus starch synthase II (AcSSII) and A. cruentus starch branching enzyme I (AcSBEI), were isolated and identified by cdna library screening. The deduced amino acid sequence of AcSSII contains a flexible arm, a conserved KTGGL motif, and a KTGGL-like motif, whereas the deduced sequence of AcSBEI consists of a catalytic (β/α) 8 -barrel and four highly conserved regions (HSHAS/GFRFDGVT/AEDVS/AESHDQ). These motifs can also be found in other SSs and SBEs of different higher plants. Southern and northern blotting analyses indicated that AcSSII and AcSBEI are encoded by low-copy genes and highly expressed in the developing seed. In vitro analysis also showed that both AcSSII and AcSBEI are active. The phylogenetic analysis of AcSSII and AcSBEI based on their amino acid sequences showed that the divergence of SSIIs in higher plants proceeded in line with the evolution of monocotyledons from dicotyledons and AcSBEI was placed in family B along with most SBEIs from other higher plants. Both AcSSII and AcSBEI exhibited, respectively, a high sequence similarity to the SSs and

5 SBEs of dicotyledons, even though the starch of grain amaranth is physically similar to cereal starch. Further research is needed to characterize the functionality of AcSSII and AcSBEI to shed light on the mechanism that confers the unusual starch properties on amaranth grains.

6 ISOLATION, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF GENES ENCODING STARCH SYNTHESIZING ENZYMES FROM GRAIN AMARANTH by Lu Bei B.Sc. Graduate School of Chinese Academy of Agricultural Sciences; M.A. China Agricultural University A thesis submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy at The University of Hong Kong January 2006

7 Declaration I declare that this thesis represents my own work, except where due acknowledgement is made, and that it has not been previously included in a thesis dissertation or report submitted to this university or to any other institution for a degree, diploma or other qualification. Signed Lu Bei i

8 ACKNOWLEDGEMENT First, I would love to express my heartiest gratitude to my supervisors, Dr. Mei Sun and Harold Corke, for their insightful advice and full support during my doctoral studying. Dr. Ravi N. Chibbar is also deeply appreciated for his kindness in offering me his lab and facilities for my study. Besides that, I benefited much from these advisors who have always encouraged me to be strong in front of difficulties. Moreover, I would thank Dr. Huang Junchao who gave me scientific suggestions and warm friendship during my study in The University of Hong Kong. And respectable scientists Dr. Monica Båga and Pooba Ganeshan in the lab in Canada have deeply inspired me by their non-stop research attitude when I worked there. In addition, I also want to acknowledge the technical assistance from Ms. Rebecca Hu. Many thanks to my labmates, Indira, Chandra, Sanjay, Marin, Rajender, and Yogi in the University of Saskatchewan, Canada, for their enthusiastic and selfless help. Many thanks to my friends Naiyee Jay, Shuhua Zou, Cindy, and Juan Wang who are in The University of Saskatchewan, Canada; friends Eugenia Law, Xue Chunyi, Sun Caiyun, Wang xinyan, Fu Guodong, Zhou hui who are in The University of Hong Kong; and many other friends I cannot name their names all here. The happy memories with them will delight me forever. Finally, I would like to thank my parents and husband, Wu Jian; without their support, I cannot be who I am now. Thank them for sharing not only happiness but also depression with me during my life. ii

9 TABLE OF CONTENTS Abstract Declaration Acknowledgement Table of contents List of figures List of tables List of abbreviations List of appendices i ii iii viii xi xii xiv CHAPTER I LITERATURE REVIEW The introduction of grain amaranth and amaranth starch Grain amaranth Amaranth starch Morphological and physicochemical characteristics of amaranth starch and starch granules Starch polymers The structure of starch granules The characteristics of amaranth starch Starch biosynthesis Proteins involved in starch biosynthesis pathway Starch biosynthesis Starch biosynthesis pathways in leaves, tubers and cereal seeds Genetic modification of starch Objectives of the research project 50 iii

10 CHAPTER II IDENTIFICATION OF STARCH SYNTHESIZING ENZYMES AND THEIR ISOFORMS FROM GRAIN AMARANTH Introduction Plant materials Experimental methods GBSSs extraction from amaranth, wheat and pea seeds SDS-PAGE gel electrophoresis and silver staining Immunoblot analysis Results GBSSs separated by SDS-PAGE gel electrophoresis Identification of GBSSs from grain amaranth starch granules by immunoblot analysis Discussion 65 CHAPTER III cdna LIBRARY CONSTRUCTION FROM GRAIN AMARANTH DEVELOPING SEEDS Introduction Plant material and methods Plant material mrna isolation from grain amaranth developing seeds cdna library construction and amplification Results The high quality of total RNA extracted from grain amaranth developing seeds The first strand cdna synthesized from mrna The synthesis and size-fraction of second strand cdnas The titer of the primary library Blue-white color selection for background determination Determining the lengths of inserts in the cdna library The titer of the amplified cdna library Discussion 82 iv

11 3.4.1 The advantages of cdna library construction and screening The classification of cdna libraries and their usages The properties of a good cdna library 86 CHAPTER IV ISOLATION, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF A PUTATIVE GENE ENCODING STARCH SYNTHASE II FROM GRAIN AMARANTH Introduction Methods cdna library screening In vivo excision of the pbluescript phagemid from the Uni-ZAP XR Vector Restriction enzyme patterns digested with different restriction enzymes Sub-cloning for sequencing Sequencing strategies Sequence alignment The structure analysis of the protein (AcSSII) Southern blotting RNA expression analysis of cdna encoding AcSSII Expression of AcSsII in E. coli mutant RH Phylogenetic analysis Results Eight positive clones isolated from cdna library Restriction enzyme patterns of eight candidate clones isolated from cdna library A candidate clone isolated from the cdna library contains a full-length cdna encoding A. cruentus starch synthase II (AcSsII) The analysis of A. cruentus starch synthase II (AcSSII) protein AcSsII is a low copy gene in grain amaranth genome and highly expressed in grain amaranth developing seeds AcSsII cdna encodes an active starch synthase Phylogenetic analysis of plant starch synthase IIs Discussion 126 v

12 CHAPTER V ISOLATION, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF A PUTATIVE GENE ENCODING STARCH BRANCHING ENZYME I FROM GRAIN AMARANTH Introduction Methods RT-PCR for the isolation of a cdna encoding partial sequence of starch branching enzyme I from A. cruentus cdna library screening In vivo excision of the pbluescript phagemid from the Uni-ZAP XR Vector Restriction enzyme patterns digested with different restriction enzymes Sub-cloning for sequencing Sequencing strategies Sequence alignment The structure analysis of the protein (AcSBEI) Southern and Northern blotting Expression of AcSbeI in E. coli mutant KV Phylogenetic analysis Results The probe amplified by RT-PCR for cdna library screening Fifteen positive clones isolated from cdna library Restriction enzyme patterns of fifteen candidate clones isolated from cdna library A positive clone isolated from cdna library contains a full-length cdna fragment encoding starch branching enzyme I The structure of the protein, AcSBEI AcSbeI is a low copy gene in grain amaranth genome and highly expressed in grain amaranth developing seeds AcSbeI cdna encodes an active starch branching enzyme Phylogenetic analysis of plant starch branching enzymes Discussion The identification of AcSSII and AcSBEI isolated from grain amaranth The structure and function motifs of AcSBEI compared with other vi

13 SBEs from different plant sources 160 CHAPTER VI GENERAL DISCUSSION AND CONCLUSIONS The properties of AcSSII and AcSBEI Are the unique physicochemical characteristics of grain amaranth starch primarily determined by starch biosynthesizing enzymes? The contributions of AcSSII and AcSBEI to future researches The applications of starch biosynthesizing enzymes in grain amaranth breeding Conclusions 182 APPENDICES APPENDIX I Nucleotide and deduced amino acid sequences of cdna encoding a putative SSII in grain amaranth 184 APPENDIX II Nucleotide and deduced amino acid sequences of cdna encoding a putative SBEI in grain amaranth 185 APPENDIX III Formaldehyde Agarose Gel Electrophoresis for the determination of total RNA concentration 186 APPENDIX IV Make T-vector 188 APPENDIX V Preparation of Media and Reagents 189 APPENDIX VI Analysis tools provided by Internet servers 193 REFERENCES 194 vii

14 LIST OF FIGURES Fig 1.1 The molecular structure of amylose and amylopectin 6 Fig 1.2 Schematic view of the hierarchical order within the starch granule 10 Fig 1.3 A cluster model for amylopectin structure 11 Fig 1.4 Fig 1.5 Fig 1.6 A potato starch granule showing growth rings and Maltese cross under a light microscope 12 Scanning electron micrographs of grain amaranth and potato starch granules 15 The catalysis characteristics of starch synthases involved in starch biosynthesis 18 Fig 1.7 The starch biosynthesis pathway from sucrose which is the product of photosynthesis 46 Fig 2.1 SDS-PAGE gel of GBSSs isolated from grain amaranth, wheat and pea seeds 60 Fig 2.2 The result of Western blotting 63 Fig 3.1 The flow chart of cdna library construction 68 Fig 3.2 Map of the Uni-ZAP XR insertion vector 69 Fig 3.3 Circular map and polylinker sequence of the pbluescript SK (+/-) phagemid 69 Fig 3.4 cdna synthesis flow chart 70 Fig 3.5 Total RNA on denatured formaldehyde RNA gel 77 Fig 3.6 The first strand cdnas for cdna library construction 78 Fig 3.7 The size-fractionated second-strand cdna 79 Fig 3.8 Determination of the lengths of inserts of a cdna library 81 viii

15 Fig 4.1 The result of cdna library screening for Amaranthus cruentus starch synthase II (AcSsII) gene isolation 100 Fig 4.2 An example of restriction patterns from a candidate clone digested with various restriction enzymes 101 Fig 4.3 Protein-to-protein alignment result of unknown protein isolated from cdna library 106 Fig 4.4 The alignment between amino acid sequences of the unknown protein and Solanum tuberosum starch synthase II 107 Fig 4.5 The amino acid sequence comparison between the unknown protein and S. tuberosum starch synthase II 108 Fig 4.6 The secondary structure of Amaranthus cruentus starch synthase II 113 Fig 4.7 The 3D prediction of core region from Amaranthus cruentus starch synthase II 116 Fig 4.8 The 3D structure of 1ruz_A 117 Fig 4.9 Secondary structure of core region from Amaranthus cruentus starch synthase II 118 Fig 4.10 The result of Southern blotting for AcSsII gene 120 Fig 4.11 The result of Northern blotting, showing different tissues hybridized with a cdna fragment from putative AcSs II gene 121 Fig 4.12 Complementation analysis of AcSSII activity in E. coli 123 Fig 4.13 Cladograms of starch biosynthesizing enzymes in higher plants and glycogen synthase in E. coli 125 Fig 5.1 The amplification of partial sbe cdna from grain amaranth by RT-PCR 137 Fig 5.2 The alignment between Query (sbe-412 cdna from grain amaranth) and Sbjct [Triticum aestivum sbe1 gene] 138 Fig 5.3 The result of cdna library screening for A. cruentus starch branching enzyme I (AcSbeI) gene isolation 140 ix

16 Fig 5.4 The restriction enzyme patterns of two candidate cdna clones isolated from grain amaranth developing seed cdna library using sbe-412 as the probe 141 Fig 5.5 Protein-to-protein alignment of unknown protein isolated from cdna library 144 Fig 5.6 The alignment between amino acid sequences of the unknown protein and Phaseolus vulgaris starch branching enzyme Fig 5.7 The amino acid sequence comparison between the unknown protein and Solanum tuberosum starch branching enzyme I 147 Fig 5.8 The secondary structure of A. cruentus starch branching enzyme I 149 Fig 5.9 The 3D prediction of core region from A. cruentus starch branching enzyme I 151 Fig 5.10 The 3D structure of 1m7x_C 152 Fig 5.11 The result of Southern blotting for AcSbeI gene 153 Fig 5.12 The result of Northern blotting showing different tissues hybridized with a cdna fragment from the putative AcSbeI gene 155 Fig 5.13 Complementation analysis of AcSBEI activity in E. coli 156 Fig 5.14 A single most parsimonious tree of SBEs in higher plants and alga, and GBEs in E. coli and human 159 Fig 5.15 The comparisons of N-terminal regions, C-terminal regions and PEGIPGVP sequences between SBEs from family A and from family B 165 Fig 6.1 Diagram of chimeric gene constructs from AcSSII (grain amaranth) and TaSSII (wheat) 178 x

17 LIST OF TABLE Table 1.1 Physical composition of storage starch of grain amaranth in comparison with corn, wheat, and potato starches 4 Table 1.2 The various isoforms of GBSS and SS found in plants 28 Table 1.3 Two families of SBEs from different plants 31 Table 3.1 Properties of cdna library constructed from grain amaranth developing seeds 87 Table 4.1 The GenBank accession number of starch synthases used in the phylogenetic analysis 98 Table 4.2 The physicochemical parameters of Amaranthus cruentus SSII, potato SSII and wheat SSII 110 Table 5.1 PCR primers designed upon conserved sequences found in sbe genes in higher plants 131 Table 5.2 The GenBank accession number of starch branching enzymes used in the phylogenetic analysis 136 Table 5.3 The physicochemical parameters of A. cruentus SBEI, kidney bean SBEI and rice SBEI 148 xi

18 LIST OF ABBREVIATIONS 3D 3-PGA Ab AcSsII AcSSII AcSbeI AcSBEI ADPG AGPase BLAST CDD cdna CDS cpm ctp Da DBE DEPC dicots DNA DP dsdna EST EtBr GBSS GRAS GS HPAEC-ENZ-PAD IPTG kb kda LB three-dimensional 3-phospho-glycerate antibody Amaranthus cruentus starch synthase II gene Amaranthus cruentus starch synthase II Amaranthus cruentus starch branching enzyme I gene Amaranthus cruentus starch branching enzyme I adenosine diphosphate glucose ADP-glucose pyrophosphorylase basic local alignment search tool conserved domain database complementary deoxyribonucleic acid coding sequence counting per minute chloroplast transit peptide dalton (molecular mass) de-branching enzyme diethyl pyrocarbonate dicotyledons deoxyribonucleic acid degree of polymerization double-stranded DNA expressed sequence tag ethidium bromide granule-bound starch synthase generally recognized as safe glucan synthase high-performance anion-exchange chromatography system equipped with a post-column enzyme reactor and a pulsed amperometric detector isopropyl-β-d-thiogalactopyranoside kilobase kilodalton Luria Bertani xii

19 monocots MOPS MOS mrna M w NC OD ORF PCR pfu Pi RNA RNAi rrna RT-PCR RVA SBE SDS-PAGE SEC SGP(s) ssdna SS SSS monocotyledons 3-(N-morpholino) propanesulfonic acid maltooligosaccharides messenger ribonucleic acid molecular weight nitrocellulose optical density open reading frame polymerase chain reaction plaques forming units orthophosphate ribonucleic acid RNA interference ribosomal ribonucleic acid reverse transcriptase - polymerase chain reaction rapid visco analyzer starch branching enzyme sodium dodecyl sulfate-polyacrylamide gel electrophoresis size-exclusion chromatography starch granule protein(s) single-stranded DNA starch synthase soluble starch synthase xiii

20 LIST OF APPENDICES APPENDIX I Nucleotide and deduced amino acid sequences of cdna encoding a putative SSII in grain amaranth 184 APPENDIX II Nucleotide and deduced amino acid sequences of cdna encoding a putative SBEI in grain amaranth 185 APPENDIX III Formaldehyde Agarose Gel Electrophoresis 186 APPENDIX IV Make T-vector 188 APPENDIX V Preparation of Media and Reagents 189 APPENDIX VI Analysis tools provided by Internet servers 193 xiv

21 Chapter I LITERATURE REVIEW 1.1 The introduction of grain amaranth and amaranth starch Grain amaranth Amaranthus belongs to the Amaranthaceae family. The genus is more than a million years old and widely distributed all over the world. Currently about 60 species are recognized in the Amaranthaceae family (National Academy of Sciences, U.S., 1984), but only three of the species are consumed by human as grains, Amaranthus hypochondriacus L., Amaranthus cruentus L., and Amaranthus caudatus L.. Grain amaranth (A. cruentus, A. hypochondriacus, or A. caudatus) was a primary food for Central American Indians but prohibited from cultivation and use by Spanish Conquerors in the early fifteenth century. However, in spite of Spanish prohibition, a handful of communities in isolated corners of Mexico continued to grow and use amaranth (Early, 1977). By 1970s, amaranth had spread around the world and become human food as grains or leaves in Africa, India and Nepal. Nowadays, amaranth is grown by a much larger number of farmers in China, Russia, parts of Eastern Europe, and South America and is reemerging as a crop in Mexico. Starting from 1970s, amaranth as a crop was researched in the United States (e.g., Marx, 1977; Jain and Hauptli, 1980). Amaranth species are C4 photosynthetic plants, whose first product of photosynthesis is a four carbon compound. The combination of their anatomical 1

22 features and C4 metabolism makes them a more efficient user of resources than most broadleaf crops when grown under high temperatures and limited rainfall conditions. It has been known that A. hypochondriacus and A. cruentus are native to Mexico and Guatemala, whereas A. caudatus is native to the Andean regions of Ecuador, Peru, and Bolivia (Sauer, 1977). Amaranthus cruentus is the earliest archaeological record of pale-seeded grain amaranth, found in Tehuacan, Puebla, Mexico, about 4000 BC (Pal and Khoshoo, 1974; Sauer, 1979). Therefore, A. cruentus is considered to be one of the oldest known food crops and probably originated in Central and South America (Grubber and van Sloten, 1981). Grain amaranths are tall plants with very broad leaves when fully grown, similar to native weedy species. The height of the plant can reach from 3 to 9 feet. The flowers are predominantly self-pollinated with purple, red, pink, orange, or green inflorescences. Seed colors of the grain species are white, tan, gold, or pink. The crop plants are indeterminate, but tend to have a dominant seedhead with fewer side branches than weedy amaranths. Both seeds and leaves of grain amaranths are edible. Each plant is capable of producing 4,000 to 6,000 tiny seeds (1-3 mm in diameter), which contain high amounts of protein (13-18%) rich in lysine and methionine, two essential amino acids that are not frequently found in all of the world s main cereal crops (Singhal and Kulkarni, 1988). The seed coat of amaranth is completely smooth and thin. In contrast to other pseudo-cereals such as quinoa and buckwheat, it is not necessary to remove the seed coat of amaranth. Hence the seeds can be used directly in most cases. 2

23 Amaranth grains or seeds have been widely used in breakfast cereals, soup, breads, pancakes, cookies, and as an ingredient in confections. It also can be milled to provide a kind of light-colored flour which has little or no gluten. As a snack, the grain is popped and has nutty-flavored taste Amaranth starch Starch as the major component of the carbohydrates is located in the perisperm of amaranth seed in the form of very small starch granules. The perisperm is in the center of amaranth seed surrounded by an embryo and the remnant of the seed is endosperm (Okuno and Sakaguchi, 1981; 1982). The starch content of amaranth grain (of total dry weight) was reported as 48% for A. cruentus and 62% for A. hypochondriacus (Becker et al., 1981; Saunders and Becker, 1984), similar to that of other starch crops such as potato, wheat, and maize. The starch granules isolated from the seed of A. hypochondriacus are the smallest found in all plant starches ( µm in diameter) and angular and polygonal in shape (Lorenz, 1981; Saunders and Becker, 1984; Stone and Lorenz, 1984), while those of A. cruentus are reported to be spherical as well as angular and polygonal (McMasters et al., 1955; Irving et al., 1981; Stone and Lorenz, 1984). In amaranth perisperm cells, starch occurs both as tiny individual granules and larger compound granules composed of hundreds of individual granules (Wilhelm et al., 1998). In the cotyledon cells the starch granules are assembled in great agglomerates consisting of several thousand single granules with a size of 80 µm (Walkowaki et al., 1997). In addition, the content of amylopectin in amaranth starch is much higher than in other crops (Table 1.1). 3

24 Table 1.1 Physical composition of storage starch of grain amaranth in comparison with corn, wheat, and potato starches Major storage Grain amaranth (Amaranthus) Corn (Zea mays) Wheat (Triticum aestivum) seed perisperm seed endosperm seed endosperm tuber Potato (Solanum tuberosum) place Granule shape spherical or angular and polygonal regular polyhedronshaped lenticular round oval Granule size µm in diameter 5-20 µm 15 µm thick and 36 µm round (mean values) µm % of Amylopectin % of Crude protein c 10.3 d 14 e - a Mean values of main species of crops b Percentage on dry weight basis c N x 5.85 d N x 5.7 e N x 6.25 Note: N is the average nitrogen content of proteins. Qian et al. (1999) reported that the fraction of amylose in A. cruentus starch is 7.8%, consistent with the report of 7.2% by Dodok (1997). However, the amylose content of A. hypochondriacus can be as low as 0.2% (Lorenz et al., 1990), and therefore they equated amaranth starch to waxy starch of cereals. Grain amaranths, quinoa, and buckwheat are known as pseudo-cereals, deriving from their production of small grain-like seeds although they are all dicotyledonous plants. Therefore, it is interesting that waxy perisperm was found in grain amaranth for the first time. Moreover, no waxy perisperm is found in other pseudo-cereals such as quinoa and buckwheat. The extremely small starch granules and high amylopectin content make the amaranth starch unique in characteristics, such as silky texture, uniform cell 4

25 structure, excellent moisture retention, good clarity, slightly cohesive properties, and stable viscosity. Amaranth starch can be used to make fat-reducing health food. More importantly, amaranth starch offers non-hazardous (environmentally safe) replacements of coating and solvent agents in tape and paper industry. Amaranth starch can be easily degraded by α-amylases because of the small starch granules. Commercial amaranth starch is a natural GRAS (generally recognized as safe) ingredient from the seed of A. cruentus. This naturally extracted powder has good dispersal ability, exhibits good water binding properties, and supports/enhances dispersions of other coating and spacing ingredients. However, since a broad diversity in starch properties is found within and among amaranth species, it should be emphasized that there is no generic or typical amaranth starch. Therefore, it is necessary to select appropriate genotypes for different applications to achieve desired functionality (Wu et al., 1995). 1.2 Morphological and physicochemical characteristics of amaranth starch and starch granules Amaranth starch granule is the smallest granule in nature. It is known that the physicochemical characteristics of granules are affected by the amount and structure of two polymers in starch, amylose and amylopectin (Bhattacharya et al., 1999; Jobling et al., 2002; Okamoto et al., 2002; Morell et al., 2003). Therefore, most of the research on amaranth grain is focused on the characteristics of starch granules and amylopectin content which affect starch physicochemical characteristics. 5

26 1.2.1 Starch polymers Starch is a complex carbohydrate polymer with high molecular weight occurring in plant storage organs such as seeds and tubers and in non-storage organs such as leaves and roots. It comprises two types of D-glucose polymer, unbranched or little-branched amylose and highly branched amylopectin (Fig 1.1). A. B. α(1,4)-linkage α(1,6)-linkage Fig 1.1 The molecular structure of A. amylose; and B. amylopectin. Amylose is the linear D-glucose and joined by α(1,4)-linkages, whereas amylopectin is a highly branched molecule in which linear chains of α(1,4)-linked glucosyl units are joined to each other by α(1,6)-linkages Amylose The molecular weight of amylose is between 5x10 5 to 10 6 and it is composed of glucosyl units connected through α(1,4) linkages. Normal amylose content of starch is typically 20-30%. Some mutant plant 6

27 genotypes, particularly of maize (Zea mays), contain starch of very high amylose content (up to 70%). However, other genotypes in a wide range of plant species, called waxy, contain starch with less than 1% amylose (maize, barley, rice, amaranth, etc.). Waxy starches typically gelatinize easily, yielding clear pastes that will not gel. A Waxy locus encoding a GBSS (granule-bound starch synthase) protein is found in most waxy mutants. These mutants provide good materials to elucidate the physiological role for starch synthesis-related enzymes (see details in section 1.3, Starch Biosynthesis). Starches with high amylose content can form hydrogen-bonded insoluble aggregations, suitable for use in adhesives, plastics and as a source of dietary-fiber starch (Doane, 1994). It is interesting to note that there is no significant difference between average molecular sizes of cereal amyloses and those of tuber and rhizome starches. A specific feature of amylose is its capability to bind iodine. When iodine is dissolved in water and dimethylsulfoxide (Me 2 SO), triiodide ions are formed. A method was developed to utilize this reaction (formation of the blue amylose-iodine complex) to provide a simple procedure for quantitative measurement of amylose in starches (Knutson, 1986). The existence of I 3 - and I 5 - in the amylose-iodine complex is measured using spectrophotometric measurements. However, brown amylopectin-iodine complexes can also form and interfere in the measurement of amylose-iodine reaction by reducing the concentration of free iodine measured by non-colorimetric methods, and may absorb at similar wavelengths to amylose-iodine complexes. Other iodine assay procedures try to overcome this 7

28 limitation, for example, the Con A method can precipitate and remove amylopectin molecules by centrifugation Amylopectin Amylopectin is a highly branched glucose chain, and it is the largest molecule commonly found in nature with a molecular weight of several millions. The branched polymer is formed of glucosyl units linked α(1,4), but additionally with 2-4% α(1,6) linked branches (Hizukuri and Takagi, 1984; Takeda et al., 1984; 1986), forming a complex structure. Amylopectin exhibits hierarchical levels of specific architectural structure (Fig 1.2) (Myers et al., 2000). 1) At the 0.1 to 1.0 nm scale, i.e. within individual chains, structure is described by branch location and chain length. A cluster model describes the three-dimensional structure of amylopectin (reviewed by Manners, 1989). The model proposes that the amylopectin molecule is made up of three broad classes of glucose chains, A, B and C. The A-chains bind in clusters only to B-chains, and B-chains bind to other B-chains or to a C-chain which has a reducing end and of which there is one per molecule (Peat et al., 1952). The ratio of A-chains to B-chains, which is also referred to as the degree of multiple branching, is an important parameter. In most plants, the ratio of A-chains and B-chains ranges from 1.0:0 to 1.5:1 which means amylopectins have more A-chains than B-chains. B-chains are subdivided into B2-, B3-, and B4-chains, with lengths corresponding to the number of glucosyl residues of two, three, and four clusters, respectively, when placed after each other in one direction. Each cluster is build 8

29 up of short chains (6-30 glucosyl residues) that also are subdivided, namely into B1-chains and A-chains, the latter representing the shortest group of chains (Hanashiro et al., 1996). 2) At the 10 nm scale, short amylopectin chains, usually free A-chains, forming double helices and associating into clusters (Jenkins et al., 1993). These clusters pack together to produce a structure of alternating crystalline and amorphous lamellar composition. Regions of amylopectin double helix formation fall within the crystalline lamellae (Oostergetel and van Bruggen, 1993). The regions between the crystalline lamellae are called amorphous lamellae, which are thought to contain some B-chains and/or C-chains and thus be less densely packed (Hizukuri, 1986). 3) The 100 nm scale is thought to be the blocklet, which is approximated as oblate spheroids with short diameters of 20 to 500 nm (Gallant et al., 1997). 4) The next structural level within the starch granules is alternating rings ranging in thickness from 120 to 500 nm (French 1984; Gidley 1992). It is thought that starch granules involve packing of amylose and other components such as lipids into a framework established by amylopectin (Myers et al., 2000). 9

30 Fig 1.2 Schematic view of the hierarchical order within the starch granule (cited and modified from Mayers et al., 2000). A) The structure of amylopectin molecule. B) The structure of amylopectin clusters which form crystalline lamellae separated by amorphous lamellae. C) The blocklet composed of crystalline lamellae and amorphous lamellae. D) The structure of growth rings in starch granule. 10

31 1.2.2 The structure of starch granules Under polarized light, starch granules in higher plants show birefringence, a characteristic of their semi-crystalline structure. In Fig 1.2B, regions of amylopectin double helix fall within the crystalline lamellae, whilst the amylopectin branch points lie in the amorphous lamellae. The amorphous region contains no ordered structures by definition and cannot be distinguished from the background. The interference pattern observed takes the form of a Maltese cross as shown in Fig 1.2D which indicates that there is an orderly arrangement of the crystalline areas within the granule (Young and Lovell, 1991). B-chains in amylopectin can be divided further by the lengths which fall in the groups of clusters (Fig 1.3) (Hanashiro et al., 1996). A chain B3 chain B1 chain Cluster repeat B2 chain Fig 1.3 A cluster model for amylopectin structure. A-chains do not carry other chains, whereas B-chains carry other A- and/or B-chains. B-chains are sub-divided into B1-, B2-, B3, and B4-chains, with lengths corresponding to the number of glucosyl residues of two, three, and four clusters. 11

32 A three-dimensional model was proposed to describe the arrangement of crystallites in potato starch granules (Oostergetel and van Bruggen, 1993). In this model it is suggested that short double helical chains in the amylopectin molecule are crystallized into 5 nm thick crystalline lamellae. These crystalline lamellae alternate with the amorphous layers in which the α(1,6) branch points are located. The crystalline lamellae have cavities each with a diameter of about 8 nm and for a more or less continuous super helical structure. The double helices forming the lamellae are packed in polymorph structure (Fig 1.4). Fig 1.4 A potato starch granule showing growth rings and Maltese cross under a light microscope (cited from A. Donald s website, poco.phy.cam.ac.uk/research/starch/ whatis.htm). Starch granules were partially digested with amylase. The region of each ring which is less susceptible to enzyme digestion is termed the semi-crystalline growth ring, with the other region being the amorphous phase. The lengths of the amylopectin branch chains can be analyzed using methods of size-exclusion chromatography (SEC) (Hizukuri 1985; 1986) or high-performance anion-exchange chromatography with a post-column enzyme reactor and a pulsed amperometric detector (HPAEC-ENZ-PAD) (Wong and Jane, 1997). Locations of branch linkages in amylopectin can also be determined by analyzing the structure of Naegeli dextrin of each starch. 12

33 Using high-performance size-exclusion chromatography (HPSEC), Klucinec and Thompson (2002) identified a shorter chain population from intracluster B-chains in several genotypes of maize. This observation provides some insight concerning the distance between adjacent branch points. In most starches, it is found that the amylopectin crystalline region of 6.3 nm to 7.4 nm is usually formed by the chain lengths with degrees of polymerization (DP) from 18 to 20 (Jane et al., 1999). Three types of polymorph detected by X-ray diffraction have been found in different starch granules. Starches of the A-type polymorph have more short branch chains (DP 14-18) and with branch linkages scattered in both amorphous and crystalline regions; those of the B-type polymorph have less short but more long branch chains (DP 45-55) with branch linkages located mainly in the amorphous region; and those of the C-type have both short and long branch chains with branching structures in between (Hizukuri 1985; 1986; 1997). A-type and B-type polymorphs differ in the geometry of their single cell units, the packing density of their double helices and in the amount of bound water within the crystal structure, A-type being more dense and binding less water than B-type. Low gelatinization temperature in starch is thought to be correlated to some physical characteristics such as short average amylopectin branch chain length (e.g., waxy rice starch), large proportions of short branch chains (DP 11-16) relative to the shoulder of DP (e.g., wheat and barley starch), and high starch phosphate monoester content (e.g., potato starch). Therefore, most A-type starches with obvious low shoulder of DP distribution have lower gelatinization temperatures. Starches of B-type polymorph but low amylose content, and those 13

34 containing substantial phosphate monoesters also exhibit lower gelatinization temperatures. Starches with amylopectin of long-branch chains display high pasting temperatures and more resistance to shear thinning (Jane et al., 1999). Although crystallinity is associated with the amylopectin component, the structural role of amylose was thought to be co-crystallinity with amylopectin within the crystalline lamellae (Kasemsuwan and Jane, 1994). Amylose may also form inclusion complexes with any lipids present within the starch granule (Robin et al., 1974). Two mechanisms to explain the disrupting effect of amylose were proposed by Jenkins and Donald (1995). One mechanism is amylose and amylopectin were co-crystalline when the starch granule was formed. And the other mechanism is perhaps that amylose may penetrate into the amorphous regions of the cluster (where the branch points are located). Both mechanisms were proposed as hypotheses that need to be tested in future The characteristics of amaranth starch Amaranth starch has extremely small size starch granules (Fig 1.5) and generally low amylose content. Lorenz (1981) reported that, compared to wheat starch, the starch of A. hypochondriacus has a much lower amylose content, lower swelling power, higher solubility, greater water uptake, lower amylograph viscosity, and higher gelatinization temperature range. Because of its lack of gluten, amaranth flour has to be blended with wheat flour in order to use it in baking. 14

35 Fig 1.5 Scanning electron micrographs of grain amaranth and potato starch granules. Left: grain amaranth starch granules (Bar: 2 µm) (from Qian et al., 1999); Right: potato starch granules (Bar: 50 µm) (from Bustos et al., 2004). Compared to maize starch, A. cruentus and A. hypochondriacus starches have higher swelling power, lower solubility, greater water uptake, lower susceptibility to α-amylase, higher amylograph viscosity, and much lower amylose content (Stone and Lorenz, 1984). In addition, high susceptibility of A. hypochondriacus and A. caudatus starch granules to amylases was reported by Tomita et al. (1981). X-ray diffraction analysis of A. hypochondriacus starch showed that it is similar to maize and rice starches, indicating A-type crystalline structure (Sugimoto et al., 1981), compared to potato starch which shows typical B-type (Oostergetel and van Bruggen, 1993). This characteristic indicates that some unknown mechanisms exist in grain amaranth starch biosynthesis that enable the starch to have the same crystalline structure type as those of monocots. A Rapid Visco Analyzer (RVA), which can measure the pasting properties of starch, was used to determine the temperature of pasting, peak viscosity, and final viscosity of amaranth starch (Qian et al., 1999). The amaranth starch in 15

36 RVA viscogram showed the normal pasting feature of cereal and root starches. However, the peak viscosity of the amaranth paste is approximately one third lower than that of other crops (Batey and Glennie-Holmes, 1999). The low viscosity of the amaranth paste could result from the short chain length of amaranth starch, because longer molecules contribute more to the viscosity of liquid, either solution or emulsion. This deduction is consistent with the studies on amylopectin structure, gelatinization and retrogradation of starches (Paredes-Lopes et al., 1994). The studies of the starch biosynthesis in plants may provide the possibility for altering starch structure in future. Although grain amaranth is a dicotyledonous plant, its starch displays some physicochemical characteristics similar to cereal starches. Therefore, amaranth as a new crop with unique starch characteristics has been noticed by agricultural scientists worldwide gradually. The information on its starch biosynthesis may help to explain how the structure and physicochemical characteristics are determined in its starch granules. 1.3 Starch biosynthesis Starch accumulates in non-storage organ such as leaves in all plants and storage organs such as the endosperm and tubers. In plant leaves, starch synthesis occurs in chloroplast and carbon can be fixed from photosynthesis during the day and mobilized at night; whereas in the cereal endosperm and tubers, starch is synthesized in a different plastid, amyloplast. In this section, the general introduction will be focused on the proteins 16

37 involved in starch biosynthesis pathway. Thereafter, two questions will be discussed: 1) what are the major starch biosynthesizing enzymes that determine starch polymer synthesis in plant organs? 2) What are the differences of starch biosynthesis pathways in leaves, tubers, and cereals? Proteins involved in starch biosynthesis pathway The complex structure of the semi-crystalline in starch granule is unexpected because only two polymers are involved in starch synthesis and only two kinds of linkages can be found. Thus starch granule proteins (SGPs), including starch biosynthesizing enzymes (AGPase, SS, SBE and DBE) and other proteins such as low molecular weight (Mw) and proteins, may play distinct roles in starch biosynthesis (Fig 1.6). Commonly, the first step of starch biosynthesis, which is considered to be a rate-limiting step, is the synthesis of ADPglucose catalyzed by ADPglucose pyrophosphorylase (AGPase). The substrates of AGPase are ATP and glucose- 1-phosphate, a hexose phosphate synthesized from sucrose in cytosol (Fig 1.6A). In plastid, ADPglucose is the substrate of starch synthesis. Granule bound starch synthase (GBSS) and soluble starch synthase (SSS) are responsible for the catalysis of linear ADPglucose chain extension by 1,4-glucose linkage (Fig 1.6B). GBSS (or GBSSI) is considered to be the only enzyme that catalyzes the amylose synthesis whereas soluble synthases (SSI, SSII, SSIII, SSIV, and SSV) are thought to be responsible for the extension of glucose chains involved in amylopectin synthesis, although starch synthases may have non-essential roles in amylose biosynthesis. 17

38 A. AGPase OP OP OP B. GBSS and SSS + ATP ADP + PPi C. BE D. DBE Fig 1.6 The catalysis characteristics of starch synthases involved in starch biosynthesis. Glucose-1-phosphate, a hexose phosphate synthesized from sucrose in cytosol, is the substrate of AGPase. The extension of amylose and amylopectin are catalyzed by granule-bound starch synthase (GBSS) and soluble starch synthases (SSS), respectively. The formation of branching of amylopectin is catalyzed by starch branching enzyme (SBE) and de-branching enzyme (DBE) simultaneously. 18

39 Starch branching enzyme catalyzes the reaction of cleaving a fragment from a linear glucan chain and transfers it to another glucose residue to form a 1,6-α-D-branch (Fig 1.6C). Two isoforms of starch branching enzyme have been found in higher plant, SBEI and SBEII (SBEIIa and SBEIIb in monocots). Comparisons of branching enzyme sequences from a wide range of species demonstrate that there are two families of branching enzymes in plants, family A and family B (Burton et al., 1995). Most SBEIs (e.g., SBEIs from kidney bean, rice, wheat, maize, barley, and potato) belong to family B but two SBEIIs from pea and sweetpotato are also grouped into this family based on their amino acid sequences. Family A includes most SBEIIs (SBEIIa and SBEIIb) along with pea SBEI, rice BE3, BE4, and others. De-branching enzyme (DBE) catalyzes the hydrolysis of α(1,6) linkages in glucans (Fig 1.6D). Isoamylase and pullulanase are the two types of starch DBE in plants. The obvious difference between the two types of DBE is their substrates specificity. The pullulanase hydrolyzes α(1,6) linkages in amylopectin and β-limit dextrin (glucans that are produced as a result of β-amylase activity during starch breakdown) but does not hydrolyze glycogen. Pullulanase shows the greatest activity on yeast pullulan. The isoamylase is inactive on pullulan but most active on amylopectin. It has activity both on glycogen and β-limit dextrin. However, both pullulanase and isoamylase are considered to be involved in determining amylopectin fine structure, and loss and reduction in these DBEs result in phytoglycogen production at the expense of amylopectin synthesis. In addition to the enzymes mentioned above, some other proteins are also 19

40 involved in starch biosynthesis pathway. For example, Sehnke et al. (2001) found that reduction of the activity of Arabidopsis protein by antisense techniques resulted in a two to four fold increase in leaf starch accumulation. Moreover, all members of starch synthase III contain the conserved protein binding motif (RYGSIP), making the enzyme inactive through binding. Therefore, protein is considered to be a regulator of key enzymes of starch biosynthesis although the exact isoform of protein involved in starch biosynthesis is not known. However, the completion of Arabidopsis genome sequencing program and the opportunities offered by functional genomics, proteomics and metabolimics might be helpful to illustrate the function of proteins involved in starch biosynthesis or starch metabolism ADP-glucose pyrophosphorylase (AGPase) ADP-glucose pyrophosphorylase (ATP: α-d-glucose-1-phosphate adenylyltransferase; EC ; AGPase) plays a very important role in starch biosynthesis because it is the key enzyme to limit the speed of starch synthesis through controlling the synthesis of ADP-glucose which is the basic sugar nucleotide utilized by starch synthases. AGPase is an allosteric enzyme that contains two large regulatory (AGPS or LSU, 51kD) and two small catalytic (AGPB or SSU, 50kD) subunits (Morell et al., 1987; Okita et al., 1990). Both subunits are regulated positively by 3-phospho-glycerate (3-PGA) and negatively by orthophosphate (Pi) (Sowokinos, 1981; Sowokinos and Preiss, 1982; Cross et al., 2004). The increase of 3-PGA to Pi ratio provides a sensitive signal that activates AGPase and stimulates the synthesis of starch (Herold, 1980; Stitt et al., 1987). 20

41 Moreover, in plant leaves and tubers, light and sugar act as inputs to trigger post-translational regulation of AGPase (Tiessen et al., 2002; Hendriks et al., 2003). Since AGPase is a step-limited enzyme in starch biosynthesis, mutants which lack AGPase activity produced less starch than normal plants (Tsai and Nelson, 1966; Lin et al., 1988; Preiss et al., 1989; Smith et al., 1989; Ball et al., 1991; van den Koornhuyse et al., 1996). On the contrary, a transgenic potato with unusually high AGPase activity produced increased starch content (Stark et al., 1992). ADP-glucose serves as the substrate for both amylose and amylopectin biosynthesis in plants. Thus ADP-glucose is usually located in plastids. However, ADP-glucose is also found in cytosol in the endosperms of many cereals such as barley (Thorbjørnsen et al., 1996a), maize (Denyer et al., 1996), rice (Sikka et al., 2001), wheat (Burton et al., 2002), or probably all grasses (Beckles et al., 2001). The difference in ADP-glucose location between non-cereals and cereals indicates that there exit two pathways for the synthesis of ADP-glucose in the cereal endosperm. It is probably caused by the optimal usage of sucrose in the cereal endosperm to accelerate starch accumulation when sucrose is plenty Starch synthases Starch synthase (EC ) catalyzes the synthesis of an α(1-4) linkage between the nonreducing end of a preexisting glucan chain and the glucosyl moiety of ADP-glucose, causing the release of ADP. Two forms of starch 21

42 synthase have been found in most organs, granule bound starch synthase I (GBSSI) which bounds tightly to starch granules and soluble starch synthase (SSS) which is in the soluble fraction of the amyloplast. GBSSI is approximately 60kDa and found to be entirely associated with the starch granule when using reagents to remove or inactivate the surface proteins of starch granules. The GBSSI protein and its activity are largely resistant to these treatments, suggesting that GBSSI is actually located within the matrix (Mu-Forster and Wasserman, 1998). The catalytic function of GBSSI was understood by the study of mutants in many species in which no amylose is synthesized. The best-known mutants of this type are the waxy mutants of cereals (Weatherwax, 1922; Ono and Suzuki, 1957; Murata et al., 1965; Nakamura et al., 1995), and equivalent mutations also affect the starch of pea embryos (Denyer et al., 1995), potato tuber (Hovenkamp-Hermelink et al., 1987), the perisperm of Amaranthus seeds (Konishi et al., 1985), and green alga Chlamydomonas cells (Delrue et al., 1992). The lack of amylose of these mutants has been shown to lie in a gene encoding GBSSI, and the activity of GBSSI cannot be complemented by other starch synthases in vivo. Therefore, GBSSI is regarded as the only enzyme that catalyzes the biosynthesis of amylose in starch granules. In higher plants, mutations that eliminate GBSSI activity do not reduce starch content, implying that the ADP-glucose not used to make amylose is used by other SSs to make amylopectin. As we known, GBSSI is located in the granule matrix whereas other SSs, which are found in soluble phase, are active close to the surface of the starch granules. However, this may not be a sufficient 22

43 explanation for the biosynthesis of amylose. GBSSI must have some other, unique properties which allow it to make long, linear polymers when other isoforms cannot. In an experiment for discovering the unique properties of GBSSI, ADP-glucose as the substrate of starch polymers synthesis was served solely to isolated starch granules from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers, respectively. Interestingly, the result of the experiment showed that ADP-glucose was added into amylopectin rather than into amylose (Denyer et al., 1996). However, when small maltooligosaccharides (maltose up to maltoheptaose, MOS) were also supplied, GBSSI elongated these to produce amylose inside the matrix (Denyer et al, 1999). Consequently, a mode of interaction between GBSSI and other SSs may explain why GBSSI alone can synthesize amylose: MOS of less than about eight glucose units can diffuse into the matrix of the granule from the surrounding soluble fraction of the amyloplast. If they encounter an isoform of starch synthase other than GBSSI, the elongation of the MOS will be limited to one or a very few glucose units. If they encounter GBSSI, the long chains may be built up. Once the chain exceeds about eight glucose units, it is too large to diffuse freely within the matrix spaces and is thus trapped to become, after further elongation, the amylose component of the starch (Smith, 2001). Another mode found in experiments with starch granules isolated from the unicellular alga Chlamydomonas suggests that in these granules, chains within the amylopectin fraction which have been elongated by GBSSI are cleaved by an unknown mechanism to form amylose (van de Wal et al., 1998). 23

44 Although there is no experimental evidence from any other plants at present, it is entirely possible that both mechanisms may operate. It is no doubt that GBSSI has a processive mode of elongation and allow it to synthesize the long, linear chains of amylose. Another form of starch synthase, soluble starch synthase (SSS), has more isoforms than GBSS (Table 1.2). The predicted amino acid sequences of both GBSS and SSS isoforms indicate that the duplication and divergence of starch synthases occurred early in the evolutionary history of dicots and monocots (Li et al., 2003). In wheat endosperm, five starch synthase activities have been identified. A 60kDa GBSS encoded by waxy gene is exclusively found in the starch granule (Clark et al., 1991; Yan et al., 2000). A 75kDa SSI and an 180kDa SSIII could be detected in soluble fraction at the mid-endosperm development stage (Li et al., 1999a; 2000). SSI is also found in the starch granule (Rahman et al., 1995). SSII is a 100/105kDa protein found in the starch granule (Denyer et al., 1995; Rahman et al., 1995) but it could also be detected in the soluble fraction of the endosperm extract during the early endosperm development stage (Li et al., 1999b). The sequence of fifth starch synthase, SSIV, has been deposited in GenBank but this enzyme seems to be predominantly expressed in the leaves and has low sequence identity with other starch synthases expressed in the endosperm. In maize, five classes of starch synthases have also been identified. In addition to GBSS, cdnas encoding SSI, SSIIa, SSIIb and dull1 SS (SSIII) have been isolated (Gao et al., 1998; Harn et al., 1998; Knight et al., 1998). All maize 24

45 soluble starch synthases possess a divergent N-terminal extension that is absent in GBSS and Escherichia coli GS. In potato tuber, the primary amino acid sequences of GBSSI and SSII isoforms have been compared and revealed that the core region ( 60kDa) was similar to all other known starch synthases and bacterial glycogen synthases. It includes an N-terminal motif (KTGGL) thought to be required for binding of ADP/ADPglucose (Edwards et al. 1999). In addition, most of the evidence for the roles of different isoforms in the biosynthesis of starch comes from studies of transgenic potatoes in which activities of one or more isoforms of the enzymes have been reduced by the expression of antisense RNA. For example, large reductions in activities of either SSII or SSIII in potato tubers alter the chain length distribution of amylopectin. And the actions of the two isoforms are shown as interaction, reflecting that they both act on the same molecule the substrate of one is the product of the other (Edwards et al., 1999; Lloyd et al., 1999). Within all isoforms found in soluble phase, SSII is a unique isoform since SSII is also found bound to starch granules in significant amounts (Dry et al., 1992; Denyer et al., 1993; Edwards et al., 1995). The major difference between GBSSI and SSII from pea and potato starches is SSII possesses a 203 amino acid domain at its N-terminus, which is not present in GBSSI (Dry et al., 1992). The domain which is present in the mature protein, is hydrophilic, carries a net positive charge, and is highly flexible. It is also comparatively rich in serine residues and has three consecutive proline 25

46 residues which mark the end of the N-terminal extension. However, the function of the domain is not clear to date. Although both GBSSI and SSII can extend a range of glucan substrates in vitro, studies of starch synthase activity on granules in different mutants of pea and studies on plant starch synthases expressed in E. coli have revealed differences in some properties of the isoforms. For example, the rug5 mutant of pea lies in the gene encoding SSII. Loss of the SSII isoform results in reduced abundance of chains of intermediate length and increased abundance of very short chains of amylopectin in the mutant embryo (Craig et al., 1998). Thus SSII is distributive in its extension of MOS, preferentially adding single glucose unit to many glucan chains. In contrast, GBSSI preferentially extends MOS in a processive manner by adding further glucose units to the same glucan chain (Denyer et al., 1996; 1999). Therefore, it is thought that GBSSI might have capability to build long chains whereas other soluble starch synthases can extent limited glucose units to the reducing end of amylose or amylopectin. Nevertheless, the functions of SSII and other soluble starch synthases are still not fully understood. Recently, some very large proteins have been isolated from potato tubers (~140kDa) by Marshall et al. (1996) and from maize (~188kDa) by Gao et al. (1998). They were demonstrated to have starch synthase activity, and have been termed starch synthase isoform III (SSIII). Suppression of SSIII expression in transformed potato lines induced obviously altered morphology of the granules, but the starch content and amylose:amylopectin ratio of the granules in the transformed lines remained relatively unchanged. Thus SSIII might have a 26

47 critical role in the structural formation of potato starch granules (Marshall et al., 1996). Furthermore, comparing the predicted amino acid sequence of SSIII with GBSSI and SSII, it s concluded that although SSIII shares some conserved regions with other starch synthases, it was also shown to have some specific gaps in SSIII sequence. These gaps are postulated to confer the specific differences in properties between the various isoforms of starch synthase. In future, functional analysis of GBSS and SSS in the starch granule will be very important for understanding starch biosynthesis during the granule development in higher plants. More mutant plants will be used in experimental systems, along with suitable techniques such as antisense RNA and plant transformation, which will promote the identification of starch synthases and characterization of their roles, importance, and interactions. 27

48 Table 1.2 The various isoforms of GBSS and SS found in plants (shown by GenBank accession number) rice (Oryza sativa) wheat (Triticum aestivum) maize (Zea mays) barley (Hordeum vulgare) Arabidopsis thaliana potato (Solanum tuberosum) taro (Colocasia esculenta) pea (Pisum sativum) kidney bean (Phaseolus vulgaris) cowpea (Vigna unguiculata) GBSS I X62134 X57233 Y16340 X03935 X07931 AC X X88789 AB GBSS Ib AJ SS I D16202 AJ AF AF AF Y10416 AY AB NM- SS II _ AF AH AC X87988 AY X88790 AB AB AF SS IIa - AJ AY SS IIb - - AF SS III AF AF AF AC X AJ SS IV AY AY AY SS V AL AJ means no particular isoform found in some plants. GBSS granule bound starch synthase SS starch synthase 28

49 Starch branching enzyme Branching of the polysaccharide chain is produced by the action of starch branching enzyme (SBE, α-1,4-glucan-6-glycosyl-transferase (EC ), formerly identified as Q enzyme), which cuts the α(1-4)-linked chains and joins them with other chains via α(1-6)-linkages (Preiss, 1988). Starch-storage tissues of cereals, pea and potato contain at least two isoforms of starch branching enzyme, SBEI and SBEII (Martin and Smith, 1995; Larsson et al., 1996). In Arabidopsis (Fisher et al., 1996), maize (Gao et al., 1997), and barley (Sun et al., 1998), SBEII can be divided into two types (SBEIIa and SBEIIb) that differ slightly in catalytic properties. Most isoforms of SBE, such as SBEI in wheat, barley, and rice endosperm, as well as SBE2 in kidney bean, are observed in the soluble fraction of amyloplast extracts (Rahman et al., 1995). Nevertheless, in kidney bean SBE1 is found to be located in the starch granule fraction (Hamada et al., 2001). Moreover, some SBEs (SBEI and SBEII in pea embryos, SBEIIs in wheat, barley, maize, and rice endosperm, and SBEIIb in maize endosperm) are found in both the soluble and granule fractions (Denyer et al., 1993; Mu-Forster et al., 1996). The SBEs bound to granules are not found to have any activity in starch biosynthesis but are simply trapped during synthesis of the starch granule. It is thought that SBE isoforms create branching chains of different lengths or branch points at different frequencies. Therefore multiple forms of SBE could 29

50 thus give rise to the branching pattern and polymodal distribution of chain lengths that underlie the cluster structure of amylopectin. A comparison of predicted amino acid sequence of SBEs from different plants indicates that they fall into two families, A and B (Table 1.3). The members of each family share a high degree of amino acid identity (90%-95%) in the central portion (~700aa) of their amino acid sequences. On the contrary, the amino acid identity in the same region between family A and family B SBEs is as low as 55%-60%. All SBEs in the plant kingdom belong to the α-amylase family whose members have the (β/α) 8 barrel structure containing the active center (Jespersen et al., 1993). Four sequences (HSHAS/GFRFDGVT/GEDVS/AESHDQ) in both family A and B SBEs were found as the most conserved domains. The high sequence diversity displayed by the SBEs is in N-terminal domain and C-terminal domain. Therefore, differences in the enzymic properties between individual SBEs would be determined by their N-terminal or C-terminal domain (Kuriki et al., 1997). This assumption has been supported partially by the analysis of chimeric enzymes between maize BEI and BEII that revealed the C-terminal domain has a role in substrate preference or maximum catalytic efficiency whilst N-terminal domain determines the size of oligosaccharide chain transferred (Hong and Preiss, 2000). 30

51 Table 1.3 Two families of SBEs from different plants Family A Family B Organs SBEs Protein ID. in GenBank SBEs Protein ID. in GenBank Kidney bean (Phaseolus vulgaris) Potato (Solanum tuberosum) Pea (Pisum sativum) SBE2 BAA SBE1 BAA SBEII CAB SBEI CAA SBEI CAA SBEII CAA Arabidopsis thaliana SBE2-1 NP SBE2-2 NP Rice (Oryza sativa) Wheat (Triticum aestivum) SBE3 BAA SBE1 AAP SBE4 BAA SBE2 AAG SBE1 CAB Barley (Hordeum vulgare) SBEIIa AAC Maize (Zea mays) SBEIIb AAC SBEIIa AAB67316 SBEI AAO SBEIIb AAC The function of SBEI and SBEII in starch biosynthesis has been analyzed through some mutants in plants. Mutants lacking an SBE isoform (SBEI), also called ae (amylose-extender) mutation, have been identified in maize (Fisher et al. 1996; Kim et al., 1998) and rice (Mizuno et al., 1993). These ae mutations correspond to the rugosus (r) gene, which confers a wrinkled phenotype in pea seeds firstly described by Gregor Mendel (Bhattacharyya et al., 1990). The observation of family A SBEs-absent mutant suggested that isoforms of family B participate in vivo in the synthesis of the long and intermediate length chains 31

52 that will span clusters, whereas isoforms of A participate in the synthesis of the shorter chains that lie wholly within clusters (Takeda et al., 1993). The same results have been obtained in transgenic potato plants with decreased family A SBE activity (Jobling et al., 1999). Although no mutants of family B SBE have been reported in higher plants, a transgenic potato expressing an anti-sense family B SBE showed no significant changes in amylose content and amylopectin structure compared with wild type (Flipse et al., 1996). Therefore, family B SBEs might have less function on the amylose content and amylopectin structure than family A SBEs. Moreover, the study of rugosus (r) mutant of pea leaves showed that the decrease in SBE activity had relatively little effect on the rate of starch synthesis but increased the amylose content (Tomlinson et al., 1997). Two types of SBEII, usually classified as SBEIIa and SBEIIb, exit in maize, barley and Arabidopsis. Preiss (1991) has summarized that SBEI is quite different from SBEIIa and IIb in molecular mass, chromatographic and enzyme kinetic properties, immunological reactivity, and amino acid composition. It has been shown that SBEI has the highest rate in branching with amylose as substrate in vitro, and that SBEIIa and IIb have higher rates than SBEI with amylopectin as substrate (Guan and Preiss, 1993). Takeda et al. (1993) suggested that SBEI preferentially transfers longer glucan chains than either SBEIIa or SBEIIb. In a model proposed by Guan and Preiss (1993), SBEI might produce slightly branched molecules, which would then serve as substrates for the action of SBEIIa and IIb. Therefore, each SBE isoform may be responsible for a unique aspect of amylopectin biosynthesis and structure. 32

53 The study on maize SBEIIa and SBEIIb indicted that they are encoded by independent genes (Fisher et al., 1996). Furthermore, the ae gene which encodes the predominant Sbe-2b-hybridizing message expressed in endosperm, also encodes the major Sbe-2b-like transcript expressed in developing embryos and tassels. The amino acid sequences of barley SBEIIa and SBEIIb have 80% identity (Sun et al., 1998). The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. This extension domain contains a high score of Ser residues and other amino acids with flexible side chains that end with a Pro-rich triplet toward the C terminus. This flexible arm is also present in the isoforms of family A SBEs and those starch synthases which are found in the soluble phase of the amyloplast or associated with the starch granule, but is missing in other starch synthases that are strictly confined to the granule (Martin and Smith, 1995). The function of the N-terminal arm was thought to be responsible for the partitioning behavior of starch synthases and SBEs (Burton et al., 1995; Martin and Smith, 1995). However, there is no conclusive information regarding the physiological function of the N-terminal arm of SBEs in family A Starch de-branching enzyme In addition to the three starch biosynthesizing enzymes (AGPase, SS and SBE), there is some evidence that starch de-branching enzyme (DBE) may have a function in starch biosynthesis, especially in amylopectin synthesis, in addition to their role in starch degradation (Pan and Nelson, 1984). In a pullulanase-type starch de-branching enzyme absent mutant, sugary-1, 33

54 amylopectin is not synthesized but replaced with a water-soluble α-1,4,α-1,6-linked glucan known as phytoglycogen, which contains more frequent and randomly located branches throughout the molecule compared with amylopectin with repeated unit structure referred to as amylopectin cluster (Nakamura et al., 1997; Wong et al., 2003). This suggests that starch de-branching enzyme directly participates in starch biosynthesis. James et al. (1995) also showed by transposon tagging that the Sugary-1 gene of maize encodes an isoamylase-like enzyme. Thereafter, it is proved that the Sugary-1 gene product possesses isoamylase activity, and that sugary-1 mutants are deficient in both isoamylase and pullulanase (Rahman et al., 1998; Kubo et al., 1999; Beatty et al., 1999). Similarly, the maize gene Su1 (Rahman et al., 1998), rice gene Sur (Kubo et al., 1999) and Arabidopsis gene Dbe1 (Zeeman et al., 1998) are also found to code for de-branching enzymes and mutations therein cause the same abnormalities in starch production. To study the role of starch de-branching enzymes in the determination of the fine structure of amylopectin, Zeeman et al. (1998) screened mutant populations of Arabidopsis and obtained two mutants lacked an isoamylase-type de-branching enzyme. The loss of this isoamylase resulted in a 90% reduction in the accumulation of starch but an increase in the highly branched water-soluble polysaccharide phytoglycogen in the contrary. In addition, both normal starch and phytoglycogen accumulated simultaneously in the same chloroplasts in the mutant lines, suggesting that isoamylase has an indirect rather than a direct role in determining amylopectin structure. 34

55 Based on these de-branching enzyme deficiency mutants, the function of de-branching enzymes in amylopectin synthesis has been proposed in two very different models. The first model, glucan-trimming model, proposes that a polymer, pre-amylopectin, is synthesized at the granule surface via starch synthase and starch branching enzyme. This highly branched pre-amylopectin (compared with amylopectin) does not have the appropriate branching structure to become organized and incorporated as part of the granule matrix. Then the pre-amylopectin is converted to amylopectin through the action of isoamylase and/or other de-branching enzymes, which trims branches until the branching structure is appropriate for crystallization onto the granule surface. The MOS released by the trimming may be converted back to ADP-glucose or may be reincorporated onto chains within the pre-amylopectin via a disproportionating enzyme. In the absence of isoamylase, most of the pre-amylopectin does not achieve an appropriate structure for crystallization. It is further acted on by synthases and branching enzyme and accumulates as phytoglycogen (Myers et al., 2000). The second model, scavenging model, proposes that isoamylose is only indirectly involved in amylopectin synthesis, which is an exclusive function of starch synthases and starch-branching enzymes. In this model, starch synthases and branching enzymes are suggested to act on small MOS in the stroma that synthesize soluble branched glucans. The action of the glucan-degrading enzymes including isoamylase and/or other de-branching enzymes may prevent the accumulation of soluble branched glucans in the stroma. Thus ADP-glucose is released from the scavenging reaction and can be the substrate of starch 35

56 synthases and starch-branching enzymes to make starch in plastids. In the absence of isoamylase, soluble glucans are elaborated by starch synthases and starch branching enzyme and accumulate as phytoglycogen (Zeeman et al., 1998) Although these models have not been confirmed experimentally, the evidence at present gives more support for the first model. It means amylopectin is the product of trimming via de-branching enzymes. It was thought for a long time that starch synthase and starch branching enzyme are the two only enzymes required to synthesize the starch polymers. This has been proved by several mutants in higher plants. However, the dbe mutants such as sugary1 mutant of maize indicated that the reduced or eliminated isoamylase activity in the mutants displayed abnormal starch which contains highly branched phytoglycogen and amounts of small starch granules (Burton et al., 2002). Therefore, the starch polymers biosynthesis is more complex than what we have known to date. Although it is not easy to design experiments to distinguish different models proposed by various researchers and prove the function of various isoforms involved in starch biosynthesis, more mutants discovered in plants and new techniques such as antisense RNA can be used in the research of starch biosynthesis. All the efforts will shed light on the amylopectin synthesis, amylose synthesis and the formation of starch granule. 36

57 Regulatory proteins involved in starch biosynthesis In cereal endosperm, some low molecular weight (Mw) proteins (~15 kda) are believed to cause endosperm softness (Greenwell and Schofield, 1986; Jolly et al., 1993; Giroux et al., 1997). Although the present evidence cannot indicate that these low Mw proteins are involved in starch biosynthesis, it has been found that one of the low Mw proteins, friabilin, is responsible for the endosperm texture (Giroux and Morris, 1997; Oda and Schofield, 1997) proteins are a group of regulation proteins that have environmentally responsive phosphorylation-related regulatory functions (Comparot et al., 2003). Generally, the regulation of the activity of several key enzymes such as AGPase, SSs or SBEs is a two-step process involving phosphorylation of the enzymes, followed by formation of a complex with proteins to complete the regulatory transition (Sehnke and Ferl, 1996; Chung et al., 1999). For example, chloroplast-localized proteins act a biological role in carbon partitioning, namely starch accumulation. It is found that reduced levels of starch granule-associated proteins result in a dramatic increase in starch accumulation (Sehnke et al., 2001). In starch metabolism, proteins act as inhibitory proteins by normally shutting down starch biosynthesis, thereby playing a key regulatory role in carbon allocation. On consensuses binding domains, one target of the granule proteins appears to be the SSIII family. SSIII members from Arabidopsis, barley, 37

58 and wheat all contain a conserved hexapeptide motif (RYGSIP) which is also found in a protein partner, nitrate reductase (Bachmann et al., 1996; Moorhead et al., 1996). Moreover, SSIII is directly involved in the production of amylopectin and has significant control over other SS isoforms. This might explain the observation that both starch accumulation and the qualitative shift in branched glucan content occurred in antisense plants (Chung et al., 1999). A mechanism (model) was proposed by Sehnke et al. (2001): starch production in continuously illuminated plants is limited through inactivation of SSs by phosphorylation and protein binding. Without proteins to complete the inactivation step, starch continues to accumulate beyond normal levels. This might be a reasonable explanation for the function of proteins in plastid at present Starch biosynthesis Starch biosynthesis as a hot topic has been researched for more than twenty years. The researches on starch synthesizing enzyme isolation, polymer chain elongation, and the synthesis of the starch granule etc. have produced a draft of complex starch biosynthesis pathway. The more we understand, the more we are curious about how such a complicated structure of starch can be made of only two simple polymers, amylose and amylopectin. 38

59 Amylose synthesis Most of storage starches contain 18-30% of the relatively unbranched glucan amylose. The amylose content is thought to be limited by the space available for the accumulation of this glucan within the crystalline, amylopectin matrix of the granule (Flipse et al., 1996). The elongation of amylose from ADP-glucose is taken place in the matrix of starch granules through the action of granule-bound starch synthase I (GBSSI). As we have known, the function of GBSSI in waxy mutants is inhibited and cannot be compensated by other starch synthases. However, in pea and Chlamydomonas, the disappearance or reduction in amylose content was found in mutants for both ADP-glucose pyrophosphorylase and phosphoglucomutase. Additionally, the Michaelis constant (Km) of GBSSI for ADP-glucose is five to ten folds higher than that of the soluble starch synthases. Therefore amylose synthesis is particularly sensitive to substantial decreases in the supply of the ADP-glucose substrate. Moreover, the structure of the starch of low ADP-glucose-containing mutants resembles that of transient starch, a form of the polysaccharide that accumulates during the day in leaf cells and is degraded at night according to the physiological needs of the plant. The restrictions in the supply of substrate in the leaf may result in less amylose than storage starches in the sink tissues. The first analysis of amylose synthesis in vitro was performed by Lelori, the Nobel Prize winner, in In his studies, non-physiological concentrations of 39

60 radiolabelled UDP-glucose were used to incubate purified starch granules. It is concluded that both amylopectin and amylose incorporated equivalent amounts of the label but that the amylose-specific radioactivity was threefold higher. It was also found that the enzyme readily added a few glucose residues to small MOS thus yielding soluble products with small extensions. Recently, Denyer and his group (1996) reported experimental conditions where the label was selectively found in amylose when starch granules from pea leaves were incubated in the presence of both radiolabelled ADP-glucose and maltotriose. In the absence of maltotriose, the entire label was detected in amylopectin, suggesting that GBSSI-catalyzed amylose synthesis requires the presence of small MOS. Therefore, it seems that MOS may trigger amylose synthesis when they are used as primers by GBSSI. van de Wal et al. (1998) performed a pulse-chase experiment on purified Chlamydomonas starch granules to test another hypothesis that GBSSI was continuously using amylopectin as a primer and extending a long outer chain. The hypothesis proposed that when the extended molecule had reached a sufficient size, an endo-type of cleavage event would terminate the mature amylose molecule. From the experiments, two distinct mechanisms of chain cleavage were postulated. 1) Amylose cleavage occurs downstream by a starch hydrolase within the granule. To achieve several rounds of amylose synthesis upon an amylopectin primer, GBSSI would have to extend another molecule from a novel accessible non-reducing end, thereby implying some freedom of movement. 2) GBSSI is postulated to be directly responsible for cleavage of the 40

61 amylose chain. The hydrolytic reaction would be triggered by steric hindrance encountered during the progress of the growing amylose chain. The presence of amylose in the pre-existing amylopectin semi-crystalline matrix is suggested by X-ray diffraction analysis of granules subjected to in vitro synthesis of amylose. Preliminary results suggest that amylose pushes amylopectin from the A- into the B-type of crystalline lattice (van de Wal et al., 1998), and the B-type allows room for infiltration of one or two amylose chains in the middle of the amylopectin crystal. Although the two hypotheses were distinguishable, they might not be independent in that both account for the synthesis of amylose. It could also be true that none of the theories provides the real explanation. Nevertheless, there is no new evidence to support or reject these two hypotheses. For more information, a detailed structural analysis of MOS fraction at the time of amylose synthesis needs to be done in future. An experiment in Arabidopsis mutants proved a suggestion that amylose is preferentially synthesized in the amorphous zones of starch granule (Blanshard, 1987). Mutant sex4 has large granules and contain alternating semicrystalline and amorphous zones similar to storage starches. Although the increase in the GBSS content is not as marked as in sex1, starch from which has lower amylose content, and starch from sex4 had the highest amylose content (Zeeman et al., 2002). Thus, amylose may be more readily synthesized in sex4 granules than in wild-type or sex1 granules. However, other factors we mentioned above such as the supply of substrates may also influence amylose synthesis or contribute to 41

62 the observed differences Amylopectin synthesis Amylopectin is the major compound in starch and composed of intermediate-size of α(1-4)-linked glucans that are clustered together by α(1-6) linkages. The major distinction from glycogen is that the basic structure of amylopectin in plant is highly ordered parallel arrays of double helical glucans. The origin of these arrays resides in the close packing of the α(1-6) linkages at the root of the unit amylopectin cluster. This allows the growth of semicrystalline granules whose dimensions often exceed those of a standard bacterial cell. It is thought that the clusters are generated by the coordinated action of elongation (starch synthases) and branching enzymes. The study on amylopectin synthesis is more difficult than on amylose synthesis because no amylopectin absent mutant has been found in higher plants. However, since some mutations lie in the genes encoding amylopectin synthesis enzymes, they are used in genetic, biochemical, and molecular biological studies to understand amylopectin biosynthesis. The distribution of amylopectin chain-length is a major property of the structure and physicochemical characteristics of amylopectin. Umemoto et al. (1999) reported clear varietal differences in chain-length distribution of amylopectin regardless of the effect of temperature on distribution, raising the possibility of a relationship between the variety and amylopectin structure. 42

63 The difference in chain length has been observed in starch from the SSII mutant of the pea embryo (Craig et al., 1998), SSII-antisensed potato tuber (Edwards et al., 1999), SGP-1 (SSIIa protein) lacking wheat endosperm (Yamamori et al., 2000; Li et al., 1999b), the SSIIa mutant of barley endosperm (Morell et al., 2003), and SSIIa mutant of rice endosperm (Umemoto et al., 2004). These observations support that ssii gene is the causative gene for differences in the chain-length distribution of amylopectin. Moreover, SSIIa deficiency in Nipponbare, a rice variety, changed the ratio of A- to B1-chains, thus suggests that active SSIIa elongates short A-chains sufficiently to allow them to be acted on by branching enzymes, resulting in these A-chains becoming B1-chains. One of mutants, the amylose-extender (ae) mutant, which is deficient in SBEIIb, results in major changes in endosperm amylopectin structure (Yuan et al., 1993; Shi and Seib, 1995; Klucinec and Thompson, 2002). The average chain length of ae amylopectin in maize endosperm is significantly longer than that of the wild-type amylopectin (Baba and Arai, 1984; Kasemsuwan et al., 1995). Two other isoforms of SBE have also been found, SBEIa and SBEIIa. The in vivo functional behavior of these three single sbe mutants showed that during amylopectin biosynthesis in maize endosperm, the function of SBEIIb is predominant to that of both SBEIa and SBEIIa, and therefore the sbe1a mutant might affect starch structure only in an ae background (Yao et al., 2004). In rice endosperm, biochemical and physicochemical analyses of ae mutant demonstrated that SBEIIb is involved in the transfer of short chains with degree 43

64 of polymerization (DP) 17, suggesting that SBEIIb plays an important role in the formation of A chains of amylopectin (Nishi et al., 2001). Meanwhile, the change in the structure of amylopectin induced by lesion of sbe1 gene was characterized by the specific α(1,4) chain-length profile in the sbe1 mutant in that chains of DP from 12 to 21 and DP 37 were depleted, whereas those of DP 10 and DP from 24 to 34 increased. These observations strongly suggest that SBEI and SBEII play distinct roles in the formation of long and short chains of amylopectin, respectively (Satoh et al., 2003). De-branching enzyme was thought to be required only to balance the excess branching activity present at the surface of the granule (Pan and Nelson, 1984). However, Mouille et al. (1996) proposed that preamylopectin, a branched precursor of amylopectin, is a natural precursor of starch synthesis, which the amorphous lamella of the amylopectin clusters is generated by selective de-branching of preamylopectin, and that amylose is synthesized downstream from amylopectin. This assumption implies that in addition to the function of the de-branching enzyme, other functions may be required to prevent further branching and to trim the crystalline lamella to generate mature 9-nm amylopectin clusters. The structure of amylopectin in higher plants is characterized by the fact that a unit structure with a constant size throughout the plant kingdom called cluster is tandem linked (Jenkins et al., 1993; Thompson, 2000), and the distinct structure may be referred to as tandem-cluster structure. On the basis of the structure of amylopectin, it is reasonable to assume that SS, SBE, and DBE 44

65 should produce three different types of branches: those formed in the amorphous and the crystalline regions of the cluster, and those that link the clusters Starch biosynthesis pathways in leaves, tubers and cereal seeds Starch synthesis occurs in chloroplast in photosynthetic organs, such as leaves, and in amyloplast in storage organs such as tubers, cereal seeds, fruits, and storage roots. In plant leaves, starch is synthesized during the day from photosynthetically fixed carbon and is mobilized at night. Starch can accumulate in large amounts over a short period in leaves, and the rate of starch synthesis can be controlled by altering the irradiance and be measured accurately by supplying 14 CO 2. However, relatively little is known about leaf starch biosynthesis, composition, and structure, compared with starches from storage organs. In tobacco leaves, an amylose content of between 15% and 20% was found (Matheson, 1996). In wild-type Arabidopsis leaves, the amylose content of the starch is approximately 15% or less, lower than those of most storage starches (Zeeman et al., 1998b). But in starch-excess mutants, increased amylose contents have been found (Critchley et al., 2001; Yu et al., 2001). Starch granules of wild-type Arabidopsis leaves are flat and discoid. The size and the shape of leaf starch granules are thought to be defined by the spaces within the chloroplast, between layers of thylakoid membranes. Except for these differences, Zeeman et al. (2002) found that the fundamental structures and 45

66 layers of organization in starch granules of Arabidopsis leaves are similar to those found in storage starches. Thus it is suggested that the mechanisms underlying the synthesis of Arabidopsis starch granules are broadly similar to those of seeds, tubers, and leaves of other higher plants. Fig 1.7 The starch biosynthesis pathway from sucrose which is the product of photosynthesis. The active enzymes and transporters involved in the pathway are as follows. (1) Suc synthase; (2) UDP-Glc pyrophos-phorylase; (3) glycolytic enzymes including phosphoglucomutase; (4) ADP-glucose pyrophosphorylase (AGPase); (5) ADP-glucose transporters; (6) hexose phosphate transporters; (7) granule-bound starch synthase I; (8) soluble starch synthases; (9) starch branching enzyme; (10) starch de-branching enzyme. Only in cells of developing cereal endosperms, ADP-glucose is also made in the cytosol via a cytosolic isoform of ADP-glucose pyrophosphorylase and enters the plastid via a specific transporter. Relatively integrated drafts of starch biosynthesis pathway in nonphoto- synthetic cells have been proposed by Myers et al. (2000) and Smith (2001), 46

67 respectively. And evidence for the pathway has been discussed in detail by Preiss (1988) and Okita (1992). In nonphotosynthetic cells of major starch-storage organs, starch is synthesized from sucrose imported from the leaves where it is made in photosynthesis (Fig 1.7). Unlike leaf starch biosynthesis which occurs in chloroplast, the synthesis of storage starch occurs in a different plastid, amyloplast. Sucrose is firstly converted into glucose 6-phosphate in cytosol, and then this hexose enters the amyloplast via a transporter in the membrane. Once inside the amyloplast, glucose 6-phosphate is converted to ADP-glucose via the enzyme ADP-glucose pyrophosphorylase (AGPase). ADP-glucose is the substrate for the biosynthesis of two starch polymers. The synthesis of ADP-glucose in the endosperm of developing cereal grain has different pathway from that in other starch-synthesizing organs. Although cereal endosperms retain the capacity for hexose phosphate transport and a plastidial AGPase, most of the flux from sucrose to ADP-glucose proceeds via the cytosolic AGPase (Thorbjørnsen et al., 1996a). AGPase is regarded as the first committed step on the pathway of starch synthesis and its activity is regulated by several metabolites. In Arabidopsis leaves, modulation of AGPase activity could control the overall rate of starch synthesis in the chloroplast during photosynthesis (Hendriks et al., 2003). However, in those storage organs such as pea and potato plants, other steps on 47

68 the pathway are of considerable importance in the control of flux in addition to AGPase (Tiessen et al., 2002). Based on these researches, it can be concluded that the distribution of starch biosynthesizing enzymes in starch biosynthesis varies considerably from one type of organ to another, and through organ development. Thus it is better to use different organs and species as materials to analyze starch biosynthesis in future. 1.4 Genetic modification of starch Starch, the dominant carbohydrate in our diets, is used as the main carbon reserve in many plants such as cereals, tubers and legumes. Especially in grain legumes, there is much genetic variation for both the total seed content and the composition of starch (Wang et al., 1998) and of raffinose family of oligosaccharides (RFOs) (Jones et al., 1999). In pea, over 30 novel starch mutants have been characterized. These mutations alter the shape of the seeds from round to wrinkle hence leading to the changes in starch content, polymer (amylose and amylopectin) composition, and physical structure of the starch granule. In humans, starch is normally consumed as part of cooked or processed food. After cooking, a proportion of the starch is recrystallized on cooling to become highly resistant to pancreatic amylase (retrograded) and cannot be digested. This kind of starch which cannot be digested by amylase is called resistant starch 48

69 (RS). RS contributes to the total unavailable carbohydrates believed to be important in combating certain forms of cancer (Aranda et al., 2001). Starch is the primary energy source in many animal diets. The high amylose content of starch provides less available energy (Aranda et al., 2001). However, the presence of the RFOs in seeds makes humans feel discomfort because they are fermented by bacteria with the release of hydrogen and methane. This occurs because higher animals, including humans, lack the enzyme (α-galactosidase) necessary to break the α(1-6) linkage that characterizes this group of compounds. It has been shown that intestinal digestion of the α-galactosides can be increased if animal diets are supplemented with exogenous α-galactosidase. The presence of α-galactosides in the colon, however, may have a beneficial effect by increasing the bi-fidobacteria population. These bacteria produce short-chain fatty acids that reduce the incidence of colon cancer in human (Aranda et al., 2001). Here we have a question. Can we alter the carbohydrate quality that makes the starch in grain fit our purposes? There are some traditional ways to manipulate carbohydrate utilization. For example, soaking and sprouting grain seeds can enhance the digestibility of starch and reduce the level of RFOs by up to 100% through the release of α-galactosidase. Another method is to genetically manipulate the level of RFO through inhibiting galactinol synthase activity (patented by Kerr et al., 1998). 49

70 We also can use mutants to manipulate starch content, composition, and granule structure genetically. This technique has been applied successfully to narbon beans (Vicia narbonensis) genetically modified by a targeted reduction in AGPase (Rolletschek et al., 2002). The effects of changes in starch granular structure on the nutritional quality of the seed have been examined in the r and rb pea mutants, which differ greatly in structural characteristics. The glycaemic index of products from the r mutant seed was predicted to be lower than from the rb seed. However, there were several other pleiotropic effects of the mutations that could have contributed to this difference. For instance, there was a large difference in amylose content and a 3-fold difference in the proportion of RS (Skrabanja et al., 1999). Genetic modification is not a new topic for many years. Many of the challenges faced in the past have been met gradually since our knowledge of the products and ability to manipulate plants has improved considerably. Researchers are utilizing existing resources and exploring the new genomic tools for modifying the nutritional components. There is no doubt that genetic modification is going to be a useful and challenging method for the development of more nutritious food supply for a growing world. 1.5 Objectives of the research project Although starch and starch biosynthesis have been researched for several decades, the packing of amylopectin and formation of starch granule still remain unclear to date. Especially in grain amaranth, the research of starch biosynthesis is almost nonexistent. 50

71 As mentioned in section 1.1 and 1.2, grain amaranth is a group of C4 photosynthetic plants which belong to dicots in the plant kingdom. However, their grains are considered as one of pseudo-cereals because the seeds contain high content of starch. Compared with cereal starch, grain amaranth starch has some unique characteristics: 1) extremely small starch granules; 2) low amylose content; 3) A-type crystalline polymorph; and 4) presence of short chains of amylopectin. These unique properties make grain amaranth starch possess low pasting viscosity, a wide range of gelatinization temperature, and high crystallinity. The analysis of starch biosynthesis indicates that some starch properties are controlled by the enzymes involved in starch biosynthesis pathway. Therefore, to analyze grain amaranth starch at the molecular level, we should focus on the analysis of amount and type of starch synthesizing enzymes in grain amaranth. It is also important to characterize the genes encoding major starch synthesizing enzymes in grain amaranth. Since the structure of amylopectin in grain amaranth starch is unusual, the study on SSII and SBE, which are two major starch synthesizing enzymes involved in amylopectin synthesis, is a crucial objective in this thesis. It is known that in some higher plants, SSII is mainly responsible for the elongation of the branching chains in amylopectin whilst SBE is the major enzyme for transferring linear chains to other chains to form amylopectin molecules. However, in grain amaranth, no isoform of starch biosynthesizing enzymes has been isolated and identified. Therefore, the objectives of this thesis will be the isolation, characterization and expression of genes encoding two major isoforms, 51

72 SSII and SBEI, involved in the process of amylopectin synthesis from grain amaranth. Chapter I in this thesis is the literature review that introduces the background of research material, grain amaranth, and the starch biosynthesizing enzymes involved in starch polymer biosynthesis. In Chapter II, GBSSs are isolated from amaranth granules. GBSSI, SSII, and SBEI are identified to be granule-bound in grain amaranth granules by Western blotting approach. In Chapter III, a cdna library is constructed from grain amaranth developing seeds and the quality of cdna library is determined by titer and other parameters. Chapter IV and V mainly describe the isolation, characterization and expression of the genes encoding two major starch biosynthesizing enzymes, SSII and SBEI, from cdna library constructed from grain amaranth developing seeds. Chapter VI is general discussion that discusses the properties of SSII and SBEI from grain amaranth and their applications in future research. From these analyses, very useful information is obtained on the structure of two genes isolated from grain amaranth. The functional characterization of amaranth SSII and SBEI sheds light on the molecular mechanisms that confer unique properties to grain amaranth starch. 52

73 Chapter II IDENTIFICATION OF STARCH SYNTHESIZING ENZYMES AND THEIR ISOFORMS FROM GRAIN AMARANTH 2.1 Introduction In Chapter I, I have discussed that there are many isoforms of starch synthesizing enzymes in plant organs. Most of them have been isolated and classified into different groups, such as starch synthase, starch branching enzyme, and starch de-branching enzyme. The common method for protein separation is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone, and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge on the polypeptide in proportion to its length, and the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size. In denaturing SDS-PAGE separations, protein migration in the gel is determined not by the intrinsic electrical charge of polypeptide(s), but by the molecular weight. The separated proteins in an SDS-PAGE gel can be transferred to a solid membrane for Western blot analysis, which is also called immunoassay. The 53

74 most popular type of probe of immobilized proteins is an antibody. The attachment of specific antibodies to specific immobilized antigens can be readily visualized by indirect enzyme immunoassay techniques, usually using a chromogenic substrate which produces an insoluble product. Therefore, one protein or its isoforms can be detected in a mixture of any number of proteins. Moreover, Western blot analysis can give information about the size of the protein. In this experiment, granule-bound starch synthases (GBSSs) were isolated from grain amaranth, wheat, and pea seeds respectively and separated on SDS-PAGE gel. The GBSSs (or isoforms) from grain amaranth and wheat were identified by Western blotting using antibodies made from the starch granule-bound synthase, soluble starch synthase and starch branching enzyme of wheat. 2.2 Plant materials Amaranth and wheat seeds were collected from mature plants and sieved to separate them from the remnant spikes. Pea seeds were also collected from mature plants. All the seeds were dried at room temperature for 24 hours and stored in a cooler at 4 C. 2.3 Experimental methods GBSSs extraction from amaranth, wheat and pea seeds In this two-step experiment, GBSSs were extracted from grain amaranth, wheat and pea seeds, respectively. 54

75 The first step was the preparation of starch granules. Mature seeds from grain amaranth, wheat and pea were soaked in water overnight at 4 C and homogenized with a pestle and mortar. The homogenate was layered on 1mL of 80% (w/v) CsCl. After centrifugation (13,000 rpm for 5 min at room temperature), the pellet was spinned down and washed twice with 1mL of wash buffer I (containing 55 mm Tris-HCl, ph 6.8, 2% SDS, 10% glycerol, and 0.5% 2-mercaptoethanol which was added just before used), resuspended in H 2 O, and washed twice with H 2 O and once with acetone. Starch granules from grain amaranth, wheat and pea were isolated and purified. The dried starch granules were stored at 4 C before use. The next step was GBSSs isolation from starch granules. 10 mg of dried starch granules were resuspended in 100 µl wash buffer II (containing 62.5 mm Tris-HCl, ph 6.8, 10% SDS, 10% glycerol, 0.05% bromophenol blue, and 0.5% 2-mercaptoethanol). For further purification, the starch granule suspension was heated in boiling water bath and cooled to form a gel-like starch solution. Immediately after centrifugation (13,000 rpm for 15 min at 4 C), the supernatant containing GBSSs was loaded on a SDS-PAGE gel for separation SDS-PAGE gel electrophoresis and silver staining GBSSs isolated from grain amaranth, wheat and pea starch granules were separated by SDS-PAGE gel electrophoresis and visualized by the silver staining method. The expected molecular weights of major GBSSs in wheat are from 60 kda 55

76 to 105 kda. To separate the GBSSs in this range, 12% running gel (see APPENDIX V) and 5% stacking gel (see APPENDIX V) were prepared for SDS-PAGE analysis. Before gel loading, the GBSS samples were mixed with an equal volume of 2 x sample buffer (see APPENDIX V), and denatured in boiling water for 5 min along with a protein marker. The samples and the protein marker were loaded on a polymerized SDS-PAGE gel. The gel was run at room temperature at 200 V (constant voltage) until the bromophenol blue dye was 1 hr out the gel, usually overnight. After electrophoresis, the gel was removed from the apparatus and soaked in pre-fixative solution A (containing 50% methanol and 10% acetic acid) for 30 minutes and then transferred to a container with pre-fixative solution B (containing 5.5% methanol and 0.75% acetic acid) and also soaked for 30 minutes. For fixing GBSSs on the gel, it was soaked in a fresh DTT solution (containing g DTT dissolved in 300 ml ddh 2 O for each gel) for 30 minutes. After fixation, the gel was transferred to a fresh staining solution [containing 1% (w/v) silver nitrate] and stained for 30 minutes. The gel was then developed in 3% Na 2 CO 3 and 10% formaldehyde for several minutes until bands were visible on the gel. Before the immunoblot analysis, the development of the gel was stopped by rinsing it with stop solution which consists of 72% (w/v) citric acid anhydrous. 56

77 2.3.3 Immunoblot analysis Immunoblot analysis consists of nitrocellulose (NC) membrane transferring, antibody interaction and development of immuno-reactive bands. The laboratory procedures of membrane transferring followed those described by Bolt and Mahoney (1997) except the performance was conducted under V (starting at ~220 ma; V/cm) at 4 C for 3.5 hr. After transferring, the marker lane was cut off from the NC membrane and the protein bands were stained for 10 min with amido black (containing 25% isopropanol and 10% acetic acid) followed by de-staining for 10 to 20 min. The remnant NC membrane containing GBSS bands was dried at room temperature and stored in a dry place. The antibodies used in the experiment were made from wheat GBSS, SSII and SBEI, which were provided by Dr. Chibbar s lab (Department of Plant Science, The University of Saskatchewan). Firstly, GBSSs from grain amaranth and wheat seeds separated by the SDS-PAGE gel were blocked onto the NC membrane using blocking buffer (containing 5% (w/v) low-fat Carnation milk in 1 x PBS; 0.1% Tween 20). Then the pre-made GBSS, SSII and SBEI antibodies were diluted 1000 times with blocking buffer, respectively, and added as primary antibodies to small plastic bags, respectively. Each plastic bag contained an NC membrane in which GBSSs from grain amaranth and wheat seeds were bound on it. After 2.5 hr incubation, the primary antibody was removed from the bag and secondary antibody (diluted goat anti-rabbit alkaline phosphatase-conjugated antibody, 1:5000) was added and incubated for 1.5 hr. The secondary antibody was then removed and a buffer containing 50 mm Tris-HCl and 150 mm NaCl (ph 7.5) was added into the bag to replace the 57

78 blocking buffer. The BCIP-NBT color substrate solution (containing 0.3 mg/ml nitroblue tetrazolium and 0.15 mg/ml BCIP) was used to develop the immuno-reactive bands on the NC membrane. When desired development had been reached, usually within min, color development was stopped by immersing the NC membrane in nanopure water. Then the NC membrane was air-dried and stored protected from light. 2.4 Results GBSSs separated by SDS-PAGE gel electrophoresis GBSSs were isolated from grain amaranth, wheat, and pea seeds, respectively, using CsCl-SDS method. Since the isolated GBSSs should be separated by SDS-PAGE and transferred to an NC membrane for Western blotting, protein isolation is a very important procedure for the whole experiment. Nowadays, a wide range of agents has been explored for the extraction of GBSSs from starch granules. The selection of agents depends on the nature of starch granules in terms of the ability of resistance to common protein solvents, such as chloroform/methanol (Schofield et al., 1987). CsCl-SDS method is an effective method for GBSSs extraction from starch granules. It is found that 80% (w/v) CsCl solution can remove most nucleic acids and polysaccharides by centrifugation (Sulaiman and Morrison, 1990). The high concentration of SDS (10%) can give maximum yields of GBSSs (Rahman et al., 1995). High SDS 58

79 solution also can improve the efficiency of higher molecular mass protein ( kda) isolation when starch granules become swelling and gelatinization at above 50º C (Rahman et al., 1995). Furthermore, low temperature (-20ºC) can help the precipitation of GBSSs with either acetone or 1:3 (v/v) diethyl ether/ethanol. In my experiment, a modified CsCl-SDS method was used for GBSSs isolation from grain amaranth, wheat and pea seeds. Firstly, crude starches were purified by layering the crushed seeds on 80% CsCl and centrifuging to pellet. Secondly, the surface proteins were then removed with 2% SDS, and internal proteins were removed with 10% SDS at high temperature (100ºC). Finally, the GBSS was concentrated by washing with acetone and collected before loading. The use of low concentration SDS (2%) prior to high concentration SDS (10%) allows less protein from the surface of starch granules remaining in GBSSs solution. Additionally, the internal proteins can be denatured and removed easily. The extremely high temperature accelerates the protein denaturation and shortens the incubation time. Using the modified CsCl-SDS method, GBSSs were isolated from grain amaranth, wheat and pea seeds, respectively, and separated by SDS-PAGE (Fig 2.1). The numbers and sizes of GBSSs are not consistent in grain amaranth, wheat and pea. 59

80 kda 212 M M Fig 2.1 SDS-PAGE gel of GBSSs isolated from grain amaranth, wheat and pea seeds, respectively. Lane 1 & 2 show the GBSSs from grain amaranth seeds (lane 1, 30 µl sample loaded and lane 2, 3 µl). Lane 3 shows the GBSSs from wheat seeds (3 µl) and lane 4 shows the GBSSs from pea seeds (3 µl). M: Protein maker (New England Biolab). In wheat GBSSs, six bands corresponding to different isoforms with molecular mass of ~60, 65, 77, 90, 100, and 105 kda, respectively, were detected. One of these isoforms, the ~60 kda protein, is granule-bound starch synthase which is encoded by waxy gene (Denyer et al., 1995). The 77 kda, 100 kda and 105 kda proteins are present in the soluble fraction of the endosperm, and the 77 kda protein is verified as SSII (Gao and Chibbar, 2000). The 90 kda protein has been shown to have starch branching enzyme activity (Nair et al., 1997). In pea embryo, the 60 kda and ~77 kda isoforms are GBSSI and SSII, respectively (Denyer and Smith, 1992; Denyer et al., 1993). Pea GBSSI and SSII have similar sizes corresponding to two isoforms from wheat. But no isoform in pea endosperm appears to be equivalent to wheat isoforms 90 kda, 100 kda, and 60

81 105 kda. This is the first report of the isolation of GBSSs from grain amaranth perisperm. As shown in Fig 2.1, 30 µl and 3 µl (10:1) of extracts were loaded in lane 1 and lane 2, respectively. However, the intensities of the bands shown in the two lanes were nearly the same. The silver staining method appeared to be insensitive to the amount of samples loaded in the SDS-PAGE gel. The strongest band from grain amaranth was ~65 kda. Some bands ranging from 55 kda to 64 kda were observed but not finely separated. A very faint band around 77 kda could be observed and two bands of 105 kda and 110 kda were found as well. In summary, some major GBSSs (such as the ~65 kda isoform in grain amaranth and ~60 kda isoforms in wheat and pea) were dominant in amounts in each species. Higher molecular mass isoforms (>77 kda) were found in grain amaranth and wheat but not in pea whilst a 90 kda protein from wheat starch granules was absent in both grain amaranth and pea. A ~77 kda isoform was found in all species in this experiment. Moreover, some lower molecular mass isoforms (<60 kda) were found in grain amaranth and pea but not in wheat starch granules. This result of SDS-PAGE analysis showed that more GBSSs were found in grain amaranth starch granules than in wheat and pea starch granules. Since no GBSS in grain amaranth starch granules had previously been identified, it was unknown which of these grain amaranth GBSSs were corresponding to which 61

82 starch biosynthesizing enzymes. Therefore, Western blotting was used in the following experiment and the grain amaranth GBSSs were identified by GBSSI, SSII and SBEI antibodies Identification of GBSSs from grain amaranth starch granules by immunoblot analysis Western blotting (or enzyme-assisted immuno-electroblotting) is usually used to detect proteins by a core technique, immunoassay. When a specific antibody is attached to a specific immobilized antigen, the attachment can be readily visualized by indirect enzyme immunoassay, usually using a chromogenic substrate which produces an insoluble product. Thus the specific protein and its molecular mass can be detected by means of its antigenicity (Harlow and Lane, 1988). The first step of Western blotting is transferring proteins to membranes from SDS-PAGE gels because it is easier to handle membranes than to handle polyacrylamide gels, and additionally polyacrylamide is not particularly amenable to the diffusion of large molecules. In the experiment, GBSSs isolated from grain amaranth and wheat starch granules were firstly separated with SDS-PAGE based on molecular mass, and then specifically detected in the immunoassay step. Three antibodies, GBSSI antibody, SSII antibody and SBEI antibody made from wheat starch synthesizing enzymes were used for immunoblot analysis, respectively. At least three isoforms of starch synthesizing enzymes were found 62

83 in grain amaranth starch granules when the GBSSs from wheat starch granules were used as positive controls, as shown in Fig 2.2. kda M GA Wh kda M GA Wh kda M GA Wh A B C Fig 2.2 The result of Western blotting. GA indicates grain amaranth and Wh wheat. Three antibodies were used for immunoblot analysis, A. GBSSI, B. SSII and C. SBEI. M is protein marker (New England Biolab). In Fig 2.2A, two proteins (~60 kda and 65 kda) in wheat starch granules were found to have the property of GBSSI. Similarly, some proteins (~55-60 kda, ~65 kda and ~110 kda) from grain amaranth starch granules have affinity to GBSSI antibody. Since the ~60 kda isoform in wheat starch granules was shown to be GBSSI, which is encoded by wax y gene (Nakamura et al., 1995), the ~65 kda isoform in grain amaranth perisperm was assumed to be GBSSI. Fig 2.2B showed that three bands (77 kda, 100 KDa and 105 kda) out of six bands in total in wheat starch granules were present in the reaction with SSII antibody but not in the reaction with GBSSI antibody. Therefore these proteins (or isoforms) could not be the isoforms of GBSSI. However, it seemed that ~60 63

84 and 65 kda proteins in wheat starch granules also had the property of soluble starch synthase although they were found in the reaction with GBSSI antibody. Similarly, the proteins ~55-60 kda, ~65 kda and ~110 kda in grain amaranth starch granules had affinities to both GBSSI antibody and SSII antibody. Only the protein ~77 kda was present in the reaction with SSII antibody. The observation of the isoforms (~65 kda in wheat and ~55-60 kda, ~65 kda, and ~110 kda in grain amaranth) found in the reactions with both GBSSI and SSII antibodies suggested that the SSII antibody used in this experiment was not very specific. In addition, a high molecular mass protein (~110 kda) in grain amaranth starch granules was found in the reaction with both GBSSI and SSII antibodies. This isoform might be an unknown starch biosynthesizing enzyme which is bound to starch granules and has both GBSSI and SSII properties. Fig 2.2C showed a ~90 kda protein from wheat endosperm with affinity to SBEI antibody. A ~105 kda protein isolated from grain amaranth starch granules was also found in the reaction with SBEI antibody. In summary, in grain amaranth perisperm, an isoform of soluble starch synthase (~77 kda) and an isoform of starch branching enzyme (~105 kda) along with a GBSSI (~65 kda) were found to be associated with starch granules. The ~77 kda and ~105 kda isoforms isolated from grain amaranth starch granules are likely to be SSII and SBEI, respectively. 64

85 2.5 Discussion In recent years, more and more proteins and their isoforms have been isolated and identified from different botanical sources using the advanced techniques such as 2D electrophoresis (SDS-PAGE and isoelectric focusing) and high performance liquid chromatography (HPLC). However, the properties of these isoforms cannot be fully identified by these isolation methods. For example, a ~77 kda protein from pea starch granules could be isolated and found to be bound to the starch granule (Fig 2.1). Therefore this isoform was nominated as pea GBSSII corresponding to pea GBSSI, a 60 kda protein isolated from pea starch previously (Smith et al., 1990). When the gene encoding pea GBSSII was identified, it was found that the peptide sequence of pea GBSSII had a divergent N-terminal extension which is more like that of most soluble starch synthase IIs but this extension is absent in all other GBSSs and in E. coli GS (Harn et al., 1998). Furthermore, pea GBSSII was also found from soluble phase and it could belong to soluble starch synthase II. Therefore, it is thought nowadays not only GBSSI can be bound to starch granules, but also some other isoforms of starch synthesizing enzymes, which can be isolated along with the starch granule. In grain amaranth and wheat, the ~77 kda isoforms were also found to be starch granule-bound. But the result of Western blotting showed that they did not have the property of GBSSI. In contrast, when the reaction was performed using SSII antibody, the ~77 kda isoforms were detected. Hence it can be concluded that the ~77 kda isoforms in grain amaranth and wheat are not GBSSIs but likely belong to SSIIs. 65

86 Similarly, it was thought that SBEs were in soluble phase that could not be isolated along with GBSSs. However, in our experiment, at least SBEI was also found to be bound to starch granules in grain amaranth and wheat. It is not known whether SSII and SBEI in granule-bound fraction have the same activities as in soluble fraction. But it has been found that some SSs and SBEs bound to starch granules have no activity in starch biosynthesis but are simply trapped during synthesis of the starch granule (Denyer et al., 1993; Mu-Forster et al., 1996). Although it is too early to predict the activities of the starch biosynthesizing enzymes in grain amaranth, at least one SSII, one SBEI, and more than one isoforms of GBSS/SSS were confirmed to be in the granule-bound phase when GBSSs were isolated from grain amaranth seeds. The result of Western blotting makes it possible to successfully isolate the genes encoding starch biosynthesizing enzyme from grain amaranth in the following experiments. 66

87 Chapter III cdna LIBRARY CONSTRUCTION FROM GRAIN AMARANTH DEVELOPING SEEDS 3.1 Introduction Complementary DNA (cdna) libraries are expressed libraries compared with genomic DNA libraries. A cdna library represents the information encoded in the mrnas and the information is obtained in a particular time from a particular tissue or organism in which the expected genes are expressed. To analyze the expression information contained in mrna, firstly, mrna molecules are converted into cdna molecules and are inserted into a self-replicating lambda or plasmid vector. Consequently, each lambda or plasmid DNA contains a cdna molecule and the recombinant DNA molecules compose a cdna library. The cdna library can be screened by hybridization using different probes. Alternatively, the inserts in a cdna library can be fully sequenced to obtain the information of gene transcription in a particular time and tissue/organism (Fig 3.1). 67

88 Choose appropriate vector for cdna library construction Isolation of total RNA Purification of poly(a)+ RNA Consider screening method and whether ultimate use requires eukaryotic or prokaryotic expression First and second strand cdna synthesis Cloning in appropriate vertor, packaging, expansion, and plating Check percentage of recombinants Screen library Sequencing of clones Sequence analysis and gene expression anaylysis Fig 3.1 Check RNA integrity by agarose/formaldehyde gel electrophoresis (Modified from Peter S. Silverstein et al.) The flow chart of cdna library construction. Lambda Uni-ZAP XR vector system (Stratagene), which combines the high efficiency of lambda library construction and the convenience of a plasmid system with blue-white color selection (Short et al., 1988), was used in this study. The Uni-ZAP XR vector can be double digested with EcoR I and Xho I and can accommodate DNA inserts from 0 to 10 kb in length (Fig 3.2). 68

89 Fig 3.2 Map of the Uni-ZAP XR insertion vector. Fig 3.3 Circular map and polylinker sequence of the pbluescript SK (+/-) phagemid. 69

90 The Uni-ZAP XR vector can be screened with either DNA probes or antibody probes and allows in vivo excision of the pbluescript phagemid (Fig 3.3), permitting the insert to be characterized in a plasmid system. The polylinker of the pbluescript phagemid has 21 unique cloning sites flanked by T3 and T7 promoters and a choice of 6 different primer sites for DNA sequencing. The outline of Stratagene s cdna synthesis protocol is given in Fig 3.4. Fig 3.4 cdna synthesis flow chart. A linker-primer including oligodt is annealed to poly(a)+ mrna and first-strand is synthesized by reverse transcriptase (RT). Second-strand synthesis is then performed using a mixture of RNaseH and DNA polymerase I. RNA fragments digested by RNaseH serve as primers for DNA synthesis by E. coli Pol I. Then the resulting double-stranded cdna with linkers and adaptors are size-fractionated and ligated into a cloning vector. The ligation mixture is then packaged followed by plating onto an appropriate E. coli host strain. 70

91 3.2 Plant material and methods Plant material Grain amaranth A. cruentus was grown in a phytotron and the developing seeds were collected for total RNA extraction mrna isolation from grain amaranth developing seeds Total RNA extraction from grain amaranth developing seeds (Hot phenol method modified from Dr. Chibbar s lab protocol) Since grain amaranth seeds contain a high content of polysaccharides, the total RNA extracted using the common total RNA extraction method such as TRIZOL reagent was always degraded. To remove polysaccharides from amaranth seeds, a modified hot phenol method was introduced in the experiment. Fresh amaranth seeds were ground in liquid nitrogen to a fine powder. The powder was mixed with an extraction buffer (containing 50 mm NaAc, ph 4.5; 20 mm EDTA; 2% SDS; and 50 mm 2-mercaptoethanol), and hot acidic phenol (65 C). Hot phenol removed most of the proteins by centrifugation and a phenol/chloroform/isoamyl alcohol (25:24:1) solution was used to extract total RNA in a water soluble phase. Then total RNA was precipitated by adding 1/10 volume of 3 M NaAc (ph 4.5) and 2.5 volume of 100% ethanol and centrifuged to form a pellet. The pellet was washed with 3 M NaAc (ph 5.2) for several times (usually three times) to remove polysaccharides from the precipitated RNA pellet. After washing, the RNA was precipitated again and dissolved in an appropriate 71

92 volume of DEPC treated water and stored at -20ºC temporarily before use. The RNA concentration was determined using a UV spectrometer and formaldehyde gel electrophoresis (see APPENDIX III: Formaldehyde Agarose Gel Electrophoresis). The total RNA concentration was estimated assuming that a 40 µg/ml total RNA solution has an absorbance of 1 at 260 nm mrna isolation from total RNA using PolyATtract mrna Isolation Systems (Promega) The total RNA extracted from grain amaranth seeds was used as the starting material for mrna isolation using the PolyATtract mrna Isolation Systems. A biotinylated oligo (dt) primer in the systems could hybridize to the 3 poly(a) region with high efficiency. The hybrids were captured and washed at high stringency using streptavidin coupled to paramagnetic particles and magnetic separation strand. The mrna was eluted from the solid phase by the simple addition of ribonuclease-free deionized water. The detailed procedures for large-scale mrna isolation from amaranth total RNA follow the protocol of PolyATtract systems I (Promega). The concentration and purity of the eluted mrna were determined using a UV spectrophotometer cdna library construction and amplification First strand cdna synthesis The first strand cdnas were synthesized from grain amaranth mrna using a 72

93 50-base oligonucleotide as linker-primer (GAGAGAGAGAGAGAGAGAGA CTAGTCTCGAGTTTTTTTTTTTTTTTTTT). The linker-primer contains a repeat GAGA sequence which protects the Xho I restriction enzyme cutting site in the primer sequence, and a poly(dt) which complements the polya tail of mrna. StrataScript RT (50 U/µL) was used to catalyze the reaction at 42ºC for an hour. The reaction mix of first-strand cdna synthesis was removed from the 42 C water bath after one-hour incubation. 5 µl of the first-strand synthesis reaction mix was loaded in a 1.0% agarose mini gel. The gel was run at 100 V for 10 minute Second strand cdna synthesis RNase H (1.5 U/µL) was used to nick the mrna strand after the first strand cdnas were synthesized and these mrna fragments were used as primers for second strand cdna synthesis and the reaction was catalyzed by DNA polymerase I (9.0 U/µL). 32 P labeled datp was added to the reaction to trace the amount of double-stranded cdna molecules Blunting the cdna termini and ligating the EcoR I adapters The double-stranded cdnas were blunted using blunting dntp mix and Pfu DNA polymerase (2.5 U/µL) followed by EcoR I adapter ligation to the ends of double-stranded cdna. During blunting and ligating, a handheld Geiger counter was used to monitor the counts of reaction to ensure that the synthesized cdnas were completely in 73

94 the solution Digesting with Xho I The double-stranded cdnas were digested with Xho I to provide both Xho I and EcoR I restriction enzyme ends so that the double-stranded cdnas could be ligated to a cloning vector. After digestion, the double-stranded cdnas were precipitated by 100% (v/v) ethanol and dissolved into 1xTE buffer (50 mm Tris-HCl and 20 mm EDTA, ph 7.5) Size Fractionating A drip column was assembled for size fractionating. The column was eluted with TE buffer and the double-stranded cdnas were immediately loaded when ~50 µl of the TE buffer remained above the surface of the resin. Once the sample entered the Sepharose CL-2B gel filtration medium, the connecting tube was filled with TE buffer. A fresh microcentrifuge tube was used to collect each fraction (three drops for one microcentrifuge tube). A minimum of 12 fractions, each containing ~100 µl, should be collected. 8 µl of each collected fraction was removed for fast agarose gel electrophoresis. The collected fraction, which contained most double-stranded cdna molecules greater than 500 bp, was extracted with phenol-chloroform and then precipitated using 100% ethanol. After precipitation, the cdna pellet was resuspended in 5 µl of sterile water and 0.5 µl of cdna was removed for quantitating. 74

95 Ethidium bromide (EtBr) plate assay - quantitating the cdna A prepared EtBr LB plate (1 µl of EtBr stock solution (10 mg/ml) per plate with 10 ml of LB agar) was used for quantitating. A DNA sample of known concentration was used to spot on EtBr LB plate as standards (200, 150, 100, 75, 50, 25, and 10 ng/µl, respectively). After spotting all of the standards, 0.5 µl of the cdna sample was immediately spotted onto the plate adjacent to the line of standards. After all spots absorbed into the plate, the lid of the plate was removed and photographed using a UV light box. The concentration of cdna sample was obtained in comparison with the concentration of the standards Ligating the cdna inserts The double-stranded cdna molecules reverse-transcribed from grain amaranth mrna were ligated into the Uni-ZAP XR vector in this step. ~100 ng of cdna sample was added in the ligation mixture containing 0.5 µl of T4 DNA ligase (4 U/µL). The reaction tube was incubated for 2 days at 4 C, longer than normal ligation reaction. After ligation was complete, 3 µl of the ligation mixture was packaged using Gigapack III Gold packaging extract Packaging reaction and the titer of primary cdna Before packaging, a 50 ml culture of XL1-Blue MRF cells (Bullock et al., 1987) from a colony isolated on a tetracycline LB agar plate (See APPENDIX V: Preparation of Media and Reagents) was grown in LB broth with supplements (See APPENDIX V: Preparation of Media and Reagents) one day in advance. 3 µl ligation mixture was used in one packaging reaction to meet a good 75

96 representational primary library size which consists of ~1 x 10 6 clones. To titer the packaging reaction, 1:10 and 1:100 dilutions of the final packaged reaction were grown on NZY agar plate (See APPENDIX V: Preparation of Media and Reagents) feed by XL1-Blue MRF cells. Plaques were visible after 6-8 hours incubation and the number of plaques in one plate determined the titer in plaque-forming units per milliliter (pfu/ml). Titer of cdna library= Number of plaques (pfu) x dilution factor Volume plated (µl) x 1000 µl/ml where the volume plated (in microliters) refers to the volume of the helper phage solution added to the cells Determining background by blue-white color selection Since the Uni-ZAP XR vector contains lac Z operator, the percentage of recombination in ligation can be calculated by the ratio of blue-white clones in a LB plate by adding IPTG as a trigger Amplifying the library The primary cdna library was amplified along with the growing of phage on NZY agar plates. The amplified cdna library was resuspended in SM buffer (See APPENDIX V: Preparation of Media and Reagents) and stored in 7% (v/v) DMSO at -80 C. The titer of the amplified library was assumed to be ~ pfu/ml. 76

97 3.3 Results The high quality of total RNA extracted from grain amaranth developing seeds The hot-phenol method for total RNA isolation, provided by the lab of Dr. Chibbar (Dept. of Plant Science, The University of Saskatchewan, Canada), was testified to be a best method for large amount total RNA extraction from grain amaranth seeds whereas polysaccharides in the seeds could not be removed using the common TRIZOL reagent. In this experiment, fresh grain amaranth seeds (1.5 g) were used as starting material and 500 µg total RNA was extracted using the hot-phenol method. The A260/A280 absorbance ratio of total RNA detected by a UV photospectrometer was above 2.0 (data not shown). The denatured formaldehyde RNA gel electrophoresis showed two bands (28S and 18S rrna) and the 28S band was about 4.5 kb and twice the intensity of the18s band whose size was around 1.9 kb (Fig 3.5). Kb M Fig 3.5 Total RNA on denatured formaldehyde RNA gel. M (marker) is kb RNA ladder (Gibco-BRL). Lanes 1 to 6 are different concentrations of the total RNA samples (0.5 µg, 1.0 µg, 1.5 µg, 2.0 µg, 2.5 µg and 3.0 µg). 77

98 The grain amaranth total RNA shown on the denatured formaldehyde RNA gel indicated that the quality of total RNA was good for mrna extraction. Using PolyATtract mrna Isolation System I (Promega), 5 µg of mrna was isolated from total RNA. Thus it was estimated that the total RNA contains approximately 1% mrna in grain amaranth developing seeds The first strand cdna synthesized from mrna The first strand cdnas were synthesized using a ZAP-cDNA synthesis kit. mrna was primed in the first-strand synthesis with a hybrid oligo(dt) linker-primer that contained a Xho I restriction site and was reverse-transcribed using Strata-Script reverse transcriptase and 5-methyl dctp. Full-length, complete first strand cdna transcripts were synthesized. The first strand cdnas appeared as a smear extending from approximately 8.0 kb to approximately 0.5 kb (Fig 3.6). The bulk of the cdnas was clustered around 3.0 kb. M FS-cDNA 3000 bp 500 bp Fig 3.6 The first strand cdnas for cdna library construction. The marker is GeneRuler 100 bp DNA ladder plus (MBI Fermentas). 78

99 3.3.3 The synthesis and size-fraction of second strand cdnas The second strand cdnas were synthesized via RNaseH which nicked the RNA bound to the first strand cdnas to produce a multitude of fragments and DNA polymerase I which used these fragments as primers for the synthesis. After some modification such as blunting the termini of the double-stranded cdnas, EcoR I adapter ligation, and dephosphorylation, the double-stranded cdnas were digested with Xho I to provide Xho I and EcoR I ends corresponding to the Uni-ZAP XR vector. The released EcoR I adapter and residual linker-primer from the 3 end of the cdnas were separated on a drip column containing Sepharose CL-2B gel filtration medium (Fig 3.7). The size-fractionated cdnas were precipitated and ligated to the Uni-ZAP XR vector. M bp 500 bp Fig 3.7 The size-fractionated second-strand cdna. The marker is Gene-Ruler 100 bp DNA ladder plus (MBI Fermentas). Lane 1 is the first selected tube and lane 2 is the second selected tube. The cdna in the first tube was used for the ligation to the vector. 79

100 3.3.4 The titer of the primary library The double-stranded cdnas reverse-transcribed from the mrna of grain amaranth developing seeds were ligated to the Uni-ZAP XR vector to form a cdna library. Two volumes (1 µl and 4 µl) of ligation reaction were packaged in a high efficiency system, Gigapack III Gold extract, and were plated on the E. coli cell line XL1-Blue MRF. The titer of the two primary cdna libraries constructed from grain amaranth developing seeds were 2.43 x 10 6 pfu/ml and 5.47 x 10 6 pfu/ml, respectively. Since a good representational primary library should consist of ~1 x 10 6 clones, both primary libraries constructed in this experiment contained enough amounts of cdnas for gene screening Blue-white color selection for background determination A background test was completed by plating several hundred plaques on a plate with IPTG and X-gal. IPTG was used to induce expression of genes regulated by the lac system because the lac repressor bound to the operator region when IPTG was absent. Therefore, the adding of IPTG promoted the release of lac repressor from lac operator and induced the gene expression in E. coli. In this experiment, the diluted (1:10) primary cdna library was grown on a NZY agar plate. On IPTG treated plates, empty plaques showed in blue, whilst recombinant plaques showed in white (clear). Only one blue plaque was found out of 350 total plaques on the plate. 80

101 Therefore, 99.7% of the plaques were recombinant which contained the cdna inserts reverse-transcribed from mrna molecules Determining the lengths of inserts in the cdna library Fifty plaques from a primary cdna library (2.43 x 10 6 pfu/ml) were cored arbitrarily from the agar plate for in vivo excision to obtain phagemids. The phagemids were digested with EcoR I and Xho I to determine the lengths of inserts. The result of agarose gel electrophoresis showed that eight clones were not completely digested with the restriction enzymes. However, the sizes of most cdna inserts were greater than 500 bp. Only a few cdna inserts were approximately 400 bp. Therefore, it could be concluded that in the primary cdna library (2.43 x 10 6 pfu/ml), approximately 95% of the inserts were > 500 bp (Fig 3.8) bp 500 bp 3000 bp 500 bp Fig 3.8 Determination of the lengths of inserts of a cdna library (2.43 x 10 6 pfu/ml). The marker is MassRuler DNA ladder (MBI Fermentas). 81

102 3.3.7 The titer of the amplified cdna library The primary cdna library constructed from grain amaranth developing seeds was amplified to obtain an amplified cdna library containing large amount of cdnas (high-titer) with stable quantity. In this experiment, the amplified cdna library from one primary cdna library (2.43 x 10 6 pfu/ml) resulted in 5 x 10 9 pfu/ml, three-fold greater than that of primary cdna library. The amplified cdna library was stable in 7% (v/v) DMSO and it could be stored at -80 C for over two years. 3.4 Discussion The advantages of cdna library construction and screening There are many advantages to construct a gene expression library, cdna library, for gene cloning. Firstly, a cdna library is an array of DNA copies of an mrna population that are propagated in a cloning vector and usually maintained in E. coli. Therefore, cdna library screening is considered as a useful technique for the isolation of the full-length cdna from a particular tissue or time in which the gene is highly expressed. Secondly, the cdna library can be phagemid library or plasmid library. Furthermore, a PCR-based cdna library has been developed. Thus different cdna libraries can meet different purposes in practice. Thirdly, since a cdna library contains most mrna molecules expressed in the organ(s), many different genes can be screened from one cdna library. 82

103 Since starch synthesizing enzymes in one organ contain many isoforms encoded by different genes, to construct a cdna library will be a best method to isolate these starch synthesizing enzyme genes. Moreover, the probes for cdna library screening can be heterologous probes other than homologous probes, thus the screening is widely used in the isolation of a cluster of genes or family genes The classification of cdna libraries and their usages cdna libraries are broadly classified as directional and random cdna library. A directional library contains cdna inserts cloned in a specific orientation relative to the transcriptional polarity of the original mrnas; whereas a random library contains cdna inserts cloned in either orientation. Directional libraries are usually constructed to drive expression of the cloned gene by a controllable promoter (contributed by the vector) and to immunologically detect the target protein (antigen) with a specific probe (an antibody). Therefore, all members of a directional library are potentially able to express antigen, whilst only fifty percent of the clones in random libraries contain cdna inserts oriented properly for expression. cdna libraries are also classified as phagemid or plasmid libraries distinguished by the different vectors used in the cdna library construction. Generally, phagemid libraries are more stable and much easier for screening than plasmid libraries. But after obtaining a pure plaque, cdna inserts should be isolated from high-quality preparations of lambda DNA which is difficult to obtain. In contrast, plasmid libraries are easy to prepare or to sequence. In recent years, however, several lambda-based in vivo excision systems have been developed. These systems utilize a helper phage that is co-infected into an appropriate strain 83

104 of E. coli along with a plaque-purified lambda clone (Hay and Short, 1992). Through a recombination event, a phagemid is excised from the lambda clone and is recovered in plasmid form, thus obviating the need for DNA preparations from phage. To date, most commercial kits utilize the method of Gubler and Hoffman (1983). But a popular kit used by most laboratories is the ZAP-cDNA synthesis kit which is produced by Stratagene Company. This kit contains a phagemid cdna synthesis system and a high-efficiency Gigapack III Gold packaging extract which can produce high-titer libraries (Kretz et al., 1989). A new method for cdna library construction is PCR-based. The principle is when the first and second strands of cdna are synthesized, the 3 linker/primer and 5 adaptor with a pair of PCR primers are involved in RT reaction. Double strand cdnas can be PCR amplified to obtain many copies. The PCR-based cdna library construction method can provide full-length cdnas and conserve the integrity of 5 end of all cdna inserts. Most of all, it needs a very small amount of total RNA or mrna as starting material. The lowest amount can be as low as 500 ng of total RNA whereas the traditional method needs as much as 5 µg of mrna. However, because PCR technique has some unstable characteristics such as template competition and wrong amplification, some low-abundance mrna molecules may be lost during the amplification. So the titer of PCR-based cdna library should be minimum 10 6 pfu/ml. Otherwise, the library may not be large enough for the screening of target clones. In conclusion, before constructing a cdna library, it is very important to choose an appropriate method or construction kit. The following factors should be considered: 1) the probe used for cdna library screening. If the probe is an 84

105 antibody, a directional library should be constructed. Otherwise, the cdna molecules in the library cannot express target proteins (antigens) and it is difficult to detect them with the specific probe (antibody). 2) the titer of the cdna library. Usually, the plasmid cdna library gives a lower titer than the phagemid cdna library. However, the procedures in plasmid cdna library construction can be easily handled. If the mrna is highly expressed in the starting material, a plasmid cdna library is able to provide enough cdna copies corresponding to the target gene although the titer of the cdna library is low. 3) the amount of total RNA or mrna of starting material. If the amount is low, a PCR-based cdna library construction method will be a best choice. In our experiment, cdna fragments were used as probes for cdna library screening. Thus both the directional and random library could be chosen. Since it is not difficult to obtain mrna from grain amaranth developing seeds, the method of PCR-based cdna library was not needed for this work. A plasmid cdna library was constructed from grain amaranth developing seeds at first. However, the titer of the plasmid library was only 10 4 pfu/ml (data not shown). The low titer could be explained if cdna molecules were not ligated into the plasmid vector efficiently, or the efficiency of transformation of plasmid vector into E. coli was low. Then the ZAP-cDNA synthesis kit was used to construct phagemid cdna libraries and the titers of the primary libraries were 2.43 x 10 6 pfu/ml and 5.47 x 10 6 pfu/ml, respectively. Therefore, the package of phagemid into phage via Gigapack III Gold packaging extract is more efficient than the transformation of the plasmid into E. coli. Moreover, since the in vivo excision system makes it 85

106 much easier to obtain the phage DNA than before, a phagemid cdna library should be the best choice for making high quality cdna library compared to other methods such as plasmid cdna library construction The properties of a good cdna library To construct a good cdna library is the prerequisite for the isolation of full-length cdna by screening. A good cdna library should be large enough to contain representatives of all sequences of interest, some of which may be derived from low-abundance mrnas. The clones in a good cdna library should contain a minimal number of small cdna inserts (often defined arbitrarily as 500 bp), whilst most cdna inserts should be near full-length copies of the mrna molecules from which they were derived. Commonly, if the starting material, mrna, is of high quality, the cdna library will satisfy all criteria in that it contains full-length cdna inserts and those cdnas derived from low-abundance mrnas. The selection of an appropriate cdna library to construct is also very important. Although most cdna library construction kits provide very detailed protocols, the researchers still need to be highly cautious in handling RNA, radioactivity and other dangerous chemicals. Especially, since cdna molecules cannot be visualized during the experiment, radioactivity must be used to detect the cdnas in some steps. The properties of cdna library constructed from grain amaranth developing seeds are summarized in Table 3.1. All criteria of a good cdna library are satisfied, as a good cdna library should guarantee at least 1 x 10 6 pfu/ml of primary clones and 87% recombinant clones, and contain an average insert size of 86

107 at least 1 kb. Table 3.1 Properties of cdna library constructed from grain amaranth developing seeds Starting Material: Grain amaranth developing seeds Starting Amount: mrna (5 µg) Primary Clones: 2.43 x 10 6 pfu/ml Amplified Clones: 5 x 10 9 pfu/ml Average Insert Size a : 1.2 kb % Recombinants: 99.7% a Average insert size determined by restriction enzyme digestion of 50 clones picked at random from library. 87

108 Chapter IV ISOLATION, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF A PUTATIVE GENE ENCODING STARCH SYNTHASE II FROM GRAIN AMARANTH 4.1 Introduction cdna library screening allows the detection of expressed genes for subsequent cloning and sequencing. Therefore it is a good method to obtain full-length cdnas which are probably candidate genes. The desired clone in cdna library can be hybridized to bind to a radioactively labeled DNA probe. Usually, the size of the probe ranges from a few nucleotides to hundreds of kilobases (kb). Long probes are usually made by cloning, whilst short probes (oligonucleotide probes) can be made by chemical synthesis. The library screening includes phage particle lifting, immobilized cdna hybridization with a labeled probe, positive plaque picking, and in vivo excision. The positive cdna clones isolated from cdna library are usually sequenced and analyzed by comparing with the sequences in a gene database to find the similarity to those known genes. In chapter II, some isoforms of starch biosynthesizing enzymes were found in grain amaranth seeds. An isoform corresponding to starch synthase II in pea embryos showed granule-bound characters and was identified as a very important isoform involved in amylopectin synthesis (Craig et al., 1998). Therefore, in our experiment, the gene encoding this isoform from grain amaranth was first isolated 88

109 by cdna library screening. Since no gene or partial gene encoding any starch synthesizing enzyme was previously isolated from grain amaranth, a wheat starch synthase II gene (GenBank ACC no. AJ269502) was used as a homologous probe to detect the gene from the cdna library of grain amaranth developing seeds. The nucleotide sequence of the cdna isolated from grain amaranth contained an open reading frame (ORF) encoding a starch synthase II precursor. Moreover, using Southern and Northern blotting methods, the cdna was found to be a single or very low copy gene in grain amaranth genome and highly expressed in developing seeds. Grain amaranth is an old crop in the world, but few molecular investigations have been carried out. The isolation of the gene encoding an isoform of starch synthase II was the first report for grain amaranth. Further analysis of gene structure and expression is needed in future studies. 4.2 Methods cdna library screening Plaque particle lifting Twenty plates (150 mm in diameter) were used for plaque lifting, and approximately 5 x 10 4 phage particles were grown in each plate. A NC membrane (Hybond -N, Amersham Pharmacia Biotech) was placed onto each plate for 2 minutes to allow the transfer of the phage particles to the membrane. The agar plates for lifting were stored at 4 C until plaque particles were picked for secondary screening. The membranes with nitrocellulose-bound DNA were 89

110 denatured by submerging the membrane in a denaturation solution (containing 1.5 M NaCl and 0.5 M NaOH) for 2 minutes, neutralized in a neutralization solution (1.5 M NaCl and 0.5 M Tris-HCl, ph 8.0) for 5 minutes, and rinsed for no more than 30 seconds with a rinsing solution (0.2 M Tris-HCl and 2 x SSC, ph 7.5). The membranes were dried briefly on a Whatman 3MM paper and the DNA was bound to the membranes by Stratalinker UV crosslinker (120,000 µj of UV energy) for ~30 seconds. The membranes were stored at 4 C before pre-hybridization and hybridization P-dCTP labeled DNA probe for cdna library screening A DNA fragment (1.5 kb long digested with Sal I and EcoR I from wheat SsII gene) was labeled with isotope 32 P using Random Primers DNA Labeling System from Invitrogen Life Technologies. Labeled-DNA was purified by NAP5 (Amersham Pharmacia Biotech) column followed the protocol provided by Amersham. And 1µL eluted probe was removed for radioactivity checking with a scintillation counter. About 80, ,000 cpm/µl column-purified probe was expected if fresh isotope and 25 ng DNA were used for labeling Pre-hybridization and hybridization The prepared membranes, as described in , were immerged into 5 x SSPE (see APPENDIX V: Preparation of Media and Reagents) and placed in a hybridization bottle (DNA side inside ). A hybridization buffer (church buffer, see APPENDIX V: Preparation of Media and Reagents) was used for 90

111 pre-hybridization and the membranes were incubated at 65 C for 4 hours or overnight. The hybridization buffer was refreshed after pre-hybridization, and the labeled probe (as described in ) was added into the bottle (note: the probe was denatured at C for 10 minutes and placed on ice for 5 minutes prior to addition to the hybridization solution). The hybridization bottle was then incubated overnight at 65 C in a hybridization oven Membrane washing and film exposure For the first screening, a low stringent condition (42 C) was used for washing. Membranes were removed individually from the bottle and put in a container containing a washing buffer (2 x SSC, 0.1% SDS). The washing buffer was refreshed three times, and was incubated each time at room temperature with gentle shaking. The membranes were then transferred to a prewarmed (42 C) washing buffer (0.1 x SSC, 0.1% SDS) and incubated at 42 C for 20 minutes on the shaker. The washing was continued until the radioactive signal from the membranes was low. After washing, the membranes were placed on Whatman 3MM paper in a large X-ray cassette holder and covered with plastic wrap. The X-ray film was exposed overnight or longer (depending on the signal performance.) at -70 C in a dark room Secondary and tertiary screening The positive plaques (may not be single plaque) were cored and put into a sterile microcentrifuge tube containing 1 ml of SM buffer (see APPENDIX V: 91

112 Preparation of Media and Reagents) with a drop of chloroform and incubated for 1-2 hours at room temperature or overnight at 4 C. Each tube might contain high amounts of plaques that should be diluted with 10 mm MgSO 4 (1/100). The diluted sample was combined to XL1-Blue MRF host cells (OD 600 =0.5), and the plating culture with cells was carefully poured onto 150 mm petridishes and incubated at 37 C for ~8 hours. When the plaques were grown on the plate, the plates were ready for secondary screening. Like primary screening, the procedures of membrane lifting, pre-hybridization and hybridization were repeated, but the membrane was washed under a more stringent condition (65 C). It was necessary to perform tertiary screening to make sure that all plaques on the plate were well separated and all were positive clones In vivo excision of the pbluescript phagemid from the Uni-ZAP XR Vector After the tertiary screening, the plaque of interest was cored from the agar plate and transferred to a sterile microcentrifuge tube containing 500 µl of SM buffer and 20 µl of chloroform. After the phage particles were released into the SM buffer, the microcentrifuge tube was incubated at room temperature for 1-2 hours or overnight at 4 C. The phage stock could be stable for up to 6 months at 4 C. The phage was excised in vivo to form a phagemid using an ExAssist helper phage with SOLR strain following the in vivo excision protocol provided by ZAP-cDNA Synthesis Kit (Stratagene). After in vivo excision, colonies appearing on the plate contained the pbluescript double-stranded phagemid with the cloned 92

113 cdna insert. To maintain the pbluescript phagemid, the colony was streaked on a new LB-ampicillin agar plate. For long-term storage, a bacterial glycerol stock was prepared and stored at -80 C. method. The phagemid DNA was extracted using a standard plasmid DNA extraction Restriction enzyme patterns digested with different restriction enzymes The cdna library for screening was an amplified library, having more than one positive clone. Some of the clones were false positive, whilst some others were from different mrna molecules but they were transcribed from the same gene. Therefore, these clones were digested with different restriction enzymes to make a restriction enzyme map so that it was not necessary to sequence all the clones. The enzymes used in this experiment were those unique restriction enzymes corresponding to the pbluescript SK (-) multiple cloning site (Fig 3.3). Fifteen restriction enzymes chosen for digestion were BamH I, EcoR I, BstX I, Cla I, Hind III, Not I, Kpn I, Pst I, Sac II, Sac I, Sal I, Sma I, Spe I, Xba I, and Xho I. Using these enzymes can prevent the digestion of the vector used, avoiding obtaining too many fragments in the analysis Sub-cloning for sequencing Positive clones were subcloned according to the method of Sambrook et al. (1989). In this experiment, a cdna insert was digested with Xho I and Spe I from 93

114 a positive clone isolated from grain amaranth cdna library and ligated to a vector to obtain a new constructed plasmid. The reconstructed plasmid was checked by several restriction enzymes digestions (Xho I, Xho I/Spe I, and some other enzymes, e.g. Hind III, which are in the sequence of insert) to confirm the cdna fragment was ligated into the vector successfully Sequencing strategies Two unique sequencing sites on the vector, M13 primer binding site and M13 reverse primer binding site, were used in the sequencing. Moreover, two more sequencing primers were designed since the unique primers on the vector were not sufficient for sequencing the full length of cdna insert for both strands. The new primers designed were 5 TGGTGCTGCAAGACAATC 3 and 5 CCAGCCTATTTCCTCCAT 3. All sequencing reactions were done at the Robert Research Institute (London, Canada) using ABI GeneAmp 9700 thermocycler. The sequences were analyzed with a sequence analysis package, DNA STAR Sequence alignment The DNA sequences were aligned using BLAST (Basic Local Alignment Search Tool) and FASTA (FAST-All from European Bioinformatics Institute). The nucleotide sequence was translated to an amino acid sequence for CDD (Conserved Domain Database) search, an analyzer which compares protein sequence with the known sequences in the database The structure analysis of the protein (AcSSII) The primary structure of AcSSII was analyzed using ProtParam (Gasteiger et 94

115 al., 2005) compared to potato SSII (CAA61241) and wheat SSII (AAD53263). The secondary structure of AcSSII was analyzed using Sspro8 program (Pollastri et al., 2002) provided by ExPASy World Wide Web server. The 3D structure of AcSSII was analyzed using 3D-JIGSAW (Bates et al., 2001) provided by ExPASy World Wide Web server. Since the AcSSII sequence was too long to be calculated, the sequence was splitted into a smaller chunk which was a 503 aa peptide Southern blotting In Southern blotting, chromosomal DNA (60 µg) was isolated from the seedlings of A. cruentus, and digested completely with restriction enzymes, Xho I, Hind III, BamH I, EcoR I and EcoR V, respectively. The restriction fragments were then loaded on an agarose gel for electrophoresis and separated by the sizes of the fragments. DNA fragments separated by gel electrophoresis were transferred to an NC membrane using a transferring apparatus, VacuGene XL, following the procedures provided by VacuGene XL Vacuum blotting System (Amersham Biosciences). The DNA fragments were fixed onto the membrane by UV-crosslinking using Stratalinker (x 2 cycles). Then the membrane was rinsed briefly with ddh 2 O to remove salt, and dried in air. The membrane was stored in a plastic bag at 4 C for a couple of days before use. The DNA probe used in Southern hybridization was a 2.1 kb long fragment from the cdna encoding A. cruentus starch synthase II (AcSsII) which was 95

116 digested with Xho I and EcoR I. The fragment was then purified and labeled with 32 P. Pre-hybridization, hybridization and washing were performed following the standard protocol of Southern blotting (Southern, 1975) RNA expression analysis of cdna encoding AcSSII Total RNA extraction and formaldehyde gel electrophoresis Total RNAs were extracted from grain amaranth leaves, roots, stems, flowers and seeds, respectively, following the hot phenol method from Dr. Chibbar s lab. RNAs (10 µg per lane) were run on formaldehyde agarose gel electrophoresis (see appendix III: Formaldehyde Agarose Gel Electrophoresis) and transblotted to an NC membrane using VacuGene XL. The RNAs were fixed on the membrane by UV-crosslinking Northern blotting The probe used in Northern blotting was the same as used in Southern blotting. RNAs on the NC membrane were hybridized with the probe using a standard method (Sambrook et al., 1989). After hybridization, the NC membrane was washed by washing buffer (2 x SSC, 0.1% SDS) at 65 C until the radioactivity background was low. The membrane was then exposed to an X-ray film Expression of AcSsII in E. coli mutant RH Construction of expression plasmids Three PCR primers were designed for amplifying two cdna fragments from 96

117 AcSsII: F1 (5 -GGCACATGTCAGTGAGTGGGTGTAGAA-3 ), F2 (5 -ACCAC ATGTACGACGCTGCCGCT-3 ), and R1 (5 -CTGATCTAGAGGCGTGAG-3 ). Primers F1 and F2 contained Nco I restriction site, which provided the sticky end that could be ligated to the same site on the expression vector. PCR products from F1/R1 and F2/R1 were cloned into T-vector (see APPENDIX IV). The cdna fragments were digested with Nco I and BamH I which were on the T-vector to yield two fragments of 2220 bp and 1580 bp, respectively. After Gel purification, the cdna fragments were cloned into pet28a expression vector at the Nco I and BamH I sites to form the plasmid pnb1 and pnb2, respectively. The reconstructed clones were sequenced to verify that no mutations had occurred during these modification steps Complementation of E. coli mutant RH98 The expression plasmids, pnb1 and pnb2, were transformed respectively into a competent strain RH98, a glycogen-deficient E. coli strain. For the complementation test of GS deficiency in RH98 by AcSsII, RH98/pNb1, RH98/pNb2, RH98/pET28(a), and wild type E. coli strain DH5α were grown on an enriched medium [containing 0.85% (w/v) KH 2 PO 4 ; 1.1% (w/v) K 2 HPO 4 ; 0.6% (w/v) yeast extract; 1% (w/v) glucose; and 1.5% (w/v) agar; ph 7.0], and grown under inductive conditions (0.1 M IPTG). Following growth at 37 C for 20 hr, the cells were stained with an iodine solution (0.03 M I 2, 0.04 M KI) Phylogenetic analysis Phylogenetic analysis of AcSSII and other SSIIs from different plants was conducted using PAUP* 4.0 beta version 10 (Swofford, 2003). The amino acid 97

118 sequences of all SSIIs except for AcSSII were downloaded from GenBank (Table 4.1). Table 4.1 The GenBank accession number of starch synthases used in the phylogenetic analysis Name Taxon Starch synthase Acce. No. Arabidopsis Arabidopsis thaliana SSII NP_ Rice Oryza sativa SS IIb AAK81729 SS IIa AAL16661 Wheat Triticum aestivum SS II AAD53263 Barley Hordeum vulgare SS II AAN28307 Maize Zea mays SS IIa AAS77569 SS IIb AAD13342 Potato Solanum tuberosum SS II CAA61241 Sweet potato Ipomoea batatas SS II AAC19119 Pea Pisum sativum SS II CAA61269 Kidney bean Phaseolus vulgaris SS IIa BAD18846 SS IIb BAD18847 Grain amaranth Amaranthus cruentus SSII - Bacteria E. coli GS AAA Results Eight positive clones isolated from cdna library The cdna library constructed from grain amaranth developing seeds was screened using a homologous probe from wheat ssii gene. Approximately 1 x 10 6 phage particles were screened in primary screening, and 42 positive clones were 98

119 found from twenty membranes in total (Fig 4.1I). Since each plate contained about 5 x 10 4 phage particles for primary screening, the phage particles were condensed on the plate and could not be separated easily. Therefore, the 42 positive clones were cored from the plates and diluted for secondary screening. Each 150 mm petridish for secondary screening contained 200 to 300 clones, much less than that in primary screening. After secondary screening, eight clones out of the 42 clones were confirmed to be positive clones. In each plate containing positive clones, it was found about one-third of the positive clones hybridized to the radiolabeled probe (Fig 4.1II). All eight clones were used for tertiary screening using 80 mm petridishes. This time all the clones on each petridish were positive (Fig 4.1III). In summary, eight candidate clones from grain amaranth cdna library hybridized with the probe made from wheat SsII gene. 99

120 I II II III III Fig 4.1 The result of cdna library screening for Amaranthus cruentus starch synthase II (acssii) gene isolation. The cdna library was constructed from grain amaranth developing seeds. The probe for screening was 1.5 kb fragment from wheat starch synthase II cdna, provided by Dr. Chibbar s lab (Dept. of Plant Science, University of Saskatchewan, Canada). I. Primary screening; II. Secondary screening; and III. Tertiary screening. 100

121 4.3.2 Restriction enzyme patterns of eight candidate clones isolated from cdna library Fifteen restriction enzymes were used to digest the eight candidate clones. Some of the restriction enzymes were unique enzymes in multiple cloning site of Uni-ZAP XR vector, but some of them were not. Therefore, most of the digestions should produce one fragment on the agarose gel. On the other hand, the digestions resulted in more than one fragment indicated that the cdna insert contained one or more digested sites. BamH I EcoR I Hind III Kpn I Sac II Sal I Spe I Xho I bp M BstX I Cla I Not I Pst I Sac I Sma I Xba I Fig 4.2 An example of restriction fragments from a candidate clone digested with various restriction enzymes. The maker is MassRuler DNA ladder (MBI Fermentas). 101

122 Fig. 4.2 shows an example that one candidate clone were digested with various restriction enzymes listed on the top of the figure. One fragment was found in the digestion with BamH I, BstX I, Hind III, Not I, Pst I, Sac II, Sal I, Sma I, Spe I, or Xba I; two fragments were found in the digestion with Cla I, Kpn I, Sac I, or Xho I; and three fragments were found in the digestion with EcoR I. Although the digestion with Sal I showed a single band, the size of the band was less than a linear DNA containing the vector and cdna insert. This band could be a coiled plasmid DNA. Since the Uni-ZAP XR vector does not contain Sal I, Hind III, and Cla I restriction enzyme sites, the digestion with these three restriction enzymes shows that 1) no Sal I restriction enzyme site exists in the vector sequence or the cdna insert; 2) one Hind III and two Cla I restriction enzyme sites exist in the sequence of the cdna insert. Since the other twelve restriction enzyme sites are all on the sequence of the Uni-ZAP XR vector, it is not surprising that the digestion with BamH I, BstX I, Not I, Pst I, Sac II, Sma I, Spe I, or Xba I shows a single band like linear DNA. Similarly, one Kpn I, Sac I, and Xho I and two EcoR I sites are found to exist in the cdna insert in addition to the site in the Uni-ZAP XR vector. Compared to this restriction enzyme pattern, the digestions of all eight candidate clones were found to be identical. Therefore, it could be concluded that all the clones were the same. To further confirm the result of restriction enzyme digestion, two of the clones were picked randomly for sequencing in the following experiment. 102

123 4.3.3 A candidate clone isolated from the cdna library contains a full-length cdna encoding A. cruentus starch synthase II (AcSSII) The sequences of the two clones randomly picked from eight candidate clones showed similar sequences at both ends of the inserts (data not shown). Therefore, only one clone was fully sequenced. The cdna insert in the sequenced clone is approximately 3.0 kb with 0.3 kb untranslated 5 end sequence and poly(a) tail. The result of sequencing shows that the cdna insert contains a 2430 bp of open reading frame (ORF) with start codon (ATG) and stop codon (TGA) (See APPENDIX I). Before the start codon, 286 bp long untranslated sequence is found whilst the poly(a) tail is located on 3 end which is 265 bp behind the stop codon. The ORF encodes a protein consisting 809 amino acids with 54 amino acids of N-terminal chloroplast transit peptide (ctp). Since starch synthesizing enzymes are encoded by nuclear genes and imported from the cytosol to amyloplast, the deduced amino acid sequence of a starch synthesizing enzyme precursor should contain an N-terminal transit peptide. The hallmark for ctp is an amino acid composition with a high score of hydroxylated and positively charged residues and only a few carboxylated residues (Gavel and von Heijne, 1990). Nucleotide-to-nucleotide sequence (nr) alignment (via BLAST in NCBI World Wide Web server) shows that the ORF of the cdna insert has some similarities compared with other ssii genes from various plants. The best score of the query sequence (unknown cdna, 2430 bp, from amaranth cdna library 103

124 screening) is 157 bits to Phaseolus vulgaris (kidney bean) pvss2 mrna (2570 bp) for starch synthase II precursor (AB126833). However, the overlapped region is only 203 bp and the identity is 84% (172/203) with no gap. In comparison with the full-length of unknown cdna, this overlapped region is too short to identify the unknown cdna. FASTA is another tool available for comparing nucleotide or protein sequences with sequence databases and calculates the statistical significance of matches. FASTA was also used to perform the same nucleotide alignment. Different result was obtained with FASTA that the best score was bits compared to Manihot esculents (cassava) starch synthase II precursor mrna (2527 bp) (AF173900). The identity was as high as 73.29% (ungapped) in 1636 nucleotide overlap ( for the unknown cdna vs for MessII gene). This result indicates that FASTA is a better program than BLASTN for identifying long regions of low similarity especially for highly diverged sequences. On the other hand, when the unknown cdna was translated to protein sequence, the alignments using both FASTA and BLASTP provided the same result showing that the protein encoded by the unknown cdna has high similarities to starch synthase II from potato (CAA61241), cassava (AAF13168) and sweet potato (AAC19119), etc. Compared with potato starch synthase II precursor, which is 767 amino acid (aa) long, the 760 aa overlap shows 63% identities (483/760), 76% positives (578/760), and 4% gaps (31/760). Moreover, in a highly conserved region shown in red letters in Fig 4.4, the percentage of 104

125 identities is as high as 82% (412/501), and the percentage of positives is 91% (459/501). The result of protein-to-protein sequence alignment indicates that the protein translated from the unknown cdna contains a conserved domain of glycosyl transferases group 1 from the conserved domain database (CDD) (Fig 4.3). It is known that the members of this family transfer UDP, ADP, GDP or CMP linked sugars. Moreover, a conserved domain of glycogen synthase is also identified. 105

126 UNKNOWN PROTEIN (Amaranthus cruentus starch synthase II?) 33.0% identity in 485 residues overlap; Score: 449.0; Gap frequency: 4.3% Unknown: 318 MNVILVAAECAPWCKTGGLGDVAGALPKALARRGHRVMVVIPRY----AEYADCQFTGVR 373 Gs: 1 MKILFVASEIFPFVKTGGLADVVGALPKALAKRGVDVRVLLPSYPKVQKEWRDLLKVVGK 60 Unknown: 374 KRYKVAGQDNEVLYFHTYIDG-VDFVFIEAPMLRNLGKDIYGGNRLDILKRMILFCKVAV 432 Gs: 61 FGVLKGGRAQLFIVKEYGKDGGVDLYLIDNPALFKRPDSTLYGYYDNAE-RFAFFSLAAA 119 Unknown: 433 EVPWHVPCGGVCYGDGNLVFI--ANDWHTALLPVYLKAYYRDNGLMKYTRSVLVIHNIAH 490 Gs: 120 ELAPL GLISWLPDIVHAHDWQTGLLPAYLKQRYRS---GYIIPTVFTIHNLAY 169 Unknown: 491 QGRGPVDDFTYVDLPPHYLDYFKLYDPVGGEHFNIFAAGLKTADRILTVSHGYAWELKTQ 550 Gs: 170 QGLFRLQYLEELGLPFEAYASFGL---EFYGQISFLKGGLYYADAVTTVSPTYAGEIYTP 226 Unknown: 551 EGGWGLHGIINDNDWKLRGIVNGIDVTEWNPELDLYLQSDGYANYSLETLKPGKAQCKAA 610 Gs: 227 EYGEGLEGLLSWRSGKLSGILNGIDYDLWNPETDPYI----AANYSAEVL-PAKAENKVA 281 Unknown: 611 LQNELGLPVREDVPLIGFIGRLDGQKGVDIIAESIPWLSGQDVQLVMLGTGRPDLEQMLR 670 Gs: 282 LQERLGLDVDLPGPLFGFVSRLTAQKGLDLLLEAIDELLEQGWQLVLLGTGDPELEEALR 341 Unknown: 671 ECESRYPDKIRGWVGFSVKTAHRITAGVDILLMPSRFEPCGLNQLYAMNYGTIPVVHAVG 730 Gs: 342 ALASRHPGRVLVVIGYDEPLAHLIYAGADVILMPSRFEPCGLTQLYAMRYGTLPIVRETG 401 Unknown: 731 GLRDTVQPFDPYNNT--GYGWTFDRAEANRLIDALGNCLLTYRQYKESWEGLQTRGMTQD 788 Gs: 402 GLADTVVDRNEWLIQGVGTGFLFLQTNPDHLANALRRALVLYRAPPLLWRKVQPNAMGAD 461 Unknown: 789 LSWDHAAEIYEE 800 Gs: 462 FSWDLSAKEYVE 473 Fig 4.3 Protein-to-protein alignment result of unknown protein (putative A. cruentus starch synthase II) isolated from cdna library. In the figure, black bar is the unknown protein (809 amino acid); red bar shows glycosyl transferase group 1 in CDD; gray bar shows glycogen synthase in CDD; and blue bar in the unknown protein sequence indicates the masked-out region with low complexity. In the text, the letters with red, blue, and black colors represent identical amino acids, positive amino acids, and different amino acids, respectively. 106

127 54 UNKNOWN MASLNSLRFMIETRCSLHNSNGSH-HLSYQRRKPVNGGGFLKFMDHDSNGRLSLYCVKSV StSSII MENSILLHSGNQFHPNLPLLALRPKKLSLI UNKNOWN SGCRRSNSMRYNRLKAIGDDLNEGEHSKEDKDENEDALDAAIEKSNKVSAMQKELLLQIA StSSII HGSSREQMWRIKRVKATGENSGEAASA----DESNDALQVTIEKSKKVLAMQQDLLQQIA *. *.: * :*:** *::.*. : **.:***:.:****:** ***::** *** UNKNOWN ERKKLVSSIRNSVINSDDAPADKDYNSPQNDALGANQILVKNEYDIS--TGAARQSDSAS StSSII ERRKVVSSIKSSLAN-----AKGTYDGGSGSLSDVDIPDVDKDYNVTVPSTAATPITDVD **:*:****:.*: * *. *:......: *.::*::: : **... UNKNOWN SKLNPDQLQVPLSSLEEKSGQAKSALKMLNLDSSQPKEILTKATETDSSISNGSPIVGKR StSSII KNTPPAISQDFVESKREIKRDLADERAPPLSRSSITASSQISSTVSSKRTLNVPPETPKS.: * * :.*.*. :. **...:* :.. *.*. * UNKNOWN EMKTIGSLSSEGNANGVDASKGGDERTMLPNFLSSTNKSSSIMDKEEK-ELGESYFKGLS StSSII SQETLLDVNSRKSLVDVPGKK IQSYMPSLRKESSASHVEQRNENLEGSSAEAN. :*:.:.*...*..* :.::.*.*.**. *:: * * KTGGL UNKNOWN ELVNDAAAEDDKPPPLAGQNVMNVILVAAECAPWCKTGGLGDVAGALPKALARRGHRVMV StSSII EETEDPVNIDEKPPPLAGTNVMNIILVASECAPWSKTGGLGDVAGALPKALARRGHRVMV *.:*.. *:******* ****:****:*****.************************* UNKNOWN VIPRYAEYADCQFTGVRKRYKVAGQDNEVLYFHTYIDGVDFVFIEAPMLRNLGKDIYGGN StSSII VAPRYDNYPEPQDSGVRKIYKVDGQDVEVTYFQAFIDGVDFVFIDSHMFRHIGNNIYGGN * *** :*.: * :**** *** *** ** **:::*********:: *:*::*::***** UNKNOWN RLDILKRMILFCKVAVEVPWHVPCGGVCYGDGNLVFIANDWHTALLPVYLKAYYRDNGLM StSSII RVDILKRMVLFCKAAIEVPWHVPCGGVCYGDGNLVFIANDWHTALLPVYLKAYYRDNGIM *:******:****.*:******************************************:* UNKNOWN KYTRSVLVIHNIAHQGRGPVDDFTYVDLPPHYLDYFKLYDPVGGEHFNIFAAGLKTADRI StSSII NYTRSVLVIHNIAHQGRGPLEDFSYVDLPPHYMDPFKLYDPVGGEHFNIFAAGLKTADRV :******************::**:********:* ************************: UNKNOWN LTVSHGYAWELKTQEGGWGLHGIINDNDWKLRGIVNGIDVTEWNPELDLYLQSDGYANYS StSSII VTVSHGYSWELKTSQGGWGLHQIINENDWKLQGIVNGIDTKEWNPELDVHLQSDGYMNYS :******:*****.:****** ***:*****:*******..*******::****** *** UNKNOWN LETLKPGKAQCKAALQNELGLPVREDVPLIGFIGRLDGQKGVDIIAESIPWLSGQDVQLV StSSII LDTLQTGKPQCKAALQKELGLPVRDDVPLIGFIGRLDPQKGVDLIAEAVPWMMGQDVQLV *:**:.**.*******:*******:************ *****:***::**: ******* UNKNOWN MLGTGRPDLEQMLRECESRYPDKIRGWVGFSVKTAHRITAGVDILLMPSRFEPCGLNQLY StSSII MLGTGRRDLEQMLRQFECQHNDKIRGWVGFSVKTSHRITAGADILLMPSRFEPCGLNQLY ****** *******: *.:: *************:******.****************** KTGGL look-alike UNKNOWN AMNYGTIPVVHAVGGLRDTVQPFDPYNNTGYGWTFDRAEANRLIDALGNCLLTYRQYKES StSSII AMKYGTIPVVHAVGGLRDTVQPFDPFNESGLGWTFSRAEASQLIHALGNCLLTYREYKKS **:**********************:*::* ****.****.:**.**********:**:* 809 UNKNOWN WEGLQTRGMTQDLSWDHAAEIYEEVLVAAKYQW StSSII WEGIQTRCMTQDLSWDNAAQNYEEVLIAAKYQW ***:*** ********:**: *****:****** Fig 4.4 The alignment between amino acid sequences of the unknown protein (putative A. cruentus starch synthase II, 809 aa) and Solanum tuberosum starch synthase II (StSSII, 767 aa). The numbers beside the arrows represent the amino acid position in the sequence. A transit peptide is shown in italic letters. A most conserved region in SSIIs from diverse sources is shown in red letters. *, : and - represent identical amino acids, positive amino acids and gaps, respectively. 107

128 In Fig 4.5, the fragment (54 aa) which is close to N-termini of this protein has no similarity to other starch synthases because it might be N-terminal chloroplast transit peptide (ctp). The region from 60 aa to 310 aa shows low similarity to other SSIIs since it is probably the flexible arm in starch synthases. The highly conserved region in the unknown protein sequence is from 306 aa to 809 aa, starting from a Pro-rich region. UNKNOWN PROTEIN (Amaranthus cruentus starch synthase II?) PPP KTGGL motif KTGGL look-alike motif StSSII (potato starch synthase II) Fig 4.5 The amino acid sequence comparison between the unknown protein (putative A. cruentus starch synthase II) and S. tuberosum starch synthase II (StSSII). The numbers in the figure represent predicted amino acid positions in the proteins. Non-significant similarity (<10%) region is shown in blue; low similarity (~50%) region is shown in green; and the red bar shows the highly conserved region in which the percentage of identities is 82% and the percentage of positives is 91%. Predicted amino acids in blue letters are thought to be conserved regions in SSIIs from diverse sources. The result of alignment indicates that the nucleotide sequence and protein sequence of the unknown cdna show a relatively high similarity to the nucleotide/amino acid sequence of other SSIIs. Therefore, the unknown protein was named as A. cruentus starch synthase II (AcSSII) and the cdna encoding this protein was named as A. cruentus starch synthase II (AcSsII) gene. 108

129 4.3.4 The analysis of A. cruentus starch synthase II (AcSSII) protein For protein analysis, information in protein databases can be used to predict certain properties about a protein, which can be useful for its empirical investigation. The identification and analysis of protein are two processes which are complementary. Some of useful analysis tools are available through the ExPASy World Wide Web server (Gasteiger et al., 2003). These tools include Compute pi/mw, a tool for predicting protein isoelectric point (pi) and molecular weight (Mw); ProtParam, for calculating various physicochemical parameters; Peptidemass, for theoretically cleaving proteins and calculating the mass of their peptides and any known cellular or artifactual posttranslational modification; PeptideCutter, for predicting cleavage sites of proteases or chemicals in protein sequences; and ProtScale, for representing any amino acid scale, such as hydrophobicity plots The primary structure of AcSSII ProtParam (Gasteiger et al., 2005) is a tool which allows the computation of various physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or for a user entered sequence. ProtParam program is used in this experiment to analyze the physicochemical parameters (primary structure analysis of protein) of A. cruentus SSII, potato SSII and wheat SSII, respectively. Table 4.2 shows the computed parameters including the molecular weight, theoretical pi, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity 109

130 (GRAVY) index. Table 4.2 The physicochemical parameters of Amaranthus cruentus SSII, potato SSII and wheat SSII Amaranthus cruentus SSII Potato SSII Wheat SSII Number of amino acid Mw (kda)* pi* Total number of negatively charged residues (Asp+Glu) Total number of positively charged residues (Arg+Lys) Atomic composition Carbon C Hydrogen H Nitrogen N Oxygen O Sulfur S Total number of atoms Instability index (II)* (Stable) (Unstable) (Unstable) Aliphatic index* Grand average of hydropathicity (GRAVY)* * See details in the text. 110

131 In Table 4.2, the molecular weight is derived from the 1997 IUPAC standard atomic weights, assuming ph=7.0. The isoelectric point is calculated using the algorithm from ExPASy s COMPUTE pi/mw program, which is provided by Elisabeth Gasteriger (also by Bjellqvist et al., 1993). The instability index (II) provides an estimate of the stability of the protein in a test tube (Guruprasad et al., 1990). A protein with an instability index smaller than 40 is predicted to be stable, and a value above 40 predicts that the protein may be unstable. The aliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine). It may be regarded as a positive factor for the increase of thermostability of globular proteins. The Grand Average of Hydropathy (GRAVY) value for a peptide or protein is calculated as the sum of hydropathy values (Kyte and Doolittle 1982) of all the amino acids, divided by the number of residues in the sequence. It is found that in Table 4.2, the physicochemical parameters among grain amaranth, potato, and wheat are very similar except for instability index (II). The data of instability index shows that AcSSII is a stable protein, whereas potato and wheat SSIIs are unstable proteins. Therefore, AcSSII is more stable in a test tube than the other two SSIIs. The above analyses of primary structure of proteins show that all SSIIs analyzed in the experiment have very similar physicochemical parameters. Therefore, it can be concluded that the physicochemical properties of different 111

132 SSIIs are almost the same although they have different amino acid sequences The secondary structure of AcSSII SSpro8 is an on-line server (Pollastri et al., 2002) which is used to predict the secondary structure from the primary sequence in this experiment. In SSpro8, a better algorithm is explored to obtain multiple alignments of homologue sequences, based on PSI-BLAST instead of BLAST. Fig 4.6 shows the secondary structure of A. cruentus starch synthase II (AcSSII) calculated by SSpro8. The four lines have the following meanings: Line 1) The 1-letter code of the primary sequence of the protein; Line 2) Secondary structure prediction: H = helix, E = strand, C = the rest; Line 3) 8-class secondary structure prediction: H: alpha-helix, G: 310-helix, I: pi-helix (extremely rare), E: extended strand, B: beta-bridge, T: turn, S: bend, C: the rest; Line 4) Prediction of relative solvent accessibility: - represents that the residue is buried and e represents that the residue is exposed. 112

133 Query_name: AcSSII Query_length: 809 Prediction: MASLNSLRFMIETRCSLHNSNGSHHLSYQRRKPVNGGGFLKFMDHDSNGRLSLYCVKSVSGCRRSNSMRY CCCHHHHHHHHHHHCCCCCCCCCCCCECECCCCCCCCCCEEECCCCCCCCEEEEECCCCCCCCCCCCCCC CCCHHHHHHHHHHEEEEECTTSCCEEEEEECCCCTTCCHHEEECTCTTSEEEEEEEEEECSCCCCSHHHH eeeeee-eee-eee-eeeeeeeeeeeeeeeeeeeeeee--e-eeeeeeee-e-e--eeeeeeeeeeeeee NRLKAIGDDLNEGEHSKEDKDENEDALDAAIEKSNKVSAMQKELLLQIAERKKLVSSIRNSVINSDDAPA CCCCHCCCCCCHHHHCCCCCCCCCHHCCCCCCCCCHHHHHHHHHHHHHHHHCCCCHCCCCCCCCCCCCCC HHHHHHCCCTCTTCCCCTCTCHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHEEECTSSCC ee-eeeeeeeeeeeeeeeeeeeeeee-eeeeeeeeeeeeeeee--ee-eeeeeeeee-eeeeeeeeeeee DKDYNSPQNDALGANQILVKNEYDISTGAARQSDSASSKLNPDQLQVPLSSLEEKSGQAKSALKMLNLDS CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHCCCCCCCCHHHCCCCCCCCCCCCCCCC CTCTCCCHHHHHTCCEEEEETCECECHHHHHCCCCHCCCCCTTGCECCCGCHHHHTHHHHHHHHHHTCCT eeeeeeeeeeeeeeee---eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee SQPKEILTKATETDSSISNGSPIVGKREMKTIGSLSSEGNANGVDASKGGDERTMLPNFLSSTNKSSSIM CCCCHCCCCCCCCCCCCCCCCCCCCCHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHH TCCHHHHHHHCCCCSCCCTSCCEEEEEEEEEEECCCCTSCCTCCCCCSTCCCCCCCCTCCCCSCCCCHHH eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee-eeeeeeeeeeeeee KTGGL motif DKEEKELGESYFKGLSELVNDAAAEDDKPPPLAGQNVMNVILVAAECAPWCKTGGLGDVAGALPKALARR HHHCCCCCCCCCCCCCCCCCCCCCHCCCCCCCCCCCCCEEEEEEECCCCHHHCCCHHHHHHCCHHHHHHC CHHHHHHHHHHHHHHHHHHHHHHHTTCSCCCCTTHHHHHEEEEHHHHCCGCCTTCHHHHHTHHHHHHHHT eeeeeeeeeee-ee-eee-eeeeeeeeeeeeeeeee--e-----ee-eee-eeeeeeeeeee-eee-eee GHRVMVVIPRYAEYADCQFTGVRKRYKVAGQDNEVLYFHTYIDGVDFVFIEAPMLRNLGKDIYGGNRLDI CCEEEEEECCCCCCCCCCCCCCEEEEEECCCEEEEEEEEEEECCCEEEEECCCCHCCCCCCCCCCCCCCH TCEEEEEECEHHHHHHHHEHEEEEEEEEECCCCEEEEEEEEETTCEEEEEECHHHHHHTHHHTTCCHHHH eee-----eee-ee-e-eeeeeeeeeeeeeeeee----e-e-ee-e------e--eeeeee-eeeee-e- LKRMILFCKVAVEVPWHVPCGGVCYGDGNLVFIANDWHTALLPVYLKAYYRDNGLMKYTRSVLVIHNIAH HHHHHHHHHHHHHHHHHCCCCCCCCCCCCEEEEECCHHHHHHHHHHHHHHCCCCCCCCCCEEEEEEEHHH HHHHHHHHHHEEECCTECCCSEEEETTSCEEEEESCHHHHHHHHHHHHHEHHTTHHHHHHHEEEEEHHHH -ee-----e---e-eee-eee-e-eeeee-----eeeeee--e---e---eeeeeeeeee-----e--ee QGRGPVDDFTYVDLPPHYLDYFKLYDPVGGEHFNIFAAGLKTADRILTVSHGYAWELKTQEGGWGLHGII CCCCCHHHHHHCCCCHHHHCCCCCCCCCCCCEEEHHHHHHEECCEEEEECHHHHHHHHCCCCCCCCHHHH TTCCCCCCEEEECCCCTGEHEEEECCTTTHHHHHHHHHHCHHHHEEHEEHTTHEEEEECCTTCCCCEEEE eeeeeeee-e--e-eee--e--e-eeeeeeee-e---ee-ee-ee--e-eeee-ee-eeeeeeee-e--- * NDNDWKLRGIVNGIDVTEWNPELDLYLQSDGYANYSLETLKPGKAQCKAALQNELGLPVREDVPLIGFIG HCCCCEEEEEEECCCCCCCCCCCCCCCCCCCCCCCCHCHHHHHHHHHHHHHHHHHCCCCCCCCCEEEEEE ECTTCEEEEEEEEEEECCCCTTCCEEEECTTCEEECHHCCCTTHHHHHHHHHHHTTCCCCTTCCEEEEEE eeeeee-e---e--e-eeeeee-eee-eeeee-eeeeeeeeeeeee-eee-eeeeeeeeeee-e RLDGQKGVDIIAESIPWLSGQDVQLVMLGTGRPDLEQMLRECESRYPDKIRGWVGFSVKTAHRITAGVDI ECCHCCCHHHHHHHHHHHHHCCCEEEEECCCCHHHHHHHHHHHHHCCCCEEEEEECCCHHHHHHHHCCCE ECTTCTTCEEEEEECCCCTTCEEEEEEECCSCCCHHHHHHHHHHSCCTGEEEEEEEEEEHHHHHHTTCCE eeeeeee-e---ee-eeeeeee-e---eeeeeee-ee--ee-eeeeeee-eee-e-e-eeeee-e-e-e- KTGGL look-alike LLMPSRFEPCGLNQLYAMNYGTIPVVHAVGGLRDTVQPFDPYNNTGYGWTFDRAEANRLIDALGNCLLTY EEECCCCCHCHHHHHHHHHCCCCHHEHCCCCCCCEEEECCCCCCCCCEEEECCCCHHHHHHHHHHHHHHH EECCCSCCCCCHHHHHHHHTTCCEEEEEETCCCCCCCCCCTTCCCCCCCCH.HHHHHHHHHHHTHHEEEH --eeeeeeeee-ee-eeeeeee-e--e--ee-eee-eeeeeeeeeeee-e-eeee-ee--ee-ee-e-ee RQYKESWEGLQTRGMTQDLSWDHAAEIYEEVLVAAKYQW HCCCHHHHHHHHHHHHHCCCCCHHHHHHHHHHHHHHHCC HHHHHHHHHCCCTTCCCCCCHHHHHHHHHHHHHHHHHCC eeeeeeeeeeeeeeeeeeeeeee-ee-eee--ee-eeee Fig 4.6 The secondary structure of Amaranthus cruentus starch synthase II (AcSSII). See text for detailed description. 113

134 The lined sequences highlight the N-terminal KTGGL motif and the C-terminal KTGGL look-alike motif. An asterisk indicates the lysine residue which is equivalent to that involved in the catalytic reaction of GS from E. coli (Furukawa et al., 1995). The aim of secondary structure prediction is to provide the location of alpha helices, and beta strands within a protein for the prediction of three-dimensional (3D) structure The 3D structure of AcSSII The prediction of 3D structure of protein is a process using a server that can build three-dimensional models for the protein based on homologues of known structure. Some experimental data can aid the structure prediction process such as disulphide bond, spectroscopic data, and site directed mutagenesis studies, post-translational modification and knowledge of proteolytic cleavage sites, etc.. If the protein has come directly from a gene prediction (in most cases), it may consist of multiple domains. Therefore, before starting to predict the structure of protein, the following should be considered: 1) whether the protein is a transmembrane protein, or whether it contains transmembrane segments? 2) whether the protein contains coiled coils? and 3) whether the protein contains regions of low complexity? If the answer to any of the above questions is yes, then it is worthwhile trying to break the protein sequence into pieces, or to ignore particular sections of the sequence. In this study, a 54 aa (1-54) chloroplast transit peptide was found in the 114

135 amino acid sequence of AcSSII (Fig 4.4) and in the same region, a low-complexity region was also found (Fig 4.3). Thus before the 3D structure prediction, these regions should be masked-out first. After alignment, a most conserved region of AcSSII, from 307 aa to 809 aa, was found (Fig 4.5). No coil was found in this peptide when it was analyzed using Paircoil program. Thus this 503 aa peptide, AcSSII-503, was splitted out for the prediction of 3D structure of AcSSII. 3D-JIGSAW (version 2.0) was used for 3D structure prediction in this analysis. It is one of the useful automated systems on-line to build three-dimensional models for proteins. Using 3D-JIGSAW (version 2.0) to predict the 3D structure of AcSSII-503 peptide, a best alignment template, 1rzu_A, was found from sequence databases (PFAM+PDB+nr) (Fig 4.7). The score of accuracy of AcSSII to 1ruz_A is , greater than 0. That means that 95% of the alignments over the two sequences are accurate. The number (1e-77) shows the expected number of random sequences with the same similarity in the databases. ACCI is relative solvent accessibility for each residue of the template; from low to high *, 1, 2, 3, 4, 5, 6, 7, 8, 9,?. # means the end of calculation. SS_QP and SS_TP are the secondary structure (H = helix, E = strand, C = the rest) of query and template which are predicted by the method of Jones et al. (1999). SS is known secondary structure of the template. 115

136 TARGET AcSSII_503 IE1 IH1 IE2 IH2 IE3 Query PPPLAGQNVMNVILVAAECAPWCKTGGLGDVAGALPKALARRGHRVMVVIPRYAEYADCQFTGVRKRYKVAGQDNEVLYF 1rzu_A MNVLSVSSEIYPLIKTGGLADVVGALPIALEAHGVRTRTLIPGYPAVKAAVTDPVKCFEFTDLLGEKADLL ACCI **1***1* * ***1146* *253714*1 SS_QP CCCCCCCCCCEEEEEECCCCCHHCCCCHHHHHHHHHHHHHHCCCEEEEEECCCCCCCCHHCCCCEEEEEECCCEEEEEEE SS_TP CEEEEEECCCCHHHCCCHHHHHHHHHHHHHHHCCCEEEEEECCCCHHHHHHHCCCEEEEEEECCCCEEEEE SS CEEEEECCCCCCCCCCCHHHHHHHHHHHHHHCCCCEEEEEEECCHHHHHHCCCCEEEEEECCCCCCCEEEE IE4 IH3 IH4 IE5 IH5 Query HTYIDGVDFVFIEAPMLRNLGKDIY----GGNRLDILKRMILFCKVAVEVPWHVPCGGVCYGDGNLVFIANDWHTALLPV 1rzu_A EVQHERLDLLILDAPAYYERSGGPYLGQTGKDYPDNWKRFAALSLAAARIGA-----GVLPGWRPDMVHAHDWQAAMTPV ACCI ****1* *****1**14* ?1715**111111****1* SS_QP EEEECCCCEEEECCHHHCCCCCCCC----CCCCHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCCCEEEEECCHHHHHHHH SS_TP EEEECCCEEEEEECHHHHCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHH-----HCCCCCCCEEEEECCHHHHHHHH SS EEEECCEEEEEEECHHHHCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHC-----CCCCCCCCCEEEEEHHHHCCHHH IE6 H LINKER query YLKAYYRDNGLMKYTRSVLVIHNIAHQGRGPVDDFTYVDLPPHYLDYFKLYDPVGGEHFNIFAAGLKTADRILTVSHGYA 1rzu_A YMRYAETPE-----IPSLLTIHNIAFQGQFGANIFSKLALPAH---AFGMEGIEYYNDVSFLKGGLQTATALSTVSPSYA ACCI 11424# *** * *3368**21874****1***21*41***1*4211 SS_QP HHHHHHCCCCCCCCCCEEEEEECCCCCCCCCHHHHHHCCCCHHHHHHHCCCCCCCCCHHHHHHHHHHHHHHHHHCCHHHH SS_TP HHHHHCCCC-----CCEEEEEECCCCCCCCCHHHHHHHCCCHH---HHCCCCCCCCCCHHHHHHHHHHCCHHHHCCHHHH SS HHHHCCCCC-----CCEEEEECCCCCCCEECHHHHHHCCCCHH---HCCCCCCEECCEEEHHHHHHHHCCEEEECCHHHH H IE1 IH1 query WELKTQEGGWGLHGIINDNDWKLRGIVNGIDVTEWNPELDLYLQSDGYANYSLETLKPGKAQCKAALQNELGLPVREDVP 1rzu_A EEILTAEFGMGLEGVIGSRAHVLHGIVNGIDADVWNPATDHLI----HDNYSAANLK-NRALNKKAVAEHFRID-DDGSP ACCI 72*43672*55* *151** SS_QP HHHCCHHCCHHHHHHHHHCCCCEEEEECCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHCCCCCCCCC SS_TP HHHHHHHCCCCHHHHHHHCCCCEEEEECCCCCCCCCCCCCCCC----CCCCCCCCHH-HHHHHHHHHHHHCCCC-CCCCC SS HHCCCHHHHCCCHHHHHCCHHHEEECCCCCCCCCCCCCCCCCC----CCCCCCCCCC-CHHHHHHHHHHHHCCC-CCCCC IE2 IH2 IE3 IH3 IE4 IH4 IE5 query LIGFIGRLDGQKGVDIIAESIPWLSGQDVQLVMLGTGRPDLEQMLRECESRYPDKIRGWVGFSVKTAHRITAGVDILLMP 1rzu_A LFCVISRLTWQKGIDLMAEAVDEIVSLGGRLVVLGAGDVALEGALLAAASRHHGRVGVAIGYNEPLSHLMQAGCDAIIIP ACCI *1*1237*2652*131117**52*2744**** *1***11*111 SS_QP EEEEEECCCCCCCHHHHHHHHHHHHCCCCEEEEECCCCHHHHHHHHHHHHHCCCCEEEEEECCHHHHHHHHHHCCEEEEC RSS_TP EEEEEECCCCCCCHHHHHHHHHHHHHCCCEEEEECCCCHHHHHHHHHHHHHCCCCEEEEEECCHHHHHHHHHHCCEEEEC SS EEEEECCCCCCCCHHHHHCCHHHHHHCCCEEEEEECCCHHHHHHHHHHHHHCCCCEEEEECCCHHHHHHHHHHCCEEEEC IH5 IE6 IE7 IE8 IH6 IH7 query SRFEPCGLNQLYAMNYGTIPVVHAVGGLRDTVQPFDPYN---NTGYGWTFDRAEANRLIDALGNCLLTYRQYKESWEGLQ 1rzu_A SRFEPCGLTQLYALRYGCIPVVARTGGLADTVIDANHAALASKAATGVQFSPVTLDGLKQAIRRTVRYYHD-PKLWTQMQ ACCI *12*13*111****41*** *1** *36** SS_QP CCCCCHHHHHHHHHHCCCCEEEECCCCCCCEEEECCCCC---CCEEEEEECCCCHHHHHHHHHHHHHHHHCCCHHHHHHH SS_TP CCCCCHHHHHHHHHHCCCCEEECCCCCCCEEEECCCCCCCCCCCCCCEEECCCCHHHHHHHHHHHHHHHCC-HHHHHHHH SS CCCCCCCCHHHHHHHHCCEEEEECCHHHHHHCCCCCHHHHHCCCCCCEEECCCCHHHHHHHHHHHHHHHCC-HHHHHHHH IH8 query TRGMTQDLSWDHAAEIYEEV---LVAAKYQW 1rzu_A KLGMKSDVSWEKSAGLYAALYSQLIS----- ACCI *35* #----- SS_QP HHHHCCCCCHHHHHHHHHHH---HHHHHHCC SS_TP HHHHCCCCCHHHHHHHHHHHHHHHCC----- SS HHHHCCCCCHHHHHHHHHHHHHHHCC----- Fig 4.7 The 3D prediction of core region from Amaranthus cruentus starch synthase II (AcSSII). See text for description. 116

137 Since two proteins, AcSSII and 1ruz_A, have the better agreement in their structural superposition, it can be concluded that AcSSII is highly similar to the template, 1ruz_A. The description of 1ruz_A in protein data bank (PDB) indicates that 1ruz_A is a chain of bacterial GS, Ec (Buschiazzo et al., 2004). Therefore, the known 3D structure of 1ruz_A becomes a reference for the 3D prediction of AcSSII (Fig 4.8). Fig 4.8 The 3D structure of 1ruz_A (Bacterial GS) (from the protein data bank). The KTGGL sequence (Fig 4.7) at the beginning of helix IH1 has shown to be in van der Waals contact with heptulose-2-phosphate located at the enzyme binding site for the natural substrate, glucose-1-phosphate (Johnson et al., 1990). Fig 4.9 is a schematic representation of the secondary structure of AcSSII core region. The sheets of the N- and C-terminal domains of AcSSII core region contain five and six strands, respectively. The domains are joined by a partly helical linker. 117

138 Three helices are found at the end of the C-terminal domain and stretch up toward the N-terminal domain, so that the two ends of the polypeptide chain are relatively close together. This structure is very similar to the 3D structure of 1ruz_A shown in Fig 4.8. IH4 N-Terminal End IE3 IE7 IE6 IE1 IE2 IE4 C-Terminal End IH5 IH1 IH2 IE5 IH3 IIH5 H Linker IIH7 IIH8 IIE6 IIE5 IIH3 IIH2 H IIH1 IIH6 IIH7 IIE7 IIE4 IIE3 IIH2 IIE2 IIE1 IIH5 IIH6 IIE8 IIH4 Fig 4.9 Secondary structure of core region from Amaranthus cruentus starch synthase II (AcSSII). α-helices are shown as circles and are numbered as IH1, IH2, in the N-terminal domain, and IIH1, IIH2, in the C-terminal domain. β-strands are shown as arrows numbered similarly as IE1, AND IIE1, in the N-terminal and C-terminal domains, respectively. The linker helices are shown as H AcSsII is a low copy gene in grain amaranth genome and highly expressed in grain amaranth developing seeds Southern blotting is performed for identification of DNA fragments that are 118

139 complementary to a known DNA sequence (probe). In this experiment, the information of A. cruentus starch synthase II (AcSsII) gene in grain amaranth genome was obtained from Southern blotting via the hybridization between gene fragment (the probe) and genomic DNA fragments digested by various restriction enzymes. Grain amaranth chromosomal DNA was isolated from grain amaranth seedlings and completely digested with Xho I, Hind III, BamH I, EcoR I and EcoR V, respectively. The probe was an 1.8 kb fragment from AcSsII cdna, which contained one Xho I restriction enzyme site at the 3 end of the fragment, and one Hind III restriction enzyme site in the middle of the fragment. The cdna fragment (probe) did not contain BamH I, EcoR I and EcoR V restriction sites. The probe apparently could not hybridize with the genomic DNA fragments digested with BamH I as no band was found in Fig However, one DNA fragment (~10.5 kb) digested with Xho I hybridized to the probe. Since the Xho I restriction enzyme site is located at one end of the cdna fragment (probe), this DNA fragment from genomic DNA may contain the full length of the probe no matter how many introns exist in it. The AcSsII cdna appears to be a single copy gene in grain amaranth genomic DNA. Moreover, three fragments, 9 kb, 3.7 kb, and 1.5 kb, were found in the digestion with Hind III. Because one site of Hind III is in the middle of the probe, there must be another Hind III site in DNA introns. And the distance between the two Hind III sites must be 1.5 kb. Similarly, three fragments and four fragments were found in the digestion with EcoR I and EcoR V, respectively. Although no EcoR I or EcoR V restriction enzyme site exists in the sequence of the probe, some of their sites may be located in the introns of genomic DNA. 119

140 Therefore, the result of hybridization shown in Fig 4.10 indicates that AcSsII gene is present in grain amaranth genome and it is a single or low copy gene. Xho I BamH I EcoR V Marker Hind III EcoR I 10.5kb 9.0kb 3.7kb 3.3kb 2.0kb 1.5kb Fig 4.10 The result of Southern blotting for acssii gene (the probe was a 1.8 kb long fragment from AcSsII cdna). Gel above: chromosomal DNA digested with restriction enzymes. The marker is MassRuler DNA ladder (MBI Fermentas). 120

141 Northern blotting was used to analyze the expression of AcSsII gene in different plant tissues, such as roots, stems, leaves, flowers and seeds (Fig 4.11). R ST L F SE rrna Fig 4.11 The result of Northern blotting, showing different tissues hybridized with a cdna fragment (1.8 kb) from putative AcSs II gene. R, ST, L, F, and SE represent the total RNA from amaranth roots, stems, leaves, flowers, and seeds, respectively. The probe used for the hybridization was the same probe used in Southern blotting. However, less stringent conditions were applied to Northern hybridization and washing because RNAs from roots, stems, and leaves showed a low affinity to the probe. The result of Northern blotting shows that the band is not detected in the hybridization with grain amaranth roots. The explanation might be that the roots used in the experiment were not mature. Thus the starch content in the seedling s roots is too low to be detected by Northern blotting. In contrast, the significant expression of AcSsII gene is detected in grain amaranth seeds, greater than that in the flowers, leaves, and stems. In addition, the expression of AcSsII gene in the flowers is greater than in the leaves and stems. This could be explained if some developing seeds were hidden in the flower and thus included in the flower sample 121

142 in the analysis. In summary, the result of Northern blotting indicates that starch synthase is more active in amaranth grain than in other organs, consistent with the importance of seed as a storage organ AcSsII cdna encodes an active starch synthase The result of amino acid alignment indicates that the predicted peptide encoded by AcSsII cdna is a starch synthase II. The peptide is 809 aa long and contains a 54 aa chloroplast transit peptide in the N end. Moreover, the fragment from 310 to 809 is a highly conserved region compared with other SSIIs. To analyze the activity of this peptide, target cdnas amplified from AcSsII cdna are cloned in pet28(a) plasmid under the control of strong bacteriophage T7 transcription and translation signals; the expression is induced by providing a source of T7 RNA polymerase in RH98, a mutant strain of E. coli that lacks the activity of GS. In Fig 4.12D, wild type DH5α colonies are stained golden yellow by iodine since this strain is able to synthesize GS; whereas the RH98/pET28(a) cells stained pale (white) due to a lack of GS (Fig 4.12C). Expression of pnb1 and pnb2 cdnas in the mutant strain changes the iodine staining from white to golden yellow (Fig 4.12A & B). Thus the result shows that both the full-length cdna and the truncated cdna are able to encode a functional starch synthase. 122

143 A C A C B D B D AcSsII cdna pnb1 pnb2 RH98/pNb1 A C RH98/pET28(a) B RH98/pNb2 D DH5á Fig 4.12 Complementation analysis of AcSSII activity in E. coli. The color development was upon iodine staining. A, GS-deficient strain RH98 transformed with pnb1; B, GS-deficient strain RH98 transformed with pnb2; C, strain RH98 transformed with pet28(a); D, GS-producing strain DH5α. Plasmid pnb1 is from AcSsII cdna in which the ctp (gray bar) is cleaved for avoiding its expression in E. coli. In plasmid pnb2, the low similarity region (green bar) is deleted. The red bar is the most conserved region in all SSIIs Phylogenetic analysis of plant starch synthase IIs The software package PAUP* 4.0 b10 (Swofford, 2003) is commonly used for inferring phylogenetic relationships based on molecular data. It can be used to analyze nucleotide or amino acid sequences, or other data types using maximum likelihood, parsimony, and distance methods. In this study, fourteen amino acid sequences from diverse sources listed in Table 4.1 were analyzed using the maximum parsimony method. The sequence analyzed consists of about 505 amino acids which are located in a conserved 123

144 region. Gaps in the sequences were treated as the 21 st amino acid to include the information on length divergence among sequences. Heuristic search was conducted with 100 replicates of random sequence addition, using the tree-bisection-reconnection (TBR) branch-swapping algorithm with MulTrees in effect. A single most parsimonious tree was obtained (Fig 4.13) with a length of 1136 steps, a consistency index (CI) of 0.841, and a retention index (RI) of The GS from E. coli was used as outgroup in the analysis. The SSIIs from higher plants were separated into two groups (Fig. 4.13). One group contained SSIIs from monocotyledons such as maize, rice, barley and wheat, whereas SSIIs from dicotyledons were placed in another group. In the monocot group, rice and maize SSIIb could be separated from their SSIIa. Wheat and barley SSIIs were found to be more closely related to rice and maize SSIIa than to their SSIIb. These relationships are similar to those shown in the dendogram of starch synthases in plants and GS in E. coli (Li et al., 2003). The study by Li et al. (2003) included more plant starch synthases, and the dendogram was generated using the GrowTree program from the Genetic Computer Group (Devereaux et al., 1984) and TreeView (Roderic D. M. Page) which are different from the methods used in this thesis. This may contribute to some topological differences between the two phylogenetic trees. The amino acid sequence of grain amaranth SSII was more similar to those of dicots than monocots, so it was placed in the dicot group. Additionally, the unrooted cladogram shows an early divergence of grain amaranth SSII from other dicots included in this study. 124

145 Fig 4.13 Cladograms of starch biosynthesizing enzymes in higher plants and glycogen synthase in E. coli. The bottom left dendogram is from Li et al. (2003) in which At* is an abbreviation of Arabidopsis thaliana. The top right cladogram is generated using PAUP* 4.0 b10 (2003). The amino acid sequences of starch synthases were downloaded from GenBank except for SSII from grain amaranth. Sweetpotato SSII Kidneybean SSIIb Potato SSII Amaranth SSII Pea SSII Wheat SSII Barley SSII Kidneybean SSIIa Arabidopsis SSII Maize SSIIa Rice SSIIa Rice SSIIb Maize SSIIb E. coli GS 125

146 4.4 Discussion Starch synthase II is a very important isoform in the catalyzing process of starch biosynthesis. First of all, the major function of SSII is to catalyze the elongation of the linear glucose chains in amylopectin synthesis. SSII is found not only in the plastid stroma but also bound to starch granules. However, SSII in granule-bound form cannot compensate for the effects of loss of GBSSI on amylose synthesis (Edwards et al., 1995). In tubers, SSII contributes mainly to total starch synthase activity because the mutant analysis by antisense suppression of SSI and SSIII has shown that these mutations in SSI and SSIII make no significant alteration in starch biosynthesis and starch accumulation in starch granules (Edwards et al., 1999). On the contrary, the mutant that lacks SSII activity results in a loss of intermediate-length chains of amylopectin, thus it indicates that this isoform plays a specific qualitative role in determining the branching length distribution of amylopectin in potato (Craig et al. 1998; Lloyd et al., 1999). Although the catalysis properties of GBSSI and SSII in vivo cannot be compensated reciprocally, both GBSSI and SSII can extend a range of glucan substrates in vitro (Denyer et al., 1996). In studies of starch synthase activity on granules in different mutants of pea and analyses of plant starch synthase expressed in E. coli, it seems that GBSSI preferentially extends MOS relative to amylopectin when supplied in a mixture and may extend them in a processive manner by adding further glucose units to the same glucan chain (Denyer et al., 1999). In contrast, SSII preferentially adds single glucose units to many glucan chains (Morell et al., 2003). It is suggested that in soluble form, GBSSI is 126

147 relatively heat-labile compared with SSII. In conclusion, GBSSI might be responsible for the elongation of linear glucan chains (amylose) by adding further glucose units to the same glucan chain whereas SSII performs its elongation ability on amylopectin by adding single glucose units to many glucan chains. These differences in activity could be determined by the specific structures of the GBSSI and SSII isoforms. The distance between GBSSIs and SSIIs shown in Fig 4.13 indicates that their separation occurred earlier than the evolution of dicotyledons and monocotyledons. The main difference between GBSSIs and SSIIs is SSII has an N-terminal extension of 23 kda. This extension domain is predicted to be hydrophilic and very flexible so that it is called as flexible arm (Edwards et al., 1995). Another difference is GBSSI has a small C-terminal extension of about 20 amino acids that is unique in comparison with other starch synthases and bacterial GSs. This C-terminal region, called GBSSI tail, is conserved in GBSSIs from a wide range of species, and is relatively hydrophilic and caries a net negative charge (Edwards et al., 1999). Besides SSII flexible arm and GBSSI tail, a core region of ~60 kda was found in all known starch synthases and bacterial GSs. It includes an N-terminal motif (KTGGL) which is thought to be required for binding of ADP/ADPglucose (Furukawa et al., 1990) and a second very similar motif (the KTGGL look-alike motif) close to the C-terminus (Harn et al., 1998). The C-terminal KTGGL look-alike was proposed to be also involved in binding ADP/ADPglucose. To define functional regions of the isoforms, Edwards et al. (1999) 127

148 constructed truncated and chimeric versions of the potato GBSSI and SSII isoforms through expression in an E. coli strain that lacks GS activity. First, it was found that the N-terminal domain, or arm, of SSII had little function in determining catalytic properties, thermosensitivity or product specificity. Therefore, it was assumed that the arm might serve a more physical role in SSIIs such as determining the partitioning of the enzyme between soluble and granule-bound phases or the affinity to the substrate, amylose and amylopectin. Edwards et al. (1999) found that most of the specific properties of GBSSI were determined by GBSSI tail that included the KTGGL look-alike motif. A large proportion of the chimeras constructed from GBSSI core region and C-terminal fragments with different sizes from SSII (i.e. GBSSI tail was replaced by fragments from SSII) were completely inactive. Therefore, GBSSI tail should be a very important structure for the activity of the enzyme. Fig 4.4 shows that AcSSII contains a flexible arm, KTGGL and KTGGL look-alike motifs which are similar to other starch synthases (Preiss and Sivak, 1996). Moreover, it is found that the catalytic domain is not included within the arm of AcSSII (Fig 4.12). Since the core region is much conserved between GBSSIs and SSIIs, the flexible arm is probably the specific region which determines the specificity of different starch synthase isoforms. The main function of SSII known is to extent the linear glucose chains of amylopectin. However, it is still unknown why SSIIs cannot synthesize amylose and why the branching chain lengths of amylopectin in some species such as grain amaranth are significantly shorter than those in other species such as wheat and potato. The amylopectin structure and branching chain length distribution are 128

149 determined not only by SSIIs which extent the chain length, but also by SBEs which transfer the linear chains to join and built a cluster. Comparing SS mutants with SBE mutants in pea and maize, it seems that SSs may be more important than SBEs in amylopectin elongation (Craig et al., 1998; Gao et al., 1998). Moreover, de-branching enzymes (DBEs), which hydrolysis the branches amylopectin, may affect the length of branching chains as well (Kubo et al., 1999). To determine the functions of these enzymes or isoforms, it is necessary to look for more mutants in various species. It is known that the amylose content in A. cruentus is lower than that in normal starch species and the branching chain length is shorter than the normal length. Therefore, mutants of high amylose content and long branching chain length will be very useful for future research. To obtain the mutants, field observation is a fundamental way, and some techniques also can be used to induce mutations. Site-directed mutagenesis and antisense suppression techniques have been successfully used in Arabidopsis and potato. Induced mutations can be used to make mutation libraries for screening target mutations. 129

150 Chapter V ISOLATION, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF A PUTATIVE GENE ENCODING STARCH BRANCHING ENZYME I FROM GRAIN AMARANTH 5.1 Introduction Starch branching enzyme catalyzes the cleavage of α(1,4)-linked chains and joins them with other chains via α(1,6)-linkages (Preiss, 1988). It is thought that the biosynthesis of amylopectin is catalyzed mainly by two synthases, starch synthase and starch branching enzyme. Therefore, to isolate sbe gene from grain amaranth is a new task when the isolation, characterization and expression analysis of AcSsII gene have been done. Moreover, the study of the two genes on their structure, function and phylogenetic placement may provide useful information for the study of starch biosynthesis in grain amaranth. Since AcSsII gene was successfully isolated from the cdna library of grain amaranth developing seeds, the same method was used in the isolation of sbe gene. The only deference in cdna library screening for the sbe gene was the probe used: a fragment amplified from mrna by reverse transcriptase-polymerase chain reaction (RT-PCR). The primers for RT-PCR were designed based on the most conserved regions found in all sbe genes from diverse sources. Furthermore, Southern and Northern blotting and immunoassay were also used for the characterization and expression analysis of the sbe gene isolated from grain amaranth. 130

151 5.2 Methods RT-PCR for the isolation of a cdna encoding partial sequence of starch branching enzyme I from A. cruentus Four pairs of primers were designed upon the conserved sequences found in sbe genes from various higher plants (Table 5.1). Table 5.1 PCR primers designed upon conserved sequences found in sbe genes in higher plants Primer Code Sequence The length of expected (5 3 ) PCR product (bp) P1 F: GGCNACNGCNGARGAYGG 1000 R: GGCCGAGGATGCTTAAACACA P2 F: CCGCGCATAAAGGCAAACA 400 R: CCCATCAAATCGGAAGCCAT P3 F: TGGGATAGCCGCCTGTTCAA 850 R: TACATTAAATCGGAAGCCAT P4 F: CCGCGCATAAAGGCAAACA R: TACATTAAATCGGAAGCCAT 1150 Note: R=A/G, Y=C/T, N=A/C/G/T Total RNA was extracted from developing seeds of grain amaranth using the hot phenol method from Dr. Chibbar s lab. The first strand cdna was amplified by ThermoScript reverse transcriptase (Invitrogen) using the following reaction: 500 ng poly-t primer was mixed with 500 ng total RNA. The mixture was incubated at 70ºC for 10 min, and quickly chilled on ice. Then 1 x First Strand Buffer, 10 mm DTT, 2 mm dntp and 15 U of ThermoScript reverse transcriptase were added to the mixture. The mixture was incubated at 42ºC for 2 min and again at 50ºC for 50 min. The first strand cdna product was stored at 4ºC before use, normally within one month. 131

152 PCR reaction was performed in a total volume of 25 µl containing 1 x PCR buffer (plus 2 mm Mg 2+ ), 1 mm dntp, 2 mm forward primer, 2 mm reverse primer, 2 µl of first strand cdna product, and 1 U of Taq DNA polymerase. The PCR reaction was conducted at 94ºC for 2 min followed by 35 cycles of 94ºC for 30 sec; 56ºC for 1 min; and 72ºC for 1 min. The PCR product was run in 1% agarose gel and the target band was isolated from the gel using a gel purification kit (Quagen Com.). The cdna fragment purified from the gel was ligated into T-vector (see APPENDIX IV: Make T-vector) and was sequenced at Robert Research Institute (London, Canada) using ABI GenAmp 9700 thermocycler. Sequence was analyzed with a sequence analysis package, DNASTAR cdna library screening The procedures of cdna library screening were similar to those described in Chapter IV (refer to section 4.2.1). The probe used for screening was the cdna fragment encoding partial SBE from grain amaranth amplified by RT-PCR In vivo excision of the pbluescript phagemid from the Uni-ZAP XR Vector The procedures of in vivo excision were similar to those described in Chapter IV (section 4.2.2). 132

153 5.2.4 Restriction enzyme patterns digested with different restriction enzymes Nine restriction enzymes were chosen for digestion: Xho I, Kpn I, Hind III, EcoR V, Pst I, Sma I, BamH I, Xba I, and EcoR I Sub-cloning for sequencing A cdna insert was digested with Xho I and EcoR I from a positive clone isolated from grain amaranth cdna library and ligated to a vector to obtain a new constructed plasmid. The reconstructed plasmid was checked by several restriction enzymes digestions (Xho I, Xho I/EcoR I, and some other enzymes, e.g. Hind III) to confirm the cdna fragment was ligated into the vector successfully Sequencing strategies The sequencing strategies and sequence analysis were similar to those described in Chapter IV (section 4.2.5). Additional primers designed for the sequencing were: 5 CCTGGCTATGGCAATTCC 3 ; and 5 CACCGGATGGAAG TTGAA Sequence alignment 4.2.6). The alignment methods were similar to those used in Chapter IV (section The structure analysis of the protein (AcSBEI) The primary structure of AcSBEI was analyzed using ProtParam (see section 133

154 4.2.7) compared to Maize SBEI (AAO20100) and kidney bean SBE1 (BAA82349). The analyses of secondary structure and 3D structure of AcSBEI were similar to those given in Chapter IV (see section 4.2.7). The AcSBEI sequence was splitted to a smaller chunk which was a 510 aa peptide for 3D structure prediction Southern and Northern blotting The procedures of Southern and Northern blotting were similar to those described in Chapter IV (see section and 4.2.9) except for the probe. The probe used in this experiment was a 1.7 kb fragment cut from AcSbeI cdna with Xho I and EcoR I Expression of AcSbeI in E. coli mutant KV Construction of expression plasmids Two PCR primers, F1 and R1, were designed for cdna fragment amplification from AcSbeI: F1 (5 -CCTGAGCTCCACGCCCTTGTAT-3 ); and R1 (5 -GCCTGCAGAGGTTCTTGATTCACAT-3 ). Primer F1 contained Nco I restriction site. PCR product from F1/R1 was cloned into T-vector. The reconstructed plasmid was digested with Nco I and Sal I to yield a fragment of 2796 bp. After gel purification, the cdna fragment was cloned into pet28a vector at the Nco I and Sal I sites to form a new plasmid, pns1. The reconstructed plasmid was sequenced to verify that no mutation had occurred during these modification steps. 134

155 Complementation of E. coli mutant KV832 The expression plasmid, pns1, was transformed into competent cells made from KV832, a branching enzyme-deficient E. coli strain. For the complementation test of branching enzyme deficiency in KV832, KV832/pNs1, KV832/pET28(a), and wild type E. coli strain DH5α were grown on an enriched medium [containing 0.85% (w/v) KH 2 PO 4 ; 1.1% (w/v) K 2 HPO 4 ; 0.6% (w/v) yeast extract; 1% (w/v) glucose; and 1.5% (w/v) agar; ph 7.0] under inductive conditions (0.1 M IPTG). Following growth at 37 C for 20 hr, the cells were stained with an iodine solution (0.03 M I 2, 0.04 M KI) Phylogenetic analysis Phylogenetic analysis of A. cruentus starch branching enzyme I (AcSBEI) and other starch branching enzymes from various plants was conducted using PAUP* 4.0 version b10 (Swofford, 2003). The amino acid sequences of all starch branching enzymes except for AcSBEI were downloaded from GenBank (Table 5.2). 135

156 Table 5.2 The GenBank accession number of starch branching enzymes used in the phylogenetic analysis Source Taxon Starch branching Acce. No. enzyme Bacteria E. coli GBE BAB69770 Human Homo sapiens GBE AAH12098 Red alga Gracilaria gracilis SBE1 AAB97471 Apple Malus x domostica SBEI AAZ20130 Mungbean Vigna radiate SBEI AAT76445 SBEII AAT76444 Pea Pisum sativum SBEI CAA56319 SBEII CAA56320 Arabidopsis Arabidopsis thaliana SBE2-1 NP_ SBE2-2 NP_ Kidney bean Phaseolus vulgaris SBE1 BAA82349 SBE2 BAA82348 Wheat Triticum aestivum SBE1 CAB40980 SBE2 AAG27623 SBEIIa AAK26822 Rice Oryza sativa SBE1 AAP68993 SBE3 BAA03738 SBE4 BAA82828 Cassava Manihot esculenta SBE CAA54308 Sweet potato Ipomoea batatas SBEII BAB64912 Potato Solanum tuberosum SBEI CAA70038 SBEII CAB40746 Sorghum Sorghum bicolor SBE AAD50279 SBEIIb AAP72267 Maize Zea mays SBEI AAO20100 SBEIIa AAB67316 SBEIIb AAC33764 Barley Hordeum vulgare SBEIIa AAC69753 SBEIIb AAC69754 Grain amaranth Amaranthus cruentus SBEI - 136

157 5.3 Results The probe amplified by RT-PCR for cdna library screening Four pairs of primers designed from conserved sequences of sbe genes were used for RT-PCR. A fragment (~400 bp) was amplified in the reaction using P2 (Fig 5.1A). This cdna was isolated from the gel and cloned into T-vector. After transformation, ten clones were picked from LB-Amp plate. Eight clones out of ten shown in Fig 5.1B had two different sizes, ~430 bp and ~412 bp. M P1 P2 P3 P4 M bp 430 bp 412 bp A B Fig5.1 The amplification of partial sbe cdna from grain amaranth by RT-PCR. The marker is MassRuler DNA ladder (MBI Fermentas). A. RT-PCR using primers designed from conserved sequences from sbe genes. B. The clones from the cdna fragment (~400 bp) amplified with P2 after transformation. 137

158 The sequences of eight clones were divided into two different groups. One group (430 bp) contained cdna fragments which had no similarity to sbe genes (data not shown) whereas the other group (412 bp) contained cdnas which had high homology to sbe genes (Fig 5.2). Thus the clone (sbe-412) was used as the probe for cdna library screening. Query 1 CCGCGCATAAAGGCAAACAATTATAATACAGTACAGCTGATGGCTGTGATGGAGCATTCC 60 Sbjct 3984 CCGCGCATAAAGGCAAACAACTACAACACAGTTCAGCTGATGGCAATCATGGAACATTCA 4043 Query 61 TATTATGCTTCTTTCGGGTATCACGTGACAAACTTCTTTGCAGTGAGTAGTAGATCTGGA 120 Sbjct 4044 TATTATGCTTCTTTTGGATACCATGTGACGAATTTCTTCGCAGTTAGCAGCAGATCAGGA 4103 Query 121 AACCCCGAGGACCTTAAGTATCTGATTGACAAGGCTCACAGCTTAGGACTACGAGTACTA 180 Sbjct 4104 ACACCAGAGGACCTCAAATATCTTGTTGACAAGGCACATAGCTTAGGGTTGCGTGTTCTG 4163 Query 181 ATGGATGTCGTTCATAGCCATGCCAGCAACAATGTTACCGATGGCTTGAATGGTTTTGAC 240 Sbjct 4164 ATGGATGTTGTCCATAGCCATGCGAGCAGTAATATGACAGATGGTCTAAATGGCTATGAT 4223 Query 241 GTTGGCCAAGGTGCACAGGAATCCTATTTCCATACCGGAGATCGTGGCTATCATAAGCTA 300 Sbjct 4224 GTTGGACAAAACACACAGGAGTCCTATTTCCATACAGGAGAAAGGGGTTATCATAAACTG 4283 Query 301 TGGGATAGTAGGTTGTTCAATTATGCAAACTGGGAGGTCTTACGGTATCTTCTTTCTAAT 360 Sbjct 4284 TGGGATAGTCGCCTGTTCAACTATGCCAATTGGGAGGTCTTACGGTATCTTCTTTCTAAT 4343 Query 361 CTGAGATATTGGATGGACGAATTCATGTTTGATGGCTTCCGATTTGATGGG 412 Sbjct 4344 CTGAGATATTGGATGGACGAATTCATGTTTGACGGCTTCCGATTTGATGGG 4393 Fig5.2 The alignment between Query (sbe-412 cdna from grain amaranth amplified by RT-PCT) and Sbjct [Triticum aestivum sbe1 gene (AJ237898)]. The percentage of identities is 83% (341/412) with no gap. The underlined sequences are 5 and 3 end primers Fifteen positive clones isolated from cdna library For the isolation of starch branching enzyme gene from grain amaranth, the cdna library constructed from grain amaranth developing seeds was used for 138

159 screening. The probe was a partial sbe (Sbe-412) which was amplified from RT-PCR. After primary cdna library screening, 86 candidate clones were cored and diluted ten times for secondary screening (Fig 5.3I). From secondary screening, fifteen out of the 86 clones were confirmed as positive clones (Fig 5.3II), and the fifteen clones were cored as single clone respectively. These fifteen clones were used for tertiary screening and diluted two times. The tertiary screening showed all clones were positive (Fig 5.3III). In conclusion, fifteen candidate clones hybridized to the sbe-412 probe were isolated from grain amaranth developing seed cdna library. 139

160 III Fig 5.3 The result of cdna library screening for A. cruentus starch branching enzyme I (acsbei) gene isolation. cdna library was constructed from grain amaranth developing seeds. The probe for screening was a 0.4 kb fragment (sbe-412) amplified from RT-PCR. I. First screening; II. Secondary screening; and III. Tertiary screening. 140

161 5.3.3 Restriction enzyme patterns of fifteen candidate clones isolated from cdna library Nine restriction enzymes were used to digest plasmid DNAs isolated from the fifteen candidate clones. Fig 5.4 shows that two different lengths of plasmid DNAs exist in the fifteen candidate clones isolated from grain amaranth developing seed cdna library. bp A M B Fig 5.4 The restriction enzyme patterns of two candidate cdna clones isolated from grain amaranth developing seed cdna library using sbe-412 as the probe. 1, Xho I; 2, Kpn I; 3, Hind III; 4, EcoR V; 5, Pst I; 6, Sma I; 7, BamH I; 8, Xba I; 9, EcoR I The maker is MassRuler DNA ladder (MBI Fermentas). Plasmid A and plasmid B in Fig 5.4 are two different cdna clones isolated from grain amaranth developing seed cdna library. In the digestion with Xho I, two patterns were found in clone A whereas three patterns were found in clone B. Similarly, in each digestion, the patterns from clone A and B were different in their fragment numbers and/or sizes, indicating that clone A and B contained different cdna inserts. 141

162 In the digestion with Xba I, two fragments, 4.5 kb and 3.5 kb long, were found from clone A whilst two fragments, 3.5 kb and 2.5 kb, were found from clone B. Therefore, the length of plasmid DNA from clone A was approximately 8.0kb, whereas the length of plasmid DNA from clone B was approximately 6.0kb. Since the length of Uni-ZAP XR vector was approximately 3.0 kb, it could be deduced that the cdna inserts in clone A and B were approximately 5.0 kb and 3.0 kb, respectively. The restriction enzyme digestion patterns showed that very different restriction enzyme sites exist in the cdna insert in clone A compared with those restriction enzyme sites in the cdna insert in clone B. Therefore, it was necessary to sequence both of clone A and B to discover of their difference A positive clone isolated from cdna library contains a full-length cdna fragment encoding starch branching enzyme I The two cdna clones, A and B, contained different lengths of plasmids, 8.0 kb and 6.0 kb, respectively. The result of sequencing showed that clone A (8.0 kb) contained a full-length of mitogen-activated protein kinase (MAPK) cascade (2.2 kb) and a truncated cdna of starch branching enzyme I gene (2.5 kb) (data not shown). This cdna clone might be a wrong integration of MAPK and SBEI mrna when the cdna library was constructed. Clone B (6.0kb) contained approximately 3.0 kb cdna insert. The result of sequencing from both ends of cdna insert showed that it contained a 2886 bp of open reading frame (ORF) with start codon (ATG) and stop codon (TGA) (see 142

163 APPENDIX II). The 2886 bp ORF encoded a protein consisting 961 amino acids with 59 amino acids of N-terminal chloroplast transit peptide (ctp). Nucleotide-tonucleotide sequence alignment using BLASTN showed the ORF of cdna insert had high similarity to other sbe genes, especially to sbei genes. For example, compared to the mrna for S. tuberosum (potato) starch 1,4-α-glucan branching enzyme (Y08786), which was 2493 bp long, the percentage of identities was 79% in 1594 bp overlap (unknown :StSBE ). Similarly, when FASTA was used, it was found that the unknown sequence showed a high similarity to S. tuberosum mrna encoding starch branching enzyme (X69805) and the identity was 76.1% (76.507% ungapped) in 2251 bp overlap (unknown :StSBE ). Using BLASTP (protein-to-protein sequence) for alignment, some conserved domains such as the isoamylase N-terminal domain, the α-amylase domain, and the conserved domain of 1,4-α-glucan branching enzyme were found (Fig 5.5). 143

164 UNKNOWN PROTEIN (Amaranthus cruentus starch branching enzyme I?) COG0296, GlgB, 1,4-alpha-glucan branching enzyme [Carbohydrate transport and metabolism]. CD-Length = 628 residues, 96.5% aligned Score = 336 bits (864), Expect = 6e-93 UNKNOWN 133 YLKFGFNREEGG---IVYREWAPAAQEAQLIGDFNGWDGT-MHSMEKNQFGVWSIRIPDS 188 GlgB 23 YEKLGAHPIENGVSGVRFRVWAPNARRVSLVGDFNDWDGRRMPMRDRKESGIWELFVPG- 81 UNKNOWN 189 DGKPAIPHNSRVKFRFKHGNGVWVDRIPAWIKYATVDPTRFAAPYDGVYWEPPPEE---S 245 GlgB APPGTRYKYELIDPSG-QLRLKADPYARRQEVGPHTAS----QVVDLPDYEWQDE 131 UNKNOWN 246 YQFKYPRPPKPKAPRIYEAHVGMSSSEPRVNSYREFADEVLPRIKANNYNTVQLMAVMEH 305 GlgB 132 RWDRAWRGRFWEPIVIYELHVG-SFTPDRFLGYFELAIELLPYLKELGITHIELMPVAEH 190 UNKNOWN 306 SYYASFGYHVTNFFAVSSRSGNPEDLKYLIDKAHSLGLRVLMDVVHSHASNNVTDGLNGF 365 GlgB 191 PGDRSWGYQGTGYYAPTSRYGTPEDFKALVDAAHQAGIGVILDWVPNHFPPD-GNYLARF 249 UNKNOWN 366 DVGQGAQESYFHTGDRGYHKLWDSRLFNYANWEVLRFLLSNVRWWLEEYRFDGFRFDGVT 425 GlgB 250 D---GTFLYEHEDPRRGEHTDWGTAIFNYGRNEVRNFLLANALYWLEEYHIDGLRVDAVA 306 UNKNOWN 426 SMLYHHHGINMGFSGNYNEYFSEATDVDAVVYLMLANRLIHNIFPDVTVVAEDVSGMPGL 485 GlgB 307 SMLYLDYSRAEG-EWVPNEY-GGRENLEAAEFLRNLNSLIHEEEPGAMTIAEESTDDPHV 364 UNKNOWN 486 CRPVSEGGVGFDYRLAMA-IPDKWIDYLKNKKDEEWSMNEVMLSLTNRRYTEKCIAYAES 544 GlgB 365 TLPVAIGGLGFGYKWNMGWMHDTLFYFGKDPVYRKYHHGE--LTFGLLYAFSENVVLPLS 422 UNKNOWN 545 HDQAIVGDKTVAFLLMDQEMYTGMSCLTEASPVVERGIALHKMIHLITMAMGGEGYLNFM 604 GlgB 423 HDEVVHGKRSLGERMPGDAWQKFANLRALAAYMW-----LHPGKPLLF--MGEEFGQGRE 475 UNKNOWN 605 GNEFGHPEWIDFPREGNGWSYKMCRRQWNLPDTDHLRYKFLNLFNGAMNLLDEKFSFLAS 664 GlgB 476 WNFFSSLDWLLLDQAVREGRHKEFRRLVRDLNALYRIPDPLH----EQDFQPEGFEWI-D 530 UNKNOWN 665 SKQIVSSANEVDKVIVFERGDLVFVFNFHPVNAYEGYKIGCDLPGRYRVALDGDALMFGG 724 GlgB 531 ADDAENSVLAFYRRLLALRHEHLVVVNNFTPVPRVDYRVGVPVAGRWREVLNTDLAEYGG 590 UNKNOWN 725 KGRVGHDVDHFTNPEGIPGVPETNFNNRPNSFKVLSPPRTCVAYYR 770 GlgB 591 SGAGNLGLPVSGEDIL WHGREWSLSLTLPPLAALVLKL 628 Fig 5.5 Protein-to-protein alignment of unknown protein (putative A. cruentus starch branching enzyme I) isolated from cdna library. The black bar is the unknown protein (961 amino acids). Blue bars in the unknown protein sequence show the masked-out regions with low complexity. The dark blue bar represents the isoamylase N-terminal domain. Red bar shows the α-amylase domain which is a (β/α) 8 -barrel domain. Grey bar shows the conserved domain of 1,4-α-glucan branching enzyme. In the text, the letters in red, blue, and black colors represent identical amino acids, positive amino acids, and different amino acids, respectively. 144

165 The amino acid sequence alignment showed that the unknown protein had high similarity to starch branching enzyme I found in apple (AAZ20130), potato (CAA70038), and kidney bean (BAA82349), etc. In comparison with kidney bean starch branching enzyme 1 (847 aa), 746 aa of the unknown protein showed 80.56% identity (80.997% ungapped). The highly conserved region of the unknown protein sequence was from 70 aa to 800 aa, containing four conserved domains in all starch branching enzymes: HSHAS, GFRFDGVT, AEDVS, and AESHDQ (Fig 5.6). Fig 5.7 shows that the first 70 amino acids close to N- terminus of the unknown protein had no similarity to other starch branching enzymes because it might be a N-terminal chloroplast transit peptide (ctp). The region from 70 to 815 aa showed high similarity to other SBEs, whereas the region from 816 to 961 aa showed very low similarity to other SBEs. In conclusion, at both nucleotide sequence level and protein sequence level, the unknown cdna insert from clone B showed high similarity to other starch branching enzyme I genes from different plants or organs, and so did the protein encoded by the cdna. Therefore, the unknown protein was named as A. cruentus starch branching enzyme I (AcSBEI) and the cdna encoding this protein was named as A. cruentus starch branching enzyme I (AcSbeI) gene. 145

166 Starch branching enzyme 1 [Phaseolus vulgaris](847 aa) % identity (80.997% ungapped) in 746 aa overlap (70-815:70-811) UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN PvSBE1 UNKNOWN MSISASHTVAIPLGSVLLSAKRGSARDGSFLTQPLALNSRSKT-LQWRNSSH MFNCLCLNPFISVSSTIACTIHTV-----RSRQHLAPQKSVDLAVGYRNPLGYGFGSGLR **.* *:*.: :* *. :.. : **:. *. :.. : SKFHALVCQGMKHCSSISAVLTESSKTMGVTEGDTENLGIFDVDPSLEPYKDHFRYRMKR RSLHEMVSSRFKGV----AVMTDDKSTISSTEEYLENIGIFSIDPSLKPYKDHFKYRLKR.:* :*.. :* **:*:...*:. ** **:***.:****:******:**:** YVDQTQLIEKYEGGLEEFAQGYLKFGFNREEGGIVYREWAPAAQEAQLIGDFNGWDGTMH YVEQKKLIEEYEGGLEEFAKGYLKFGFNREEGGIVYREWAPAAQEAQIIGDFNGWDGSNH **:*.:***:*********:***************************:*********: * SMEKNQFGVWSIRIPDSDGKPAIPHNSRVKFRFKHGNGVWVDRIPAWIKYATVDPTRFAA QMEKDQFGVWSIKIPDVDGNPAIPHSSRVKFRFRHGDGVWVDRIPAWIKYATVDPTRFAA.***:*******:*** **:*****.*******:**:*********************** pro-rich motif PYDGVYWEPPPEESYQFKYPRPPKPKAPRIYEAHVGMSSSEPRVNSYREFADEVLPRIKA PYDGVYWDPPLSERYQFKYPRPPKPKAPRIYEAHVGMSSSEPRINSYREFADEILPRIRA *******:**.* *****************************:*********:****:* NNYNTVQLMAVMEHSYYASFGYHVTNFFAVSSRSGNPEDLKYLIDKAHSLGLRVLMDVVH NNYNTVQLMAGMEHSYYASFGYHVTNFYAVSSRSGTPEDLKYLIDKAHSLGLQVLMDVIH ********** ****************:*******.****************:*****:* SHASNNVTDGLNGFDVGQGAQESYFHTGDRGYHKLWDSRLFNYANWEVLRFLLSNVRWWL SHASNNITDGLNGFDVGQTSQDSYFHTGDRGYHKLWDSRLFNYANWEVLRFLLSNLRWWL ******:*********** :*:*********************************:**** EEYRFDGFRFDGVTSMLYHHHGINMGFSGNYNEYFSEATDVDAVVYLMLANRLIHNIFPD EEFEFDGFRFDGITSMLYHHHGINIAFTGDYNEYFSEATDVDAVVYLMLANCLIHSILPD **:.********:***********:.*:*:********************* ***.*:** VTVVAEDVSGMPGLCRPVSEGGVGFDYRLAMAIPDKWIDYLKNKKDEEWSMNEVMLSLTN ATVIAEDVSGMPGIGHQVSGGGIGFDYRLAMAIPDKWIDYLKNKNEYSWSMKEISWSLTN.**:*********: : ** **:*********************::.***:*: **** RRYTEKCIAYAESHDQAIVGDKTVAFLLMDQEMYTGMSCLTEASPVVERGIALHKMIHLI RRYTEKCVSYAESHDQAIVGDKTVAFLLMDEEMYSGMSCLVDASPIVERGIALQKMIHFI *******::*********************:***:*****.:***:*******:****:* TMAMGGEGYLNFMGNEFGHPEWIDFPREGNGWSYKMCRRQWNLPDTDHLRYKFLNLFNGA TMALGGEGYLNFMGNEFGHPEWIDFPREGNGWSYEKCRRQWNLVDTDHLRYKFMNAFDRA ***:******************************: ******* *********:* *: * MNLLDEKFSFLASSKQIVSSANEVDKVIVFERGDLVFVFNFHPVNAYEGYKIGCDLPGRY MNLLDDKFSFLKSTKQIVSSAHDEDKVIVFERGDLIFVFNFHPENTYEGYKVGCDLPGKY *****:***** *:*******:: ***********:******* *:*****:******:* RVALDGDALMFGGKGRVGHDVDHFTNPEGIPGVPETNFNNRPNSFKVLSPPRTCVAYYRV RVALDSDAWKFGGHGRVGHGVDHFTSPEGIPGVPETNFNNRPNSFKVLSPARTCVVYYRV *****.** ***:*****.*****.************************.****.**** EDYPELSVSTSTTVANILEETGGAKDDMTEPEEHVLVEESVSSSSVAEVLEDEQADEAED DENQEGSNDSLVG----LEDTFAAADVAKIPDKSASIE :: * *.:. **:*.* *. *::. :* DTPEPKESTLEEESVSSSNIADVLKEEQVDEAENDISEVEESNLGEEIVSSSTVEVRAQK SEYSNNLDGVKETSTSAQISVESEV---INL.*.*::. *:*:.. :*.* * : EAVEAEDDSLKPEVPGPSEKSSEPSIEKAVDRTRPDDSTINRISARGFDISKPLLSLLHF DKVGIVAASLDREI : * **. *: PLVRGFLFSV PvSBE Fig 5.6 The alignment between amino acid sequences of the unknown protein (putative A. cruentus starch branching enzyme I) and Phaseolus vulgaris starch branching enzyme 1 (PvSBE1). The sequence in red color is the highly conserved region in which the percentage of identities is % (80.997% ungapped) in 746 aa overlap (70-815:70-811). The lined sequences in green color are the most conserved domains in all SBEs from various higher plants. *, : and - represent identical amino acids, positive amino acids and gaps, respectively. 146

167 UNKNOWN PROTEIN (Amaranthus cruentus starch branching enzyme I?) Pro-rich HSHAS GFRFDGVT AEDVS AESHDQ StSBEI (potato starch branching enzyme 1) Fig 5.7 The amino acid sequence comparison between the unknown protein and Solanum tuberosum starch branching enzyme I (StSBEI). The numbers in the figure represent the amino acid positions. Red bar shows % identity (80.997% ungapped) in 746 aa overlap (70-815:70-811). The amino acids in green color are thought to be conserved regions in all SBEs from various higher plants The structure of the protein, AcSBEI The primary structure of AcSBEI ProtParam program was used in this experiment to analyze the physicochemical parameters (primary structure analysis of protein) of A. cruentus SBEI, kidney bean SBE1, and rice SBEI, respectively. The results of computed parameters are listed in Table 5.3. The computed parameters indicate that all SBEIs analyzed in the experiment have very similar physicochemical parameters. Therefore, although SBEIs from different higher plants compose different amino acid sequence, their physicochemical properties are almost the same. This conclusion is consistent with the analysis on SSIIs in Chapter IV. 147

168 Table 5.3 The physicochemical parameters of A. cruentus SBEI, kidney bean SBEI and rice SBEI A. cruentus SBEI Kidney bean SBEI Rice SBEI Number of amino acid Mw (kda) pi Total number of negatively charged residues (Asp+Glu) Total number of positively charged residues (Arg+Lys) Atomic composition Carbon C Hydrogen H Nitrogen N Oxygen O Sulfur S Total number of atoms Instability index (II) (Stable) (Stable) (Stable) Aliphatic index Grand average of hydropathicity (GRAVY) The secondary structure of AcSBEI Fig 5.8 shows the secondary structure of AcSBEI predicted by SSpro8. The secondary structure of AcSBEI provides the location of alpha helices, and beta strands within the protein for the prediction of 3D structure. 148

169 Query name: AcSBEI Query length: 961 Prediction: MSISASHTVAIPLGSVLLSAKRGSARDGSFLTQPLALNSRSKTLQWRNSSHSKFHALVCQGMKHCSSISA CCEEEECCCCCCCCCECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEE CCECECEEEEECTHHHHHHHHTTCCTTSCECCCCCEECCCCHEEEEECCTHHHHHHHEHHTCCHCCHHHE eeeeeeeeee-eeee-e-eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee--eeeee-eeeee VLTESSKTMGVTEGDTENLGIFDVDPSLEPYKDHFRYRMKRYVDQTQLIEKYEGGLEEFAQGYLKFGFNR EECCCCCCEEECCCCCCCCEEEECCCCCHCHHHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHCCCE EEEECCTTCCCCCCCCTTCEEEECCTTCCCCHHHHHHHHHHHHHHHHHHHHHTTHHHHHHHHHHHHTCCT eeeeeeeeeeeeeeeeeee---eeeeeeeeeeee-eee-eeeeeeeee-eeeeeeeee--eee-e-eeee EEGGIVYREWAPAAQEAQLIGDFNGWDGTMHSMEKNQFGVWSIRIPDSDGKPAIPHNSRVKFRFKHGNGV CCCCEEEEEECCCCCEEEEEEECCCCCCCCCCCECCCCCEEEEEECCCCCCCCCCCCCEEEEEEECCCCC HTTCEEEHHHHHHHHHHHHHECCTTCCSCHCHHHHTCEEEEEEEECCTTSCCCCCTTCEEEEEEETTTEE eee----ee--eeeee-ee-eeeeeeeeeeeeeeeeee-----e-eeeeeeeeeeeeee-e-e-eeeeee WVDRIPAWIKYATVDPTRFAAPYDGVYWEPPPEESYQFKYPRPPKPKAPRIYEAHVGMSSSEPRVNSYRE EECCCCHHHHHCCCCCCCCCCCCCCCCCCCCCHHHHCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCHHH EEECCCEEEEHHECCTCEEECCTTTEEECCCCCGEEEECCCCCCCCCCCEEEEEEETCCSSSCCCCCHHH e-ee-eee-ee-eeeeeeee-eeeeeeeeeeeeeeeeeeeeeeeeeeeee-ee-eeeeeeeeeeeeeeee FADEVLPRIKANNYNTVQLMAVMEHSYYASFGYHVTNFFAVSSRSGNPEDLKYLIDKAHSLGLRVLMDVV HHHHHHHHHHHCCCCEEEEECCECCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHCCCEEEEEEC HHHHHHHHHHHTTHHHHHHHHHHHHHEEEETTEEEEEEEEEECSCSCHHHHHHHHHHHHHHTCEEHHHHH --ee-eee-eeeeeee-e-----ee-ee-e-eee-ee----eeeeeeeee-ee--ee-eee--e---e-- HSHASNNVTDGLNGFDVGQGAQESYFHTGDRGYHKLWDSRLFNYANWEVLRFLLSNVRWWLEEYRFDGFR CCCCCCCCCCCHHHCCCCCCCCCCEEECCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCCEE ECCCCSCCCTTCTTCCTTCTCEEEEECCSCTTHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCTTEE eee-eeeeeeeeeeeeeeee-ee-e-eeeeeeeee-eeee--ee-e-e--e---ee-ee--ee-e-ee-e FDGVTSMLYHHHGINMGFSGNYNEYFSEATDVDAVVYLMLANRLIHNIFPDVTVVAEDVSGMPGLCRPVS ECHHHHHHCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHCCCEEEEEECCCCCCCEECECC ETTHHHHHHHHTTEECTCCSCCHHHHHHHHHHHHHHHHHHHHHHHHHHCTTEEEEEEECTTCTTCCCCCC -ee------e-e--e---eeeeeee-ee-ee-e ee--ee-eee-e---ee-eeeeee-eeee EGGVGFDYRLAMAIPDKWIDYLKNKKDEEWSMNEVMLSLTNRRYTEKCIAYAESHDQAIVGDKTVAFLLM CCCCCCCEEHCCCCHHHHHHHHCCCCCCCCCCCCEEEEECCCCCCCCEEEECCCCCCEEECCCEEEEEEE TTCCHHHHHHHHHCCHHHHHHHHTCCCHHHHHHHHHHHHHCHHHHHHHHHHHHHHHHHEEHHHHHHHHHH eee-e-e-e--ee-eee--ee-eeeeeeeeeeee---eeeeeeeeee--ee-eeeeee-eeeee DQEMYTGMSCLTEASPVVERGIALHKMIHLITMAMGGEGYLNFMGNEFGHPEWIDFPREGNGWSYKMCRR CCHHCCCHHHCCCCCCCHHHHHHHHHHHHHHHHHCCCCCEEEECCCCCCCCCCCCCCCCCCCCCHHHCCC HHHHHHTCCEHCCHCHHHHHHHHHHHHHHHHHHHHTTCEEEETTCCTTSCCGEECCCCTTCHHHHHHHHH eeeeeeeee--eeeeee-ee----ee e-e---e--eeeeeeeee-e-eeeeeeeeee--ee QWNLPDTDHLRYKFLNLFNGAMNLLDEKFSFLASSKQIVSSANEVDKVIVFERGDLVFVFNFHPVNAYEG CCCCCCHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCEEEEECCCCCCEEEEECCCEEEEEEECCCCCCCC HHCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHHCHHHEEECGTCCCEEEEEEETCEEEEEECCCECEEET eeeeeeeee-eee--e--ee--eeeeee-eeeeeeeeeeeeeeeeee----eeee-----e-eeee-ee- YKIGCDLPGRYRVALDGDALMFGGKGRVGHDVDHFTNPEGIPGVPETNFNNRPNSFKVLSPPRTCVAYYR CEECCCCCCCEEEEECCCCHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEECCCCEEEEEEE EEEECCCTTEEEEEECTCEEEETCTCCECCCCCTCCCTTTCCTCCCCCTTCCCTCEEEECCCCEEEEEEE ee---eeeee-e---eee----eeeee-eee-ee-eeeeeeeeeeeeeeeeeeeeeeeeeeeee-e-eee VEDYPELSVSTSTTVANILEETGGAKDDMTEPEEHVLVEESVSSSSVAEVLEDEQADEAEDDTPEPKEST CCCHCCCCCCCCCCCCCCHHHCHCHHCCCCCCCCHCCCCCCCCCCCECCCCCCCCCCCCHHHCCCCCCCC EECCCCEEECCHHHHHHHHHHHTCCCCTCCCCCGEEEEEEEECCHHHHHHHHHHHHHHHCCCCCCCCCCC -eeeeeeeeeeeee--e--eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee LEEESVSSSNIADVLKEEQVDEAENDISEVEESNLGEEIVSSSTVEVRAQKEAVEAEDDSLKPEVPGPSE CCCEEEEHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCEEECCCCEEEECHHHHHHCHCCCCCCCCCCCCC EEEEEECCHHHHHHHHHHHHHHHHTCCCHHEECCTHHEEECCCHHEEEHHHHHHHHHCTCCCCCCCCCCC eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee KSSEPSIEKAVDRTRPDDSTINRISARGFDISKPLLSLLHFPLVRGFLFSV CCCCCCHHHHHCCCCCCCCCHHHEECCCCCCCHHHHHHHCCCCHCEEEECC CCCCCCHHHHHHTCCCCSCEEEEEEETTCCCCHHHHHHHTCHHEHEEEECC eeeeee-eeeeeeeeeeeee-ee-eeeeee-eeee-ee-e-ee-e--eeee Fig 5.8 The secondary structure of A. cruentus starch branching enzyme I (AcSBEI). 149

170 The 3D structure of AcSBEI For the analysis of 3D structure of AcSBEI, a 59 aa (1-59) chloroplast transit peptide was splitted out. A more conserved region (from 261 aa to 770 aa) of 510 aa long was amplified from AcSbeI cdna for the prediction of 3D structure. The peptide was consequently named as AcSBEI-510. No coil was found in this peptide when using Paircoil program to analyze. Using 3D-JIGSAW (version 2.0) to predict the 3D structure of AcSBEI-510 peptide, a best alignment template, 1m7x_C, was found from sequence databases (PFAM+PDB+nr) (Fig 5.9). The score of accuracy of AcSBEI to 1m7x_C was , greater than 0. Thus it indicates that 95 percent of alignments over the two sequences are accurate. The E-value number (1e-71) shows the expected number of random sequences with the same similarity in the databases. Since the two proteins have the better agreement in their structural superposition, it can be concluded that AcSBEI is highly similar to the template, 1m7x_C. The description of 1m7x_C in PDB indicates that 1m7x_C is a chain of bacterial glycogen branching enzyme, Ec (Abad et al., 2002). The bacterial glycogen branching enzyme has a preference for transferring chains of 5 to 16 glucose units and catalyzing the formation of the α(1,6)-glucosidic linkages in glycogen by scission of a 1,4-α-linked oligosaccharide from growing α(1,4)- glucan chains and the subsequent attachment of the oligosaccharide to the α(1,6) position. 150

171 TARGET AcSBEI_510 query m7x_C HLRPYETLGAHADTMDGVTGTRFSVWAPNARRVSVVGQFNYWDGRRHPMRLRKESGIWELFIPGAHNGQLYKYEMIDANG ACCI 5421*452*127556#956112** **** SS_QP SS_TP CCCCCCCCCCCCCCCCCCCCEEEEEECCCCCEEEEEEECCCCCCCCCCCCCCCCCCEEEEEECCCCCCCEEEEEEECCCC SS CCCHHHCCCEEEEEECCEEEEEEEEECCCCCCEEEEECCCCCCCCCCCCEEECCCCEEEEEEECCCCCCEEEEEEECCCC query IYEAHVGMS---SSEPRVNSYREFADEVLPRIKANNY 1m7x_C NLRLKSDPYAFEAQTASLICGLPEKVVQTEERKKANQFDAPISIYEVHLGSWRRHTDNNFWLSYRELADQLVPYAKWMGF ACCI *****111** *54*173*152*66111 SS_QP CEEEECCCC---CCCCCCCCCHHHHHHHHHHHHHCCC SS_TP CCCCCCCCCCCCCCCCCCCCCCCHHCCCCHHHCCCCCCCCCEEEEEEECCCCCCCCCCCCCCCHHHHHHHHHHHHHHCCC SS CEEEECCCCCCCCCCCEECCCCCCCCCCCHHHHHCCCCCCCCEEEEECHHHCCCCCCCCCCCCHHHHHHHHHHHHHHCCC query NTVQLMAVMEHSYYASFGYHVTNFFAVSSRSGNPEDLKYLIDKAHSLGLRVLMDVVHSHASNNVTDGLNGFDVGQGAQES 1m7x_C THLELLPINEHPFDGSWGYQPTGLYAPTRRFGTRDDFRYFIDAAHAAGLNVILDWVPGHFPT DD ACCI 21*** *14*321** **42*283811** SS_QP CEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHCCCEEEEEEECCCCCCCCHHHHHHHCCCCCCCCE SS_TP CEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHCCCEEEEEEECCCCCC CC SS CEEEECCCEECCCCCCCCCCCCEEEEECCCCCCHHHHHHHHHHHHHCCCEEEEEECCCCCCC CC query YFHTGDRGYHKLWDSRLFNYANWEVLRFLLSNVRWWLEEYRFDGFRFDGVTSMLYHHHGINMGFSGNYNEYFSEATDVDA 1m7x_C FALAEFDGTNLYEHSLIYNYGRREVSNFLVGNALYWIERFGIDALRVDAVASMIYR ENLEA ACCI *133*114?65*116484*22111*1***1122*11**11122*16115? * SS_QP EEECCCCCCCCCCCCCEECCCCHHHHHHHHHHHHHHHHHHCCCEEHHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCH SS_TP CCCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCEEEHHHHHHHHHC CCCHH SS CCCCCHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHCCCCCEEEECCHHHHHCC CCHHH query VVYLMLANRLIHNIFPDVTVVAEDVSGMPGLCRPVSEGGVGFDYRLAMAIPDKWIDYLKNKKDEEWSMNEVMLSLTNRRY 1m7x_C IEFLRNTNRILGEQVSGAVTMAEESTDFPGVSRPQDMGGLGFWYKWNLGWMHDTLDYMKLDPVYRQYHHDKLTFGILYNY ACCI * * ** * *4 SS_QP HHHHHHHHHHHHHHCCCCEEEEECCCCCHHHHHCCCCCCCCCEEEEECCCCHHHHHHHCCCCCHHHHHHHHHHHHHCCCC SS_TP HHHHHHHHHHHHHHCCCCEEEEECCCCCCCEEECCCCCCCCCCCEECCCCHHHHHHHHHCCCCCCHHHCCCCHHHHHHHH SS HHHHHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCCEEECHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCHH query TEKCIAYAESHDQAIVGDKTVAFLLMDQEMYTGMSCLTEASPVVERGIALHKMIHLITMAMGGEGYLNFMGNEFGHPEWI 1m7x_C TENFV-LPLSHDEVVHGKK SILDRMPGDAWQKFANLRAYYGWMWAFPGKK-LLFMGNEFAQGREW ACCI *1*1* *1*1**1**1****1-***111** SS_QP CCCEEEEECCCCCCCHHHEEEECCCCCHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHCCCCCEEECCCCCCCCCCC SS_TP HHCEE-ECCCCCEEEECCC CCCCCCCCHHHHHHHHHHHHHHHHHHCCCCC-CCCCCCCCCCCCCC SS HCCEE-EEECHHHCCCCCC CCCCCCCCCHHHHHHHHHHHHHHHCCCCCEE-EEECCHHHCCCCCC query DFPREGNGWSYKMCRRQWNLPDTDHLRYKFLNLFNGAMNLLDEKFSFLASSKQIVSSANEVDKVIVFER-----GDLVFV 1m7x_C NHDASLD---WHLLEGGDNWHHGVQRLVRDLNLTYRHHKAMHE-LDFDPYGFEWLVVDDKERSVLIFVRRDKEGNEIIVA ACCI **34**33**42* * ****** **1 SS_QP CCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCEEEEECCCCCCEEEEEC-----CCEEEE SS_TP CCCCCCC---CCCCCCCCCHHHHHHHHHHHHHHHHHHCHHHHH-HCCCHHHEEEEECCCCCCCEEEEEEECCCCCEEEEE SS CCCCCCC---CCCCCCCCCHHHHHHHHHHHHHHHHCCCCCCCC-CCCCHHHEEEEECCCCCCCCCEEEEECCCCCEEEEE query FNFHPVNAYEGYKIGCDLPGRYRVALDGDALMFGGKGRVGHDVDHFTNPEGIPGVPETNFNNRPNSFKVLSPPRTCVAYY 1m7x_C SNFTPVP-RHDYRFGINQPGKWREILNTDSMHYHGSNAGNGGTVH SDEIASHGRQHSLSLTLPPLATIWLV ACCI ** ** ** ? #272* *1*13 SS_QP EECCCCCCCCCCEECCCCCCEEEEEECCCCHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEECCCEEEEEE SS_TP EECCCCC-CCCEEECCCCCCEEEEEECCCHHHHCCCCCCCCCCEE CCCCCCCCCCEEEEEEECCCCEEEEE SS EECCCCC-EEEECCCCCCCCEEEEEEECCCCHHHCCCCCCCCCEE CCCCCCCCCCCCCCEEECCCEEEEEE query R--- 1m7x_C REAE ACCI 4389 SS_QP C--- SS_TP ECCC SS EECC Fig 5.9 The 3D prediction of core region from A. cruentus starch branching enzyme I (AcSBEI). 151

172 The known 3D structure of 1m7x_C shown in Fig 5.10 was used as reference for the 3D prediction of AcSBEI. Fig 5.10 The 3D structure of 1m7x_C (Bacterial glycogen branching enzyme) (from the protein data bank) AcSbeI is a low copy gene in grain amaranth genome and highly expressed in grain amaranth developing seeds The information of A. cruentus starch branching enzyme I (AcSbeI) gene in grain amaranth genome is obtained from Southern blotting. Grain amaranth chromosomal DNA was isolated from grain amaranth seedling and digested completely with Xho I, Hind III, BamH I, EcoR I and EcoR V, respectively. The probe was a 1.7 kb fragment from AcSbeI cdna, digested with Xho I and EcoR I. The probe contained one EcoR V restriction enzyme site in the middle of the 152

173 fragment but BamH I and Hind III restriction enzyme sites were absent. The Southern blotting result shown in Fig 5.11 indicates that AcSbeI gene is present in grain amaranth genome. A EcoR V Xho I Hind III BamH I EcoR I Probe EcoR V Xho I AcSbeI cdna B Xho I BamH I EcoR V Marker Hind III EcoR I C 6.0 kb 4.5 kb 3.3 kb 3.0 kb 1.2 kb 0.8 kb Fig 5.11 The result of Southern blotting for acsbei gene (the probe is a 1.7kb fragment from AcSbeI cdna). A, The schematic picture of restriction enzyme digestion sites on AcSbeI cdna; B, chromosomal DNA digested by restriction enzymes. C, The result of Southern blotting. The marker is MassRuler DNA ladder (MBI Fermentas). 153

174 One DNA fragment (~4.5 kb) in the digestion with Xho I is hybridized to the probe. In AcSbeI cdna, a fragment which is approximately 2.0 kb between two Xho I restriction enzyme sites contains the sequence of probe, thus it indicates that the ~4.5 kb pattern from genomic DNA contains the sequence of probe and some introns between two Xho I restriction enzyme sites. Moreover, the full-length of AcSbeI gene should be greater than 4.5 kb and AcSbeI is a single copy gene in grain amaranth genomic DNA. Two DNA fragments, which are approximately 4.0 kb and 0.8 kb, are found in the digestion with EcoR V. It is known that two EcoR V restriction enzyme sites are on the sequence of AcSbeI cdna and the length is ~2.0 kb between them. Therefore, the ~4.0 kb DNA fragment should be corresponding to the cdna fragment digested with EcoR V. Similarly, two fragments are found in the digestion with Hind III, BamH I, and EcoR I respectively. Since there is no restriction enzyme site of each of them in the sequence of probe, it indicates that one more Hind III, BamH I, or EcoR I restriction enzyme site is in the introns of AcSbeI gene. Northern blotting was used to analyze the expression of AcSbeI gene in different plant tissues, such as roots, stems, leaves, flowers and seeds (Fig 5.12). The probe for the hybridization was the same as used in Southern blotting (the 1.7 kb fragment digested with Xho I and EcoR I). However, less stringent conditions were used for Northern hybridization and washing than those used in Southern blotting. 154

175 R ST L F SE rrna Fig 5.12 The result of Northern blotting showing different tissues hybridized with a cdna fragment (1.7 kb) from the putative AcSbeI gene. R, ST, L, F, and SE represent the total RNA from amaranth roots, stems, leaves, flowers, and seeds, respectively. The result of Northern blotting using AcSbeI cdna as probe was very similar to the result when AcSsII cdna was used as probe in section No AcSbeI expression was detected in grain amaranth roots, whereas AcSbeI was highly expressed in grain amaranth seeds. Moreover, it was found that AcSbeI was also highly expressed in grain amaranth flowers because some developing seeds might be included in the flower sample and the expression of these genes in developing seeds would affect the result of Northern hybridization AcSbeI cdna encodes an active starch branching enzyme The result of sequence alignment indicates that the predicted peptide encoded by AcSbeI cdna is a starch branching enzyme I. The peptide is 961 aa long and contains a 59 aa chloroplast transit peptide in the N end. Moreover, the fragment from 60 aa to 961 aa is a highly conserved region compared with other SBEIs. To analyze the activity of this peptide, a target cdna amplified from AcSbeI cdna was cloned in pet28(a) plasmid under the control of strong 155

176 bacteriophage T7 transcription and translation signals; expression was induced by providing a source of T7 RNA polymerase in KV832, a mutant strain of E. coli that lacks of the activity of glycogen branching enzyme (Fig 5.13). AcSbeI pns1 A C B KV832/pNs1 KV832/pET28(a) A B C DH5á Fig 5.13 Complementation analysis of AcSBEI activity in E. coli. The color development in the experiment was upon iodine staining. A, GBE-deficient strain KV832 transformed with pns1; B, strain KV832 transformed with pet28(a); C, GBE-producing strain DH5α. Plasmid pns1 was from AcSbeI cdna in which the ctp (green bar) was cleaved for avoiding its expression in E. coli. As shown in Fig 5.13C, wild type DH5α colonies were stained golden yellow by iodine since this strain is able to synthesize glycogen branching enzyme; whereas the KV832/pET28(a) cells were not stained (pale white) due to a lack of glycogen branching enzyme (Fig 5.13B). The expression of pns1 cdnas in the mutant strain changed the iodine staining from white to golden yellow (Fig 5.13A). Thus the result shows that the full-length cdna of AsSbe1 is able to encode a functional starch branching enzyme. 156